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The Vaccinia Virus 39-kDa Protein Forms a Stable Complex with the p4a/4a Major Core Protein Early in Morphogenesis
Abstract
The vaccinia virus (VV) 39-kDa protein, the product of the A4L gene, is a highly antigenic protein of the viral core. Pulse-chase and immunoprecipitation experiments have shown that the 39-kDa protein interacts with p4a (encoded by the A10L gene), the precursor of the most abundant virion protein. This interaction is maintained with the processed 4a form that arises during virion maturation. The controlled disruption of mature viral particles showed that the 39-kDa and 4a proteins are tightly bound within the virion. Immunoelectron microscopy showed that both proteins first localize within the cytoplasm and later accumulate inside the viral factories, reaching these locations via a mechanism apparently unrelated to cellular membranes. Double labeling experiments showed a colocalization of both proteins in all virus-induced structures.
... Beyond these morphological descriptions of the core architecture, identities of proteins forming the core wall and palisade layer were derived from studies using 1) immunogold labeling of intact VACV MV and purified cores (Vanslyke and Hruby 1994, Roos et al. 1996, Risco et al. 1999, Pedersen et al. 2000, 2) biochemical and proteomic studies via extraction and partitioning of MV components (Jensen et al. 1996, Chung et al. 2006, Mirzakhanyan and Gershon 2019, Bidgood et al. 2022 and 3) genetic experiments via recombinant viruses and inducible mutants of selected protein candidates (Risco et al. 1999, Heljasvaara et al. 2001, Rodriguez et al. 2006, Jesus et al. 2014. Specifically, protocols to isolate intact cores from native virus particles ( Figure 1A), using non-ionic detergent under reducing conditions, allowed a more straightforward biochemical and ultrastructural description (Dubochet et al. 1994, Bidgood et al. 2022. ...
... Beyond these morphological descriptions of the core architecture, identities of proteins forming the core wall and palisade layer were derived from studies using 1) immunogold labeling of intact VACV MV and purified cores (Vanslyke and Hruby 1994, Roos et al. 1996, Risco et al. 1999, Pedersen et al. 2000, 2) biochemical and proteomic studies via extraction and partitioning of MV components (Jensen et al. 1996, Chung et al. 2006, Mirzakhanyan and Gershon 2019, Bidgood et al. 2022 and 3) genetic experiments via recombinant viruses and inducible mutants of selected protein candidates (Risco et al. 1999, Heljasvaara et al. 2001, Rodriguez et al. 2006, Jesus et al. 2014. Specifically, protocols to isolate intact cores from native virus particles ( Figure 1A), using non-ionic detergent under reducing conditions, allowed a more straightforward biochemical and ultrastructural description (Dubochet et al. 1994, Bidgood et al. 2022. ...
... In immunogold labeling experiments A10 and A4 were detected at the outer surface of the core wall , Roos et al. 1996, Risco et al. 1999, Pedersen et al. 2000. Proteins A3 and A4 were named as components of the palisade layer (Wilton et al. 1995. ...
Poxviruses are among the largest double-stranded DNA viruses with members such as Variola virus, Monkeypox virus and the famous vaccination strain Vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core, found in all infectious poxvirus forms, has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to the individual architectural features of the core. Hence, which proteins constitute which layers of the core, such as the so-called palisade layer and the inner core wall has remained enigmatic.
Here, we have performed a multi-modal cryo-electron microscopy (cryo-EM) approach to elucidate the structural determinants of the VACV core. In combination with molecular modeling using AlphaFold, we unambiguously identify trimers formed by the cleavage product of A10 as the key component of the palisade layer. This allows us to place previously-obtained descriptions of protein interactions within the core wall into perspective and to provide a substantially revised model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely identical among Poxviridae , implying that our structural observations should be generalizable over most, if not all members of this important virus family.
One sentence summary
Single-particle cryo-EM, cryo-electron tomography, and AlphaFold modeling reveal the structural architecture of the poxvirus core and identify trimers of protein A10 as the key component of the palisade layer.
... Beyond these morphological descriptions of the core architecture, identities of proteins forming the core wall and palisade layer have been derived from studies using (1) immunogold labeling of intact VACV MV and purified cores 9,10,13-16 ; (2) biochemical and proteomic studies via extraction and partitioning of MV components 5,[17][18][19] ; and (3) genetic experiments via recombinant viruses and inducible mutants of selected protein candidates 16,[20][21][22][23] . Specifically, protocols to isolate intact cores , showing previously described structural entities, such as the palisade layer, inner core wall, pore-like structure and viral genome. ...
... The position of structural proteins within the core Immunoprecipitation experiments showed that A10 and A4 form a stable complex even before proteolytic cleavage and MV formation 16 . Moreover, previous studies using immunolabeling speculated that the spike protein in the palisade layer was A4 (refs. ...
Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses.
... Prior studies suggest that A3 is a component of the inner layer of the core wall, while A10 forms the external layer of the core wall in a complex with A4 Ichihashi et al., 1984;Risco et al., 1999). Given these observations, we were interested in analyzing whether A10 is contained in viral particles formed in the absence of A3. ...
... A10, like L4, binds to microtubules (Ploubidou et al., 2000). However, the decrease in A10 accumulation was not as striking as observed for L4 and this might be explained by the retention of A10 in the factories by A4, as A10 and A4 form a complex at the beginning of morphogenesis (Risco et al., 1999). The presence of A3 in viral factories in the absence of A10 has not been analyzed. ...
Maturation of the vaccinia virion is an intricate process that results in the organization of the viroplasm contained in immature virions into the lateral bodies, core wall and nucleocapsid observed in the mature particles. It is unclear how this organization takes place and studies with mutants are indispensable in understanding this process. By characterizing an inducible mutant in the A3L gene, we revealed that A3, an inner core wall protein, is important for formation of normal immature viruses and also for the correct localization of L4, a nucleocapsid protein. L4 did not accumulate in the viral factories in the absence of A3 and was not encapsidated in the particles that do not contain A3. These data strengthen our previously suggested hypothesis that A3 and L4 interact and that this interaction is critical for proper formation of the core wall and nucleocapsid.
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... Immunogold labeling for electron microscopy demonstrated that after treatment with 3 M NaCl, cores were depleted of the proteins F17 and A4 from the surface, but the proteins A10 and H1 were still attached to the particle (Fig. 5B). These results are consistent with previous observations suggesting that A4 protein constitutes the palisade structure of the core wall and showing that A4 interacts with the core wall protein A10 Risco et al., 1999). Interestingly, the NaCl/ DTT treatment exposed some A3 on the surface of the core. ...
... In accordance with this model are our results with the immunogold labeling of A4, where the gold particles uniformly decorate the exterior of the core wall. The partial removal of A4 from the core wall after NaCl/DTT treatment was unexpected because this protein forms a stable complex with A10 that was not removed during the procedure (Chung et al., 2006;Risco et al., 1999). Interesting, after NaCl/DTT and hypertonic shock the core wall protein A3 could be visualized by immunogold labeling, suggesting that after peeling off the A4 layer, the A3 layer became visible. ...
The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion.
Published by Elsevier Inc.
... Less is known as to how cores assemble. A core protein 39 kDa (A4L gene) interacts with the major core protein 4a (A10L gene) by an unknown mechanism [23, 43]. Through the use of a two-hybrid analysis it has been identified that out of the about 200 different proteins of VV, there are 37 protein-protein interactions, some of them self-interacting [24]. ...
... The different recombinant viruses were purified by banding through sucrose and by CsCl gradients. The virus particles in our purified preparations were largely IMVs, as determined by EM, by sedimentation on CsCl gradients, after reactivity with a specific monoclonal antibody, and as described from previous studies using this virus purification procedure [43, 51]. To partition the virion proteins in different compartments, we sequentially treated the purified virus preparations with detergents . ...
Vaccinia virus (VV) is one of the largest and most complex of animal viruses, with a virion that contains about 100 different polypeptides. Assembly of the viral proteins occurs in discrete cytoplasmic sites leading to formation of two infectious forms, an abundant (>90%) intracellular mature virus (IMV) with an envelope, and a minor extracellular enveloped virus (EEV) with an extra membrane acquired from the trans-Golgi network. It has been shown that while EEV contains in the outer membrane cellular proteins probably acquired during virus release from cells, however, IMV exclude host proteins during assembly. Since VV recombinants (VVr) expressing genes of interest are candidates as potential vaccines against pathogens and cancer, it becomes important to know if VVr can acquire foreign proteins during morphogenesis. In this investigation we show that purified virions (IMVs) from VVr can incorporate foreign proteins into different sites in the virus particle. By sequential fractionation of virion compartments with detergents, we found foreign proteins in the lipid envelope (cytokine IL-12 and CS antigen of Plasmodium yoelii), as part of a protein matrix-like membrane (HIV-1 gp41 of env), or more closely associated with the core containing the DNA complexes (HIV-1 gp160; a multiepitopic protein with the V3 loop of HIV-1 Env from different isolates, and beta-galactosidase). Similar findings were observed with purified virions derived from the WR strain as well as from the highly attenuated modified vaccinia virus Ankara (MVA) strain. These observations should be taken into consideration when VVr are used in clinical trials or in other vaccination approaches.
... Hsp90 IS IMPORTANT FOR VACCINIA VIRUS GROWTH to be present on incoming cores, virosomes, and virion progeny, staining of A4L protein in cells will faithfully reflect these viral structures in infected cells (49,57). For technical convenience, we used a recombinant vaccinia virus that expresses A4L core protein fused to an ECFP to facilitate a direct visu-alization of virosomes and intracellular virions in virus-infected cells (Fig. 6g, k, and s). ...
... p4a protein is 102 kDa in size and is cleaved at sites between amino acids (aa) 614 and 615 and aa 697 and 698 to generate core protein 4a and p25, respectively (66,68). Both p4a and 4a proteins interact with A4L protein during virion morphogenesis (57). A recombinant virus that expresses the A10L gene under isopropyl--D-thiogalactopyranoside (IPTG) regulation indicated that core proteins p4a-4a are important for virion morphogenesis (25). ...
Molecular chaperones assist protein folding, and some chaperones are induced by heat, nutrient depletion, or pathogen invasion.
This study investigates the role played by Hsp90 in the life cycle of vaccinia virus. The titer of vaccinia intracellular
mature virions (IMV) was reduced by 2 orders of magnitude in RK13 cells treated with geldanamycin (GA), which blocks the ATPase
activity of Hsp90. GA does not affect expression from the viral early promoter, but treatment with GA delays DNA replication
and intermediate gene transcription and reduces expression from the viral late promoter. Vaccinia virus infection does not
induce Hsp90 expression; however, intracellular distribution of Hsp90 is altered in virus-infected cells. Hsp90 is restricted
to the cytoplasm of mock-infected cells; in contrast, Hsp90 is transiently associated with virosomes in virus-infected cells
although it is not incorporated into IMV. In addition, Hsp90 interacts with viral core protein 4a, the mature form of the
A10L gene product, in virus-infected cells. In conclusion, these results suggest that a cellular chaperone protein, Hsp90,
is important for vaccinia virus growth in cultured cells and that viral core protein 4a associates with Hsp90-containing complexes
in the infected cells.
... The phenotype observed on repression of A10 mimics to a great degree the phenotype of mutants affected in components of the seven-protein complex described previously. The A10 precursor and the 60-kDa cleavage product form stable complexes with another abundant virion core protein, A4 (Risco et al., 1999). The implications of this interaction are discussed in Section IX.B.3 along with the role of A4 in MV formation. ...
... Consistent with these results, anti-A4 stains the surface of isolated cores, leading Roos et al. (1996) and Cudmore et al. (1996) to conclude that A4 comprises, at least in part, the "spike" or palisade layer of the outer core wall. The A4 protein forms a complex with both the A10 precursor protein, p4a, and the 62-kDa proteolysis product, 4a (Risco et al., 1999). Nevertheless, inducible A10 mutants present a significantly different phenotype than A4 mutants. ...
Poxviruses comprise a large family of viruses characterized by a large, linear dsDNA genome, a cytoplasmic site of replication and a complex virion morphology. The most notorious member of the poxvirus family is variola, the causative agent of smallpox. The laboratory prototype virus used for the study of poxviruses is vaccinia, the virus that was used as a live, naturally attenuated vaccine for the eradication of smallpox. Both the morphogenesis and structure of poxvirus virions are unique among viruses. Poxvirus virions apparently lack any of the symmetry features common to other viruses such as helical or icosahedral capsids or nucleocapsids. Instead poxvirus virions appear as "brick shaped" or "ovoid" membrane-bound particles with a complex internal structure featuring a walled, biconcave core flanked by "lateral bodies." The virion assembly pathway involves a remarkable fabrication of membrane-containing crescents and immature virions, which evolve into mature virions in a process that is unparalleled in virology. As a result of significant advances in poxvirus genetics and molecular biology during the past 15 years, we can now positively identify over 70 specific gene products contained in poxvirus virions, and we can describe the effects of mutations in over 50 specific genes on poxvirus assembly. This review summarizes these advances and attempts to assemble them into a comprehensible and thoughtful picture of poxvirus structure and assembly.
... It is interesting to report that the A12L immunoprecipitates turned out to be VV core (A4L, A10L, L4R, and F17R) and membrane (A17L, A14L, A27L) proteins. The gene product of A4L, a 39 kDa core protein, associates with a 60 kDa cleavage product (4a) of A10L, and stimulates proper progression of IV to IMV [19,20], are participated in correct viral genome packaging, which is an essential step for assembling mature virions. On the other hand, A27L, a 15 kDa VV envelope protein also incorporates with A17L just like A14L, and responsible for envelopment of IMV particles [17,21,18] . ...
... Recent studies of early morphogenic processing events have provided the participation of the membrane proteins such as A17L, A14L and A27L in early development of IV particles as well as IEV particles, recruiting nascent viral membranes to the viral foci, inducing their stable attachment to the surfaces of viral factories, and developing envelopment of IEV particles [23] . Unlike these membranous proteins, the association of A4L with A10L plays a role in the correct assembly of nucleoprotein complex and organization of IV content with the membranes while F17R (a DNA-binding phosphoprotein), and L4R (a DNA-binding protein) are proposed to work for the correct viral genome packaging and efficient transcription [20,242526. These participations of the A12L-associated proteins throughout the progression of IV to IMV and IEV particles suggest that the A12L may also be involved in multiple stages of virus morphogenesis. ...
Vaccinia virus (VV) undergoes a proteolytic processing to evolve from immature virus particles into intracellular mature virus particles. Most of structural core protein precursors such as p4a, p4b, and p25K are assembled into previrions and then proteolytically processed to yield core proteins, 4a, 4b, and 25 K, which become components of a mature virus particle. These structural rearrangements take place at a conserved cleavage motif, Ala-Gly-X (where X is any amino acid) and catalyzed by a VV encoded proteinase, the I7L gene product. The VV A12L gene product, a 25 kDa protein synthesized at late times during infection is cleaved at an N-terminal AG/A site, resulting in a 17 kDa cleavage product. However, due to the distinct characteristics of A12L proteolysis such as the localization of both the A12L full-length protein and its cleavage product in mature virions and two putative cleavage sites (Ala-Gly-Lys) located at internal and C-terminal region of A12L ORF, it was of interest to examine the A12L proteolysis for better understanding of regulation and function of VV proteolysis. Here, we attempted to examine the in vivo A12L processing by: determining the kinetics of the A12L proteolysis, the responsible viral protease, and the function of the A12L protein and its cleavage events. Surprisingly, the A12L precursor was cleaved into multiple peptides not only at an N-terminal AG/A but also at both an N- and a C-terminus. Despite the involvement of I7L proteinase for A12L proteolysis, its incomplete processing with slow kinetics and additional cleavages not at the two AG/K sites demonstrate unique regulation of VV proteolysis. An immunoprecipitation experiment in concert with N-terminal sequencing analyses and mass spectrometry led to the identification of VV core and membrane proteins, which may be associated with the A12L protein and suggested possible involvement of A12L protein and its cleavage products in multiple stages in virus morphogenesis.
... In addition, A4 has been suggested to link the viral membrane to the palisade as it is present in the membrane fraction after detergent treatment of purified virions (21). Moreover, A10 and A4 form a stable complex that can be cross-linked in purified IMVs (22,23). Recent observations suggest that three A10/A4 heterodimers are consistent with the mass and shape of a single palisade trimer, based on a low-resolution subtomogram-averaged map of the palisade and AlphaFold2 structural predictions (11). ...
Poxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.
... It is conserved in all known poxviridae with no identifiable sequence or structural homo logs outside this family. Conditional mutants under nonpermissive conditions exhibit an interruption of normal virion morphogenesis, reduced virus yield and an accumu lation in the cytoplasm of abnormal immature virus (IV)-like particles lacking the normal, dumbbell-shaped core morphology (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). During virion morphogenesis, P4a is proteolytically processed at two sites (at residues 614 and 697) marked by the diamino acid AG| (9,11,(17)(18)(19)(20)(21)(22)(23). ...
Vaccinia virus, the prototypical poxvirus and smallpox/monkeypox vaccine, has proven a challenging entity for structural biology, defying many of the approaches leading to molecular and atomic models for other viruses. Via a combination of deep learning and cross-linking mass spectrometry, we have developed an atomic-level model and an integrated processing/assembly pathway for a structural component of the vaccinia virion, protein P4a. Within the pathway, proteolytic separation of the C-terminal P4a-3 segment of P4a triggers a massive conformational rotation within the N-terminal P4a-1 segment that becomes fixed by disulfide-locking while removing a steric block to trimerization of the processing intermediate P4a-1+2. These events trigger the proteolytic separation of P4a-2, allowing the assembly of P4a-1 into a hexagonal lattice that encloses the nascent virion core.
IMPORTANCE
An outstanding problem in the understanding of poxvirus biology is the molecular structure of the mature virion. Via deep learning methods combined with chemical cross-linking mass spectrometry, we have addressed the structure and assembly pathway of P4a, a key poxvirus virion core component.
... Protein A4 interacted with p4a but not p4b (Fig 5a), consistent with prior immunoprecipitation and immunogold EM co-localization studies showing a stable interaction between p4a and A4 [41]. p4a-A4 XL were, with one exception, between the N-terminal~half of A4 and residues 170-350 of p4a fragment 1. ...
We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an ‘attachment protein’ sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus.
... (13)(14)(15) For vaccinia virus, proteolytic maturation of the most abundant major structural protein p4a leads to release of the 62, 23, and 9 kDa products, and is essential for the formation of mature infectious VV progeny. (14,15) Furthermore, the p4a major core protein forms a stable complex with a vaccinia virus 39 kDa protein (16) encoded by the VV A4L gene early in morphogenesis, which shares similarities with the ORFV080 gene. As a diagnostic core reagent, 5F2D8 may prove to be highly valuable for further examinations regarding ORFV086 protein proteolysis, viral assembly, the processes of orf pathogenicity, and orf disease control. ...
Orf is caused by the orf virus (ORFV) and is a non-systemic, widespread disease afflicting sheep, goats, wild ruminants, and humans. Recent outbreaks in sheep and goats in Jilin and other northern Chinese provinces raise concerns about orf control in China. Thirty-five hybridoma clones were constructed from splenocytes of BALB/c mice immunized with natural orf virus protein. These hybridomas were used to produce antibodies targeting ORFV proteins. Immunological characterization of these monoclonal antibodies (MAb) showed that the 5F2D8 hybridoma line produced MAb that can recognize the 100, 70, and 20 kDa bands from total viral lysate. This hybridoma was further characterized by immunoprecipitation and peptide sequencing. The results indicate that 5F2D8 specifically recognizes orf virus encoded protein ORFV086, a late expression virion core protein that plays important roles in progeny virus particle assembly, morphogenesis, and maturity. Further experiments demonstrate that this MAb did not react with other viral proteins of ORFV orthopoxviruses, but reacted strongly to different field isolates of orf viruses from China. Additionally, this anti-ORFV086 MAb possesses ORFV neutralizing capability. Sequence alignments and phylogenetic analysis determined that ORFV086 of NA1/11, clustered together with NZ2 and IA82, is highly conserved and has structural similarities with the Vaccinia virus core protein P4a. As such, this MAb has great potential as a diagnostic tool for orf viruses, in the further exploration of orf pathogenesis, and in disease control and prevention.
... (ii) Core proteins. The homologues of CPXV074 and CPXV136 in VACV are E8R and A4L, respectively, and both the E8 and A4 proteins are involved in virion assembly and are also packaged in the viral core (20)(21)(22). ...
Unlabelled:
Cowpox viruses (CPXV) cause hemorrhagic lesions ("red pocks") on infected chorioallantoic membranes (CAM) of embryonated chicken eggs, while most other members of the genus Orthopoxvirus produce nonhemorrhagic lesions ("white pocks"). Cytokine response modifier A (CrmA) of CPXV strain Brighton Red (BR) is necessary but not sufficient for the induction of red pocks. To identify additional viral proteins involved in the induction of hemorrhagic lesions, a library of single-gene CPXV knockout mutants was screened. We identified 10 proteins that are required for the formation of hemorrhagic lesions, which are encoded by CPXV060, CPXV064, CPXV068, CPXV069, CPXV074, CPXV136, CPXV168, CPXV169, CPXV172, and CPXV199. The genes are the homologues of F12L, F15L, E2L, E3L, E8R, A4L, A33R, A34R, A36R, and B5R of vaccinia virus (VACV). Mutants with deletions in CPXV060, CPXV168, CPXV169, CPXV172, or CPXV199 induced white pocks with a comet-like shape on the CAM. The homologues of these five genes in VACV encode proteins that are involved in the production of extracellular enveloped viruses (EEV) and the repulsion of superinfecting virions by actin tails. The homologue of CPXV068 in VACV is also involved in EEV production but is not related to actin tail induction. The other genes encode immunomodulatory proteins (CPXV069 and crmA) and viral core proteins (CPXV074 and CPXV136), and the function of the product of CPXV064 is unknown.
Importance:
It has been known for a long time that cowpox virus induces hemorrhagic lesions on chicken CAM, while most of the other orthopoxviruses produce nonhemorrhagic lesions. Although cowpox virus CrmA has been proved to be responsible for the hemorrhagic phenotype, other proteins causing this phenotype remain unknown. Recently, we generated a complete single-gene knockout bacterial artificial chromosome (BAC) library of cowpox virus Brighton strain. Out of 183 knockout BAC clones, 109 knockout viruses were reconstituted. The knockout library makes possible high-throughput screening for studying poxvirus replication and pathogenesis. In this study, we screened all 109 single-gene knockout viruses and identified 10 proteins necessary for inducing hemorrhagic lesions. The identification of these genes gives a new perspective for studying the hemorrhagic phenotype and may give a better understanding of poxvirus virulence.
... After the PBS wash, coverslips were blocked with a PBS solution containing 20% bovine serum albumin. Then, cells were incubated (1 h, at 37uC) with rabbit anti-gp120 polyclonal antibody and with mouse C3 monoclonal antibody against 14 Kda VV protein, generated in the laboratory of Dr Esteban as previously described [45]. Coverslips were washed extensively with PBS and incubated (1 h at 37uC) with secondary anti-rabbit immunoglobulin conjugated with Alexa-488 and with secondary anti-mouse immunoglobulin conjugated with Alexa-647 (Invitrogen). ...
The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors.
As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B.
This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate.
... The remainder of the determinants mapped within late VACV gene products are present within proteins that may associate with other VACV proteins (e.g. A10L that associates with A4L [36]) to facilitate their exit from factories. These data validate our hypothesis that late VACV proteins that remain within virus factories are not immunogenic whereas those that can leave can generate T CD8+ responses, likely via the cross-presentation pathway. ...
Virus-specific CD8(+) T cells (T(CD8+)) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector T(CD8+). Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated T(CD8+) response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the T(CD8+) response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines.
... Processing of P4a at these sites should theoretically also yield an intervening 9-kDa polypeptide (amino acids 615 to 697), but its possible localization and fate remain unclear. We have recently described that in the virion, the mature 4a protein forms a complex with the 39K core protein, the product of A5L gene (41), and this interaction may be crucial for the assembly of the core structure. ...
The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/ repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-β-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.
... This suggestion is consistent with the fact that J1R can be efficiently extracted from purified IMV by detergent in the presence of DTT. This behavior has also been observed with other vaccinia virus membrane proteins, such as L1R, A17L, A27L, and A14L (39,44,47,63). Although J1R has two hydrophobic regions that may serve as transmembrane regions, computer programs such as TMpred have suggested otherwise. ...
Vaccinia virus, a member of the poxvirus family, contains a conserved J1R open reading frame that encodes a late protein of 17.8 kDa. The 18-kDa J1R protein is associated mainly with the membrane fraction of intracellular mature virus particles. This study examines the biological function of J1R protein in the vaccinia virus life cycle. A recombinant vaccinia virus was constructed to conditionally express J1R protein in an isopropyl-beta-D-galactopyranoside (IPTG)-inducible manner. When J1R is not expressed during vaccinia virus infection, the virus titer is reduced approximately 100-fold. In contrast, J1R protein is not required for viral gene expression, as indicated by protein pulse-labeling. J1R protein is also not required for DNA processing, as the resolution of the concatemer junctions of replicated viral DNA was detected without IPTG. A deficiency of J1R protein caused a severe delay in the processing of p4a and p4b into mature core proteins 4a and 4b, indicating that J1R protein participates in virion morphogenesis. Infected cells grown in the absence of IPTG contained very few intracellular mature virions in the cytoplasm, and enlarged viroplasm structures accumulated with viral crescents attached at the periphery. Abundant intermediate membrane structures of abnormal shapes were observed, and many immature virions were either empty or partially filled, indicating that J1R protein is important for DNA packaging into immature virions. J1R protein also coimmunoprecipited with A45R protein in infected cells. In summary, these results indicate that vaccinia virus J1R is a membrane protein that is required for virus growth and plaque formation. J1R protein interacts with A45R protein and performs an important role during immature virion formation in cultured cells.
... The core wall is probably composed, in large part, of vaccinia virus proteins p4a and p4b (32), and the pegs may contain vaccinia virus protein p39/A4L (3, 33) as indicated by EM immunolocalization studies. A tight association of p39 with p4a/p4b has been reported (35). ...
Vaccinia virus, the basis of the smallpox vaccine, is one of the largest viruses to replicate in humans. We have used in situ atomic force microscopy (AFM) to directly visualize fully hydrated, intact intracellular mature vaccinia virus (IMV) virions and chemical and enzymatic treatment products thereof. The latter included virion cores, core-enveloping coats, and core substructures. The isolated coats appeared to be composed of a highly cross-linked protein array. AFM imaging of core substructures indicated association of the linear viral DNA genome with a segmented protein sheath forming an extended approximately 16-nm-diameter filament with helical surface topography; enclosure of this filament within a 30- to 40-nm-diameter tubule which also shows helical topography; and enclosure of the folded, condensed 30- to 40-nm-diameter tubule within the core by a wall covered with peg-like projections. Proteins observed attached to the 30- to 40-nm-diameter tubules may mediate folding and/or compaction of the tubules and/or represent vestiges of the core wall and/or pegs. An accessory "satellite domain" was observed protruding from the intact core. This corresponded in size to isolated 70- to 100-nm-diameter particles that were imaged independently and might represent detached accessory domains. AFM imaging of intact virions indicated that IMV underwent a reversible shrinkage upon dehydration (as much as 2.2- to 2.5-fold in the height dimension), accompanied by topological and topographical changes, including protrusion of the satellite domain. As shown here, the chemical and enzymatic dissection of large, asymmetrical virus particles in combination with in situ AFM provides an informative complement to other structure determination techniques.
... One of the VV major core proteins, A10L has been shown to be essential in virus replication and its absence in virus assembly resulted in defective virus morphology such as IV-like particles, which lacked granular viral materials and consequently produced the irregular-shaped virus parti- cles [7]. These morphogenic defects suggested that A10L protein is required for the correct organization of the nucleocomplex within the IVs [7,8]. L4R, a DNA binding protein, plays an essential role in virus replication, being involved in an early stage of infection such as early transcription or unpackaging viral core and DNA [9,10]. ...
Like the major vaccinia virus (VV) core protein precursors, p4b and p25K, the 25 kDa VV A12L late gene product (p17K) is proteolytically maturated at the conserved Ala-Gly-Ala motif. However, the association of the precursor and its cleavage product with the core of mature virion suggests that both of the A12L proteins may be required for virus assembly. Here, in order to test the requirement of the A12L protein and its proteolysis in viral replication, a conditional lethal mutant virus (vvtetOA12L) was constructed to regulate A12L expression by the presence or absence of an inducer, tetracycline. In the absence of tetracycline, replication of vvtetOA12L was inhibited by 80% and this inhibition could be overcome by transient expression of the wild-type copy of the A12L gene. In contrast, mutation of the AG/A site abrogated the ability of the transfected A12L gene to rescue, indicating that A12L proteolysis plays an important role in viral replication. Electron microscopy analysis of the A12L deficient virus demonstrated the aberrant virus particles, which were displayed by the AG/A site mutation. Thus, we concluded that the not only A12L protein but also its cleavage processing plays an essential role in virus morphogenic transition.
... Antibodies used in this study were as follows: anti-CD63, 1B5 (Fraile-Ramos et al., 2001 ), was described previously; rabbit antibodies against VPS4A and VPS4B (von Schwedler et al., 2003) and against ALIX/AIP1 (Cabezas et al., 2005) were provided by W. Sundquist (University of Utah, UT) and H. Stenmark (the Norwegian Radium Hospital, Oslo, Norway) respectively; anti-bactin clone AC-15 was purchased from Sigma Aldrich (Madrid, Spain), anti-Tsg101 clone 4A10 from GeneTex (San Antonio, TX), and anti-clathrin heavy chain, TD.1 from BD Bioscience (Palo Alto, CA); rabbit antibody against vaccinia virus wild-type strain Western Reserve (Risco et al., 1999) was prepared in house; rabbit anti-HIV-1 Gag antiserum raised against the p24 portion of Gag, ARP432, was obtained from the AIDS Reagent Project of the United Kingdom Medical Research Council (Potters Bar, UK); anti-VSV G-protein, P5D4, was originally obtained from T. Kreis (University of Geneva, Geneva, Switzerland); anti-HRP was purchased from Jackson ImmunoResearch (supplied by Vitro S.A., Madrid, Spain). Goat anti-mouse antibody labelled with Alexa 594 was from Molecular Probes Europe BV. ...
The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold-labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV-infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag-derived virus-like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment.
... Alcamí et al., 1993;Alonso et al., 2001;Andrés et al., 1997Andrés et al., , 2001Borca et al., 1996;Brookes et al., 1998a,b;Carrascosa et al., 1986;Chacó n et al., 1995;Cobbold et al., 1996;Galindo et al., 2000;García-Beato et al., 1992;Heath et al., 2001;Hingamp et al., 1992;Jouvenet and Wileman, 2005;Jouvenet et al., 2004;Martinez-Pomares et al., 1997;Moura Nunes et al., 1975;Rodríguez et al., 2006;Rouiller et al., 1998;Sanz et al., 1985;Simón-Mateo et al., 1997;Sun et al., 1996;Vigário et al., 1967 Contents of cellular origin Ubiquitin, hsp70 chaperone, g-tubulin, Pericentrin, p21, mdm1 Surrounded by: ER membranes, vimentin, p230 Golgin, mitochondria, and tubulin. Granja et al., 2004;Heath et al., 2001;Hingamp et al., 1992;Jouvenet and Wileman, 2005;Netherton et al., 2004Netherton et al., , 2006Rojo et al., 1998;Rouiller et al., 1998;Stefanovic et al., 2005 Poxviridae Almazán et al., 2001;Beaud and Beaud, 1997;Betakova et al., 2000;Chiu et al., 2005;Cudmore et al., 1996;da Fonseca et al., 2000;Davis and Mathews, 1993;De Silva and Moss, 2005;Domi and Beaud, 2000;Krijnse-Locker et al., 1996;Murcia-Nicolas et al., 1999;Nerenberg et al., 2005;Ojeda et al., 2006;Palacios et al., 2005;Pedersen et al., 2000;Reckmann et al., 1997;Resch et al., 2005;Risco et al., 1999;Roper, 2006;Salmons et al., 1997;Senkevich et al., 2002;Sodeik et al., 1995;Szajner et al., 2004a,b,c;Tolonen et al., 2001;Vanslyke and Hruby, 1994;Welsch et al., 2003;Wolffe et al., 1995;Yeh et al., 2000;Yuwen et al., 1993 (continued) Contents of cellular origin HMG20A viral genome binding protein, hSP90; transient association, Ubiquitin, ying-yang 1 transcription factor, TBP transcription factor, SP1 transcription factor, RNA polymerase II, SUMO-1, ERGIC-53 c Surrounded by: vimentin and mitochondria. Broyles et al., 1999;Dales and Siminovitch, 1961;Hsiao et al., 2006;Hung et al., 2002;Husain and Moss, 2003;Nerenberg et al., 2005;Oh and Broyles, 2005;Palacios et al., 2005;Risco et al., 2002;Wilton and Dales, 1989 Iridoviridae, Ranavirus ...
Virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the "cytopathic effect" that has been the hallmark of virus infection in tissue culture for many years. Recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. In this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. In many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. Interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. It is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. The recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. Disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release.
The recent global outbreak of mpox, caused by monkeypox virus (MPV) emerged in Europe in 2022 and rapidly spread to over 40 countries. The Americas are currently facing the highest impact, reporting over 50,000 cases by early 2023. In this study, we analyzed 880 MPV isolates worldwide to gain insights into the evolutionary patterns and initial introduction events of the virus in Mexico. We found that MPV entered Mexico on multiple occasions, from the United Kingdom, Portugal, and Canada, and subsequently spread locally in different regions of Mexico. Additionally, we show that MPV has an open pangenome, highlighting the role of gene turnover in shaping its genomic diversity, rather than single-nucleotide polymor-phisms (SNPs), which do not contribute significantly to genome diversity. Although the genome contains multiple SNPs in coding regions, these remain under purifying selection, suggesting their evolutionary conservation. One notable exception is amino acid position 63 of the protein encoded by the Cop-A4L gene, which is intricately related to viral maturity, which we found to be under strong positive selection. Ancestral state reconstruction indicated that the ancestral state at position 63 corresponds to the amino acid valine, which is present only in isolates of clade I. However, the isolates from the current outbreak contained threonine at position 63. Our findings contribute new information about the evolution of monkeypox virus.
The recent global outbreak of Monkeypox, caused by the Monkeypox virus (MPVX) emerged in Europe in 2022 and rapidly spread to over 40 countries. The Americas are currently facing the highest impact, reporting over 50,000 cases by early 2023. Here, we analyze 880 MPXV isolates worldwide to gain insights into the evolutionary patterns and initial introduction events of the virus in Mexico. We found that MPXV entered Mexico on multiple occasions, from the United Kingdom,: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. subsequently spread locally in different regions of Mexico. Additionally, we show that MPXV has an open pangenome, highlighting the role of gene turnover in shaping its genomic diversity, rather than SNPs variations, which do not contribute significantly to genome diversity. Although the genome presents multiple SNP sites in coding regions, these remain under purifying selection, suggesting their evolutionary conservation. One notable exception is the amino acid site 63 of the protein encoded by the Cop-A4L gene, intricately related to viral maturity, for which we picked up a strong signal of positive selection. Ancestral state reconstruction deduced that the ancestral state at site 63 corresponds to the amino acid valine, present only in isolates of clade I. However, the isolates of the current outbreak evolved for threonine at site 63. Finally, our findings contribute to the knowledge of the evolutionary processes of the Monkeypox virus.
Use of recombinant viral vectors encoding nonnative Ags is an attractive mechanism for the generation of protective Ab, CD4+ T cell (TCD4+), and CD8+ T cell (TCD8+) responses in vivo following immunization. However, the life cycle and tropism of the viral vector, and its interactions with various components of the immune system, must be fully understood to maximize the efficacy of any vaccination strategies. Ab and TCD4+ responses typically target native Ags driven by late promoters in vaccinia virus (VACV)-based vectors. However, it has been demonstrated that model Ags driven by late promoters in recombinant VACV vectors do not stimulate TCD8+ responses, whereas identical Ags driven by early promoters stimulate strong responses. Conversely, TCD8+ can be generated against some natural late VACV Ags. We explored this dichotomy by investigating the Ag presentation pathways responsible for presentation of natural late VACV Ags in mice. We found that all of the late VACV Ags we examined could be cross-primed (i.e., presented by uninfected professional APC), as well as directly presented by infected dendritic cell populations. However, one Ag was only presented by professional APC populations and was not the target of a protective TCD8+ response. Therefore, there is no generalized blockade in Ag presentation of late VACV Ags, and expression of nonnative Ags driven by a late promoter allows production of large quantities of Ag that may allow simultaneous targeting of both TCD4+ and Ab responses, as well as TCD8+ responses, in the future.
PhD thesis "Expression Libraries as Tools for the Development of Subunit Vaccines and Novel Detection Molecules for Orthopoxviruses"
Smallpox constitutes a major bioterrorism threat, which underscores the need to develop antiviral drugs for rapid response in the event of an attack. Viral processivity factors are attractive drug targets in being both specific and essential for their cognate DNA polymerases to synthesize extended strands of DNA. An in silico model of the vacinnia virus processivity factor, comprised of the A20 and D4 heterocomplex, was constructed and used for lead optimization of an indole-based scaffold identified earlier from a high-throughput screening. Based on this model, a new class of potent antivirals against vaccinia virus was designed and synthesized, of which two (24a and 24b) exhibited superior improvement over the parent scaffold (IC50 = 42 and 46 vs. 82000 nM, respectively). The ability of 24a to suppress vaccinia DNA synthesis is supported by the inhibition of late viral gene expression, as well as by the diminished incorporation of bromodeoxyuridine into viral replication factories.
Many viruses reorganise cellular membrane compartments and the cytoskeleton to generate subcellular microenvironments called virus factories or 'viroplasm'. These create a platform to concentrate replicase proteins, virus genomes and host proteins required for replication and also protect against antiviral defences. There is growing interest in understanding how viruses induce such large changes in cellular organisation, and recent studies are beginning to reveal the relationship between virus factories and viroplasm and the cellular structures that house them. In this review, we discuss how three supergroups of (+)RNA viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large DNA viruses.
The aim of this study was to define the effects on antigen-presenting cells of the expression of HIV antigens from an attenuated poxvirus vector. We have analyzed the transcriptional changes in gene expression following infection of human immature monocyte-derived dendritic cells (DC) with recombinant modified vaccinia virus Ankara (MVA) expressing the genes encoding the gp120 and Gag-Pol-Nef antigens of HIV type 1 clade B (referred to as MVA-B) versus parental MVA infection. Using microarray technology and real-time reverse transcription-PCR, we demonstrated that the HIV proteins induced the expression of cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex (MHC) genes. Levels of mRNAs for interleukin-1, beta interferon, CCR8, and SCYA20 were higher after HIV antigen production. MVA-B infection also modulated the expression of antigen processing and presentation genes: the gene for MICA was upregulated, whereas those for HLA-DRA and HSPA5 were downregulated. Indeed, the increased expression of the gene for MICA, a glycoprotein related to major histocompatibility complex class I molecules, was shown to enhance the interaction between MVA-B-infected target cells and cytotoxic lymphocytes. The expression profiles of the genes for protein kinases such as JAK1 and IRAK2 were activated after HIV antigen expression. Several genes included in the JAK-STAT and mitogen-activated protein kinase signaling pathways were regulated after HIV antigen expression. Our findings provide the first gene signatures in DC of a candidate MVA-B vaccine expressing four HIV antigens and identified the biological roles of some of the regulatory genes, like that for MICA, which will help in the design of more effective MVA-derived vaccines.
The HIV epidemic in Argentina is characterized by the high prevalence of infections caused by subtype B and BF variants. In this study, the Nef protein was used as a tool to study the impact of HIV-1 BF variants in the design of future vaccines. DNA and MVA vectors expressing Nef of the CRF12_BF recombinant form of HIV-1 were generated and characterized. After the administration of single DNAprime/MVAboost immunization schedules in Balb/c mice we found that NefBF delivered from these vectors generated a response of high specificity with low cross-reactivity against subtype B. But, when a more potent response was induced after 3 priming DNA doses and a booster with MVA virus, cross-reactivity against NefB was detected, although of lower magnitude than the NefBF specific. These results will be pivotal for vaccines designs in our region, indicating that antigens from these viral variants must be considered for a future vaccine.
Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early
phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell.
After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules,
filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze
fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support
the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment
(ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around
viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents
protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose
that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories
and the first VV form through a unique mechanism of viral morphogenesis from cellular elements.
The myxoma virus M131R gene encodes a catalytically inactive homolog of cellular Cu-Zn superoxide dismutase (SOD1) and this 17,786 Da protein is a major virion component. We have used matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS) to study the effect(s) of deleting the gene on virion composition and structure. This approach confirmed that the M131R gene product is an abundant virion component. This conclusion was based upon the ready detection of a 1805.3 Da peptide released from the N-terminus of the myxoma SOD1 protein by mild trypsin treatment, as well as the detection of a 17,790 Da protein in HPLC fractionated virus extracts, which subsequently yielded M131R-encoded tryptic peptides. Neither peptide nor protein was detected in particles bearing a genome encoding an M131RDelta deletion mutation. Curiously, more proteins and tryptic peptides were detected when M131RDelta mutant virions were subjected to MALDI-TOF MS analysis compared with wild-type virus particles. This suggested that particles assembled in the absence of myxoma SOD protein are structurally unstable. Plaque analysis confirmed this conjecture by showing that SOD-deficient MYX particles are unusually heat labile and trypsin sensitive. Mutant Shope fibroma virus exhibited the same phenotype. Thus a previously unappreciated feature of MALDI-TOF MS is that the method can sometimes detect alterations in virion stability.
Genome replication and assembly of viruses often takes place in specific intracellular compartments where viral components concentrate, thereby increasing the efficiency of the processes. For a number of viruses the formation of 'factories' has been described, which consist of perinuclear or cytoplasmic foci that mostly exclude host proteins and organelles but recruit specific cell organelles, building a unique structure. The formation of the viral factory involves a number of complex interactions and signalling events between viral and cell factors. Mitochondria, cytoplasmic membranes and cytoskeletal components frequently participate in the formation of viral factories, supplying basic and common needs for key steps in the viral replication cycle.
The poxvirus strains NYVAC and MVA are two candidate vectors for the development of vaccines against a broad spectrum of diseases. Although these attenuated virus strains have proven to be safe in animals and humans, little is known about their comparative behavior in vitro. In contrast with MVA, NYVAC infection triggers greater cytopathic effect in a range of permissive and nonpermissive cell lines. The yields of NYVAC cell-associated virus in permissive cells (BHK-21) were slightly reduced compared with those of MVA infection. During the course of infection in HeLa cells, there is a translational block induced by NYVAC late in infection, which correlated with a marked increase in phosphorylation levels of the initiation factor eIF-2alpha. In contrast to MVA, the synthesis of certain late viral proteins was only blocked in NYVAC-infected HeLa cells. Electron microscopy (EM) analysis revealed that morphogenesis of NYVAC in HeLa cells was blocked at the stage of formation of immature viral forms. Phase-contrast microscopy, EM, flow cytometry, and rRNA analyses demonstrated that contrary to MVA, NYVAC infection induces potent apoptosis, a phenomenon dependent on activation of caspases and RNase L. Apoptosis induced by NYVAC was prevented when the virus gene C7L was placed back into the NYVAC genome, recovering the ability of NYVAC to replicate in HeLa cells and maintaining the attenuated phenotype in mice. Overall, our findings demonstrate distinct behavior between NYVAC and MVA strains in cultured cells, as well as a new role for the C7L viral gene as an inhibitor of apoptosis in NYVAC infection.
Although recombinants based on the attenuated poxvirus vectors MVA and NYVAC are currently in clinical trials, the nature of the genes triggered by these vectors in antigen-presenting cells is poorly characterized. Using microarray technology and various analysis conditions, we compared specific changes in gene expression profiling following MVA and NYVAC infection of immature human monocyte-derived dendritic cells (MDDC). Microarray analysis was performed at 6 h postinfection, since these viruses induced extensive cytopathic effects, rRNA breakdown, and apoptosis at late times postinfection. MVA- and NYVAC-infected MDDC shared upregulation of 195 genes compared to uninfected cells: MVA specifically upregulated 359 genes, and NYVAC upregulated 165 genes. Microarray comparison of NYVAC and MVA infection revealed 544 genes with distinct expression patterns after poxvirus infection and 283 genes specifically upregulated after MVA infection. Both vectors upregulated genes for cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex genes. mRNA levels for interleukin 12beta (IL-12beta), beta interferon, and tumor necrosis factor alpha were higher after MVA infection than after NYVAC infection. The expression profiles of transcription factors such as NF-kappaB/Rel and STAT were regulated similarly by both viruses; in contrast, OASL, MDA5, and IRIG-I expression increased only during MVA infection. Type I interferon, IL-6, and Toll-like receptor pathways were specifically induced after MVA infection. Following MVA or NYVAC infection in MDDC, we found similarities as well as differences between these virus strains in the expression of cellular genes with immunological function, which should have an impact when these vectors are used as recombinant vaccines.
The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2'-5'-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.
Live recombinants based on attenuated modified vaccinia virus Ankara (MVA) are potential vaccine candidates against a broad spectrum of diseases and tumors. To better understand the efficacy of MVA as a human vaccine, we analyzed by confocal and electron microscopy approaches MVA-induced morphological changes and morphogenetic stages during infection of human HeLa cells in comparison to other strains of vaccinia virus (VV): the wild-type Western Reserve (WR), Ankara, and the New York City Board of Health (NYCBH) strains. Confocal microscopy studies revealed that MVA infection alters the cytoskeleton producing elongated cells (bipolar), which do not form the characteristic actin tails. Few virions are detected in the projections connecting neighboring cells. In contrast, cells infected with the WR, Ankara, and NYCBH strains exhibit a stellated (multipolar) or rounded morphology with actin tails. A detailed transmission electron microscopy analysis of HeLa cells infected with MVA showed important differences in fine ultrastructure and amounts of the viral intermediates compared to cells infected with the other VV strains. In HeLa cells infected with MVA, the most abundant viral forms are intracellular immature virus, with few intermediates reaching the intracellular mature virus (IMV) form, at various stages of maturation, which exhibit a more rounded shape than IMVs from cells infected with the other VV strains. The "IMVs" from MVA-infected cells have an abnormal internal structure ("atypical" viruses) with potential alterations in the core-envelope interactions and are unable to significantly acquire the additional double envelope to render intracellular envelope virus. The presence of potential cell-associated envelope virus is very scarce. Our findings revealed that MVA in human cells promotes characteristic morphological changes to the cells and is able to reach the IMV stage, but these virions were not structurally normal and the subsequent steps in the morphogenetic pathway are blocked.
Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.
The release of vaccinia virus from RK-13 cells and its specific inhibition by N(1)-isonicotinoyl-N(2)-3-methyl-4- chlorobenzoylhydrazine (IMCBH) was studied. Intracellular naked vaccinia virus (INV) was wrapped by intracytoplasmic membranes, forming an intracellular double-membraned virion. Wrapped virions migrated to the cell surface, where the outer virion membrane presumably fused with the plasma membrane, releasing virus surrounded by the inner membrane, referred to as extracellular enveloped vaccinia virus (EEV). At no time was there any evidence that vaccinia virus acquired an envelope by budding of naked virus from the cytoplasmic membrane. Naked virus and double-membraned virus each constituted about one-third of intracellular virus at 8 and 12 h postinfection (p.i.). Beginning at 16 h p.i., the proportion of intracellular virus occurring as double-membraned virus steadily decreased to 1% at 24 h while the proportion of naked virus rose to 87%. IMCBH inhibited the formation of the double-membraned virion and the appearance of EEV while not affecting the production of INV. IMCBH had no effect on INV infectivity or polypeptide composition, on vaccinia virus-specified membrane-associated proteins or glycoproteins, or on hemadsorption. The presence of IMCBH until 4 h p.i. did not decrease the amount of EEV at 48 h p.i., whereas less than 10% of the normal 48-h EEV yield was obtained if the drug was present during the first 16 h p.i. Cell cultures infected at very low multiplicities showed a rapid virus dissemination in the absence of the drug, whereas the presence of IMCBH very effectively inhibited this spread. We conclude that vaccinia virus is liberated via a double-membraned intermediate as an enveloped virion and that it is this extracellular enveloped virus that is responsible for dissemination of infection.
Vaccinia virus (VV) is a potent immunogen, but the nature of VV proteins involved in the activation of the immune response of the host is not yet known. By screening a lambda gt11 expression library of rabbitpox virus DNA with serum from humans vaccinated against smallpox or with serum from VV-immunized animals, we identified several VV genes that encode highly antigenic viral proteins with molecular masses of 62, 39, 32, 25, 21, and 14 kDa. It was found that VV proteins of 62, 39, 25, and 21 kDa are part of the virus core, while proteins of 32 and 14 kDa are part of the virus envelope. All of these proteins were synthesized at late times postinfection. Proteins of 62 and 25 kDa were produced by cleavage of larger precursors of 95 kDa (p4a) and 28 kDa, respectively. The 21-kDa protein was the result of a cleavage of p4a, presumably at amino acid Gly-697. DNA sequence analysis, in comparison with the known nucleotide sequence of VV, provided identification of the corresponding open reading frames. Expression of the viral genes in Escherichia coli was used to monitor which of the viral antigens elicit immunodominant responses and the location of antigenic domains. Three viral antigens of 62, 39, and 32 kDa exhibited immunodominant characteristics. The most antigenic sites of 62 and 39 kDa were identified at the N terminus (amino acids 132 to 295) and C terminus (last 103 amino acids), respectively. Immunization of mice with the 62-, 39-, or 14-kDa antigenic proteins conferred different degrees of protection from VV challenge. Proteins of 32 and 14 kDa induced cellular proliferative responses in VV-infected mice. Our findings demonstrate the nature of VV proteins involved in the activation of host immune responses after vaccination, provide identification of the viral gene locus, and define structural and immunological properties of these antigenic VV proteins.
Sequence analysis of the vaccinia virus strain Western Reserve genome revealed the presence of an open reading frame (ORF), SalL4R, which has the potential to encode a transmembrane glycoprotein with homology to C-type animal lectins (G. L. Smith, Y. S. Chan, and S. T. Howard, J. Gen. Virol. 72:1349-1376, 1991). Here we show that the SalL4R gene is transcribed late during infection from a TAAATG motif at the beginning of the ORF. Antisera raised against a TrpE-SalL4R fusion protein identified three glycoprotein species of Mr 22,000 to 24,000 in infected cells. Immunogold electron microscopy demonstrated that SalL4R protein is present in purified extracellular enveloped virus particles but not in intracellular naked virus (INV). A mutant virus was constructed by placing a copy of the SalL4R ORF downstream of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible vaccinia virus promoter within the thymidine kinase locus and subsequently deleting the endogenous SalL4R gene. The growth kinetics of this virus demonstrated that SalL4R was nonessential for the production of infectious INV but was required for virus dissemination. Consistent with this finding, the formation of wild-type-size plaques by this mutant was dependent on the presence of IPTG. Electron microscopy showed that without SalL4R expression, the inability of the virus to spread is due to a lack of envelopment of INV virions by Golgi-derived membrane, a morphogenic event required for virus egress.
There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV). To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation. The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene. Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation. By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions. Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface. The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion. These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane. By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.
A novel method has been developed to study the functional roles of Individual vaccinia virus gene products that is neither
limited by the possible essentiality of the target gene nor by the availability of conditional lethal mutants. The system
utilises the E.coli lac repressor protein, the operator sequence to which it binds and the specific inducer IPTG. It allows the generation of
recombinant viruses in which the expression of any chosen gene, and hence virus replication, can be externally controlled.
In principle, this system is broadly applicable to the functional analysis of genes in any large DNA virus. This approach
has demonstrated that the gene encoding the 14 kDa membrane protein of vaccinia virus is non-essential for the production
of infectious intracellular virus particles, but essential for the envelopment of intracellular virions by Golgi membrane
and for egress of mature extracellular viral particles. This is the first vaccinia virus protein shown to be specifically
required for these processes. In vivothis system may prove useful as a means of attenuating recombinant vaccinia virus vaccines by preventing virus spread without
reducing the amount of the foreign antigen expressed in each infected cell. Attenuation of other live virus vaccines may be
developed in a similar way.
Little is known about the nature of poxvirus proteins involved in the host immune response. Screening a lambda gt11 expression library of genomic rabbit poxvirus DNA with hyperimmune rabbit anti-vaccinia virus serum and selection of monospecific antibodies identified a highly antigenic viral protein of about 39,000 molecular weight (39K protein). The same-size protein of vaccinia virus was also identified with a monoclonal antibody (MAb B6) obtained from hybridomas generated after fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Structural analysis revealed that the 39K protein is an acidic polypeptide, that it can exist in two molecular forms because of intramolecular disulfide linkages, and that it is part of the virus core. This protein shares antigenic determinants with a cytoplasmic component(s) from uninfected cells. Functional studies revealed that the 39K protein is synthesized at late times postinfection and appears to be required for virus assembly. This protein is highly conserved in members of the Orthopoxvirus group, but in cowpox virus, a 41K virion protein was specifically recognized by antibodies that reacted against the vaccinia virus 39K protein. Significantly, during long-term passages of Friend erythroleukemia cells persistently infected with vaccinia virus, some virus mutants were found to increase or decrease by about 2 kilodaltons the size of the 39K protein. Mapping analysis localized sequences encoding the 39K protein in a rifampin-sensitive gene cluster between the two major core-associated viral polypeptides, 4a and 4b. The fact that the 39K core protein of vaccinia virus elicits strong humoral immune response, induces antibodies that react against a host component(s), and is subjected to genetic variability suggests that this protein has important biological functions.
Vaccinia virus (VV) A17L gene encodes a 23-kDa protein that is proteolytically cleaved to generate a 21-kDa product that is incorporated into the viral particles. We have previously shown that the 21-kDa protein forms a stable complex with the VV 14-kDa envelope protein and suggested that the 21-kDa protein may serve to anchor the 14-kDa protein to the envelope of the virion (D. Rodríguez, J. R. Rodríguez, and M. Esteban, J. Virol. 67:3435-3440, 1993). To study the role of the 21-kDa protein in virion assembly, in this investigation we generated a VV recombinant, VVindA17L, that contains an inducible A17L gene regulated by the E. coli repressor/operator system. In the absence of the inducer, shutoff of the A17L gene was complete, and this shutoff correlated with a reduction in virus yields of about 3 log units. Although early and late viral polypeptides are normally synthesized in the absence of the A17L gene product, proteolytic processing of the major p4a and p4b core proteins was clearly impaired under these conditions. Electron microscopy examination of cells infected in the absence of isopropylthiogalactopyranoside (IPTG) revealed that virion morphogenesis was completely arrested at a very early stage, even prior to the formation of crescent-shaped membranes, which are the first distinguishable viral structures. Only electron-dense structures similar to rifampin bodies, but devoid of membranes, could be observed in the cytoplasm of cells infected with VVindA17L under nonpermissive conditions. Considering the most recent assembly model presented by Sodeik et al. (B. Sodeik, R. W. Doms, M. Ericsson, G. Hiller, C. E. Machamer, W. van't Hof, G. van Meer, B. Moss, and G. Griffiths, J. Cell Biol. 121:521-541, 1993), we propose that this protein is targeted to the intermediate compartment and is involved in the recruitment of these membranes to the viral factories, where it forms the characteristic crescent structures that subsequently result in the formation of virions.
The binding domains of four monoclonal antibodies (MAbs) specific for the M protein of the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) have been located in the 46 carboxy-terminal amino acids of the protein by studying the binding of MAbs to recombinant M protein fragments. Immunoelectron microscopy using these MAbs demonstrated that in a significant proportion of the M protein molecules, the carboxy terminus is exposed on the external surface both in purified viruses and in nascent TGEV virions that recently exited infected swine testis cells. The same MAbs specifically neutralized the infectivity of the PUR46-MAD strain, indicating that the C-terminal domain of M protein is exposed on infectious viruses. This topology of TGEV M protein probably coexists with the structure currently described for the M protein of coronaviruses, which consists of an exposed amino terminus and an intravirion carboxy-terminal domain. The presence of a detectable number of M protein molecules with their carboxy termini exposed on the surface of the virion has relevance for viral function, since it has been shown that the carboxy terminus of M protein is immunodominant and that antibodies specific for this domain both neutralize TGEV and mediate the complement-dependent lysis of TGEV-infected cells.
Previous studies have suggested that cleavage of vaccinia virus core protein precursors occurs within the consensus tripeptide motif -A-G decreases X-. As an approach to delineate the sequence and structural features of the precursor polypeptides that are responsible for directing site-specific scission within this element, site-directed mutagenesis procedures were employed in concert with an in vivo trans-processing assay of the P25K: FLAG reporter plasmid. The results obtained suggest that residue occupancy at the P1' site (following the nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1976) Biochem. Biophys. Res. Commun. 27, 157-162), the positions at the amino- and carboxyl-proximal residues are indicated as P1, P2, etc., and P1', P2', etc., respectively) was extremely permissive, with only a proline substitution blocking cleavage. In contrast, the permissible occupancy of the P1 (serine or alanine) and P2 (cysteine, serine, or asparagine) sites was extremely restricted. Analysis of P1/P2 double mutants supported this conclusion and suggested additional levels of combinatorial stringency. Insertion or deletion of sequences immediately adjacent (amino- or carboxyl-terminal) to the -A-G-X- motif completely abrogated cleavage, suggesting the presence of additional important structural determinants. Mutation of the conserved proline or basic amino acid residues in these regions had no effect on cleavage, whereas it appeared that the presence of a hydrophobic residue in the P4 site was required.
During the assembly of vaccinia virus, the intracellular mature virus becomes enwrapped by a cellular cisterna to form the intracellular enveloped virus (IEV), the precursor of the extracellular enveloped virus (EEV). In this study, we have characterized the origin of this wrapping cisterna by electron microscopic immunocytochemistry using lectins, antibodies against endocytic organelles, and recombinant vaccinia viruses expressing proteins which behave as Golgi resident proteins. No labelling for endocytic marker proteins could be detected on the wrapping membrane. However, the wrapping membrane labelled significantly for a trans Golgi network (TGN) marker protein. The recycling pathway from endosomes to the TGN appears to be greatly increased following vaccinia virus infection, since significant amounts of endocytic fluid-phase tracers were found in the lumen of the TGN, Golgi complex, and the wrapping cisternae. Using immunoelectron microscopy, we localized the vaccinia virus membrane proteins VV-p37, VV-p42, VV-p21, and VV-hemagglutinin (VV-HA) in large amounts in the wrapping cisternae, in the outer membranes of the IEV, and in the outermost membrane of the EEV. The bulk of the cellular VV-p37, VV-p21, and VV-p42 were in the TGN, whereas VV-HA was also found in large amounts on the plasma membrane and in endosomes. Collectively, these data argue that the TGN becomes enriched in vaccinia virus membrane proteins that facilitate the wrapping event responsible for the formation of the IEV.
The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses.
Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.
The mechanism by which the 14-kDa fusion protein of vaccinia virus (VV) is anchored in the envelope of intracellular naked virions (INV) is not understood. In this investigation, we demonstrate that the 14-kDa protein interacts with another virus protein with an apparent molecular mass of 21 kDa. Microsequence analysis of the N terminus of the 21-kDa protein revealed that this protein is encoded by the VV A17L gene. The 21-kDa protein is processed from a 23-kDa precursor, by cleavage at amino acid position 16, at the consensus motif Ala-Gly-Ala, previously identified as a cleavage site for several VV structural proteins. The 21-kDa protein contains two large internal hydrophobic domains characteristic of membrane proteins. Pulse-chase analysis showed that within 1 h after synthesis, the 14-kDa protein forms a stable complex with the 21-kDa protein. Formation of the complex was not inhibited by rifampin, indicating that the interaction between these two proteins occurs prior to virion morphogenesis. Immunoprecipitation analysis of disrupted virions showed the presence of the 21-kDa protein in the viral particle. Release of the 14-kDa-21-kDa protein complex from INV required treatment with the nonionic detergent Nonidet P-40 and a reducing agent. The protein complex consisted of 14-kDa trimers and of 21-kDa dimers. Since the 14-kDa fusion protein lacks a signal sequence and a large hydrophobic domain characteristic of membrane proteins, our findings suggest that the 21-kDa protein serves to anchor the 14-kDa protein to the envelope of INV.
The entry and intracellular transport of Moloney-murine leukemia virions inside mouse NIH 3T3 cells have been followed by electron microscopy techniques. Five viral proteins--matrix (MA, p15), capsid (CA, p30), nucleocapsid (NC, p10), integrase (IN), and the envelope glycoprotein (SU, gp70)--were located by immunolabeling using gold probes. After entering the cells, viral particles were frequently detected inside cytoplasmic vesicles of variable size. Their viral envelope was apparently lost during intracytoplasmic transport. When the unenveloped viral cores reached the nuclear membrane or its vicinity, they were disrupted. Two of the immunolabeled proteins, NC and IN, were detected entering the nucleus of non-dividing cells, where both were targeted to the nucleolus. However, MA and CA were found only in the cytoplasm. NC is a nucleic acid-binding protein which contains potential nuclear localization signals. We suggest that NC could enter the nucleus as part of a nucleoprotein complex, associated with IN, and possibly, also with viral DNA.
We have recently provided morphological evidence that a key event in the assembly of vaccinia virus is the formation of a novel cisternal domain of the intermediate compartment (IC) between the endoplasmic reticulum and the Golgi complex (Sodeik, B., Doms, R. W., Ericsson, M., Hiller, G., Machamer, C. E., van't Hof, W., van Meer, G., Moss, B., and Griffiths, G. (1993) J. Cell Biol. 121, 521–541). This tightly apposed cisternal domain incompletely surrounds the spherical immature virus that matures into the first of the two distinct infectious forms of vaccinia, the intracellular mature virus (IMV). In this study we describe the characterization of an abundant membrane protein of the IMV, the gene product of A17L, a 21-kDa protein that has recently been shown to be essential for the formation of the viral membranes (Rodriguez, D., Esteban, M., and Rodriguez, J. R. (1995) J. Virol. 69, 4640-4648). Upon translation in vitro, p21 associated with rough microsomal membranes in a co-translational manner. Using NH2- and COOH-terminal specific antibodies, we show that both in vitro as well as in vivo, p21 adopts a topology where the NH2 and COOH termini are cytoplasmically orientated. Immunocytochemical experiments demonstrated that p21 is a component of the inner of the two cisternal membranes of the immature virus as well as of membranes of the IC, identified using antibodies against Rab1.
Taken together, these data provide the first molecular evidence in support of our assembly model; they show that an essential membrane protein of the IMV inserts into the rough endoplasmic reticulum, but gets efficiently targeted to the IC and membranes of the viral factory.
We introduce a novel approach for combining immunogold labelling with cryoelectron microscopy of thin vitrified specimens. The method takes advantage of the observation that particles in suspension are concentrated at the air-water interface and remain there during the subsequent immunogold labelling procedure. Subsequently, a thin aqueous film can be formed that is vitrified and observed by cryoelectron microscopy. In our view, a key early step in the assembly of vaccinia virus, the formation of the spherical immature virus, involves the formation of a specialized cisternal domain of the intermediate compartment between the endoplasmic reticulum and the Golgi. Using this novel cryoelectron microscopy approach, we show that in the intracellular mature virus (IMV) the core remains surrounded by a membrane cisterna that comes off the viral core upon treatment with dithiothreitol, exposing an antigen on the surface of the viral core. Complementary protease studies suggest that the IMV may be sealed not by membrane fusion but by a proteinaceous structure that interrupts the outer membrane. We also describe the structure and membrane topology of the second infectious form of vaccinia, the extracellular enveloped virus, and confirm that this form possesses an extra membrane overlying the IMV.
Molluscum contagiosum virus (MCV) commonly causes asymptomatic cutaneous neoplasms in children and sexually active adults as well as persistent opportunistic acquired immunodeficiency syndrome (AIDS)-associated disease. Sequencing the 190-kilobase pair genome of MCV has now revealed that the virus potentially encodes 163 proteins, of which 103 have homologs in the smallpox virus. MCV lacks counterparts to 83 genes of the smallpox virus, including those important in suppression of host responses to infection, nucleotide biosynthesis, and cell proliferation. MCV possesses 59 genes that are predicted to encode previously uncharacterized proteins, including major histocompatibility complex class I, chemokine, and glutathione peroxidase homologs, which suggests that there are MCV-specific strategies for coexistence with the human host.
Coronaviruses are enveloped RNA viruses involved in a variety of pathologies that affect animals and humans. Existing structural models of these viruses propose a helical nucleocapsid under the virion envelope as the unique internal structure. In the present work, we have analyzed the structure of the transmissible gastroenteritis coronavirus. The definition of its organization supports a new structural model for coronaviruses, since a spherical, probably icosahedral, internal core has been characterized. Disruption of these cores induces the release of N-protein-containing helical nucleocapsids. Immunogold mapping and protein analysis of purified cores showed that they consist of M and N proteins, M being the main core shell component. This surprising finding, together with the fact that M protein molecules are also located in the virion envelope, indicates that a reconsideration of the assembly and maturation of coronaviruses, as well as a study of potential M-protein subclasses, is needed.
We describe herein the characterization of p39, the product of the A4L gene of vaccinia virus. By immunolabelling of thawed cryosections from infected HeLa cells, we show that this protein is initially located in the central region, or viroplasm, of the viral factories, as well as in the immature virions, with very small amounts of labelling observed on the surrounding membranes. The localization of p39 changes dramatically during the transition of the immature virion to the intracellular mature virus (IMV), coincident with the appearance of the core structure in the center of the IMV, with p39 located between this core and the surrounding membranes. Complementary biochemical data, such as partitioning into the Triton X-114 detergent phase and stripping of the viral membranes with Nonidet P-40 and dithiothreitol, suggest that p39 is associated with the innermost of the two membranes surrounding the core. Sodium carbonate treatment also indicates that p39 is associated with membranes, even at the early stages of viral assembly. However, following in vitro translation of p39 in the presence of microsomal membranes, we failed to detect any association of the independently expressed protein with membranes. We also failed to detect any posttranslational acylation of p39 with myristate or palmitate, suggesting that p39 does not achieve its membrane association through lipid anchors. Therefore, p39 is most likely membrane associated through an interaction with an integral membrane protein(s) present in the innermost of the two membranes surrounding the IMV. These data, together with our recent data showing that p39 colocalizes with the spike-like protrusions on the IMV core (N. Roos, M. Cyrklaff, S. Cudmore, R. Blasco, J. Krijnse-Locker, and G. Griffiths, EMBO J. 15:2343-2355, 1996), suggest that p39 may form part of this spike and that it possibly functions as a matrix-like linker protein between the core and the innermost of the two membranes surrounding the IMV.
The vaccinia virus (VV) A17L gene encodes a 21- to 23-kDa virion component that forms a stable complex with the 14-kDa envelope protein (A27L gene). In a previous report, we described the construction of a VV recombinant, VVindA17L, in which the expression of the A17L gene is inducibly regulated by isopropyl-beta-D-thiogalactoside (IPTG). We demonstrated that shutoff of the A17L gene results in a blockade of virion morphogenesis at a very early stage (D. Rodríguez, M. Esteban, and J. R. Rodríguez, J. Virol. 69:4640-4648, 1995). In the present study, we show that virus growth is restored if the inducer is provided not later than 6 h postinfection. Immunofluorescence and immunoelectron microscopy analysis of VVindA17L-infected cells revealed that in the absence of the 21- to 23-kDa protein, the 14-kDa protein is distributed throughout the cytoplasm. After IPTG addition, the 14-kDa protein can be detected around viral factories and immature virions; at later times, it localizes in the external membranes of intracellular mature virions. Immunoelectron microscopy with anti-21- to 23-kDa antibodies showed that soon after induction, the protein accumulates in membranes of the rough endoplasmic reticulum and in the nuclear envelope. With time, the protein localizes in viral crescents and subsequently associates to the membranes of immature and intracellular mature virions. These results are consistent with a model in which the 21- to 23-kDa protein would be synthesized at the endoplasmic reticulum, from where the protein could be translocated to the membranes of the intermediate compartment to generate the precursors of the viral membranes. Also, these results argue that 14-kDa envelope protein becomes posttranslationally associated to viral membranes through its interaction with the 21-kDa protein.
Vaccinia virus (VV) membrane biogenesis is a poorly understood process. It has been proposed that cellular membranes derived from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) are incorporated in the early stages of virion assembly. We have recently shown that the VV 21-kDa (A17L gene) envelope protein is essential for the formation of viral membranes. In the present work, we identify a 15-kDa VV membrane protein encoded by the A14L gene. This protein is phosphorylated and myristylated during infection and is incorporated into the virion envelope. Both the 21- and 15-kDa proteins are found associated with cellular tubulovesicular elements related to the ERGIC, suggesting that these proteins are transported in these membranes to the nascent viral factories. When synthesis of the 21-kDa protein is repressed, organized membranes are not formed but numerous ERGIC-derived tubulovesicular structures containing the 15-kDa protein accumulate in the boundaries of the precursors of the viral factories. These data suggest that the 21-kDa protein is involved in organizing the recruited viral membranes, while the 15-kDa protein appears to be one of the viral elements participating in the membrane recruitment process from the ERGIC, to initiate virus formation.
The use of two-dimensional gel electrophoresis has identified the gene products A14L (p16) and A13L (p8) as abundant membrane proteins of the first infectious form of vaccinia virus, the intracellular mature virus (IMV; O. N. Jensen, T. Houthaeve, A. Shevchenko, S. Cudmore, T. Ashford, M. Mann, G. Griffiths, J. Krijnse Locker, J. Virol. 70:7485-7497, 1996). In this study, these two proteins were characterized in detail. In infected cells, both proteins localize not only to the viral membranes but also to tubular-cisternal membranes of the intermediate compartment, defined by the use of antibodies to either rab1A or p21, which colocalize with rab1A (J. Krijnse Locker, S. Schleich, D. Rodriguez, B. Goud, E. J. Snijder, and G. Griffiths, J. Biol. Chem. 271:14950-14958, 1996). Both proteins appear to reach this destination via cotranslational insertion into the rough endoplasmic reticulum, as shown by in vitro translation and translocation experiments. Whereas p16 probably spans the membrane twice, p8 is inserted into the membrane by means of its single NH2-terminal hydrophobic domain, adopting a topology which leaves the C terminus exposed to the cytoplasm. Combined immunocytochemical and biochemical data show that p16 is a member of the inner of the two IMV membrane layers, whereas p8 localizes to both the inner and the outer membrane. These findings are discussed with respect to our model of IMV membrane assembly.
The immunodominant, 39,000-molecular weight core protein (39K protein) of fowlpox virus (FP9 strain), equivalent to the vaccinia virus A4L gene product, contains highly charged domains at each end of the protein and multiple copies of a 12-amino-acid serine-rich repeat sequence in the middle of the protein. Similar repeats were also detected in other fowlpox virus strains, suggesting that they might confer a selective advantage to the virus. The molloscum contagiosum virus homolog (MC107L) also contains repeats, unlike the vaccinia virus protein. The number of repeats in the fowlpox virus protein does not seem to be crucial, since some strains have a different number of repeats, as shown by the difference in the size of the protein in these strains. The repeat region could be deleted, indicating that it is not essential for replication in vitro. It was not possible to delete the entire 39K protein, indicating that it was essential (transcriptional control signals for the flanking genes were left intact). The repeat region is partly responsible for the immunodominance of the protein, but the C-terminal part of the protein also contains highly antigenic linear epitopes. A role for the 39K protein in immune system modulation is discussed.
anddistinguished fromspontaneous TK-mutantsbytheaddition ofa (3-gal indicator totheagaroseoverlay. Plaques thatexpressed ,8-gal stained darkbluewithin several hoursat3rC. Alternatively, TK-selection could beeliminated, andrecombinant plaques could bereadily identified solely by their bluecolor. Thereverseprocedure, inwhichthestarting virus expresses(3-gal (i.e., formsblueplaques) andthedesired recombinant hasdeleted theentire (-gal gene (i.e., formswhiteplaques), isanother alternative. Eachprotocol wastested byconstructing vaccinia virus recombinants that expresshepatitis Bvirus surface antigen. Vaccinia virus, themostthoroughly studied memberof thepoxvirus family, wassuccessfully usedasalive vaccine toeradicate smallpox. Medical interest invaccinia virus subsequently declined butwasrestimulated whenlive re- combinants wereshowntobecapable ofexpressing foreign genes (8,18)andofprotectively immunizing animals against infections withinfluenza virus (26), herpes simplex virus types 1(6,19)and2(6), hepatitis B virus (14), rabies virus (31) andvesicular stomatitis virus (10). Theability toincor- porate large amountsofforeign DNA without a lossof infectivity (25), thecorrect processing ofexpressed proteins (6,10,24,26,31), andthewidehostrange ofvaccinia virus alsomakethis expression system ofspecial valueforre- search purposes. Onenovel application hasbeentoidentify target antigens forcell-mediated immuneresponses tovi- ruses (1,33). Tofacilitate theuseofvaccinia virus asacloning and expression vehicle, Mackett etal.(9)constructed plasmid vectors thatcontain defined vaccinia virus promoters, re- striction sites fortheinsertion offoreign coding sequences, andflanking segments ofthevaccinia virus thymidine kinase (TK)gene.Vaccinia virus promoters, whicharefound immediately upstream ofearly andlate vaccinia virus genes (2,5,9,30), arenecessary because ofthespecificity ofthe viral transcription system (21)andthecytoplasmic site of replication. Flanking segments ofvaccinia virus DNA are usedforhomologous recombination (16, 23,28), themost practical wayofinserting foreign sequences into thelarge 187,000-base-pair
The emphasis of this paper is on developing suitable intervening variables and constraint approximations for structural reliability analysis. Traditionally, these procedures are used in structural optimization, whereas this research work adopts these concepts to safety index and failure probability computations. The use of these concepts enables the development of an efficient and stable iteration algorithm for identifying the most probable failure points (MPPs) of the limit state functions. An approximate second-order failure probability is calculated at this MPP with no extra computations of the limit state function and gradients. The efficiency and accuracy of the proposed algorithm are demonstrated by several examples with highly nonlinear, complex, explicit/implicit performance functions.
The serological relationships between several members of the poxvirus group were investigated by a variety of techniques, including complement fixation, gel diffusion and ring precipitation, hemagglutinin inhibition, pock and plaque neutralization, and staining with fluoroscein-coupled antibody.The existence of close relationships between vaccinia, cowpox, rabbit-pox, and ectromelia; and between myxoma and fibroma; was confirmed. No crossing could be demonstrated between these subgroups, or between either of them and fowlpox or monkey tumor poxvirus, except when an alkaline extract of vaccinia or myxoma viruses (the “NP antigen”) was used. The “NP antigen” appears to contain a group antigen common to all viruses of the poxvirus group. In addition to fixing complement and precipitating when tested against both homologous and heterologous “NP antigens,” antisera to the “NP antigen” of vaccinia and myxoma neutralized the homologous but not the heterologous virus, when tested by pock and plaque neutralization tests.
Rifampicin was used to block vaccinia virus assembly and accumulate structural proteins that have not undergone maturational processing. Two of these proteins were purified and shown by tryptic peptide analysis to be higher molecular weight precursors of the major core components. In contrast, rifampicin did not prevent the formation from its precursor of another virion protein located outside the virus core.
Specific missense mutations of the vaccinia virus D13L gene confer resistance to the effects of rifampicin on virion morphogenesis. We constructed a recombinant vaccinia virus in which elements of the Escherichia coli lac operator system were used to regulate the D13L gene. Replication of the recombinant vaccinia virus was dependent on addition of the inducer isopropyl beta-D-thiogalactoside (IPTG) and the virus yield was decreased by more than 99% when IPTG was omitted. Under the nonpermissive condition, transcription of the D13L gene was reduced and synthesis of the 65,000-Da protein product was inhibited by more than 95%. Consequently, virion morphogenesis was blocked at an early stage and uncoated membrane precursors of the immature viral envelope and uncleaved precursors of the major core proteins accumulated. The phenotype of the conditional lethal mutant virus, in the absence of IPTG, closely resembled that of wild-type virus in cells treated with rifampicin.
The most abundant vaccinia virus (VV) core protein found within the virion is protein 4a, which represents approximately 14% of the particle's dry weight. The 4a protein is synthesized as a 102.5-kDa precursor, which is proteolytically processed to a 62-kDa product concomitant with virion assembly. To identify the pathway by which P4a is converted into 4a, immunological reagents which are specific for subregions of the P4a precursor were developed and used in concert with peptide mapping and protein sequencing procedures. The results obtained suggest that the 891 amino acid P4a precursor is cleaved at two locations, between residues 614 and 615 and 697 and 698. Both the large amino-terminal 4a protein (residues 1-614) and the carboxy-terminal-derived 23-kDa protein (residues 698-891) become major virion constituents. The location and fate of the small internal peptide (residues 615-697) is not known. Interestingly, an analysis of the predicted amino acid sequences at the sites of cleavage within the P4a precursor indicated the presence of an Ala-Gly decreases Thr motif flanking the 697-698 site and an Ala-Gly decreases Ser motif flanking the 614-615 site. Since both of these signals are quite similar to the Ala-Gly decreases Ala signal previously identified as the cleavage point within the VV P4b and P25K core protein precursors (VanSlyke et al., 1991.J. Gen. Virol. 72, 411-416), this suggests that processing of all three core protein precursors may be coordinately linked and/or catalyzed by the same proteinase during viral assembly.
A conditional lethal vaccinia virus mutant, which constitutively expresses the Escherichia coli lac repressor and has the lac operator controlling the F18R gene (the 18th open reading frame of the HindIII F fragment of the vaccinia virus strain WR genome) encoding an 11-kDa protein, was previously shown to be dependent on the inducer isopropyl-beta-D-thiogalactoside (IPTG) for replication (Y. Zhang and B. Moss, Proc. Natl. Acad. Sci. USA 88:1511-1515, 1991). Further studies indicated that the yield of infectious virus could be regulated by titration with IPTG and that virus production was arrested by IPTG removal at appropriate times. Under nonpermissive conditions, an 11-kDa protein reactive with antiserum raised to a previously described DNA-binding phosphoprotein (S. Y. Kao and W. R. Bauer, Virology 159:399-407, 1987) was not synthesized, indicating that the latter is the product of the F18R gene. In the absence of IPTG, replication of viral DNA and the subsequent resolution of concatemeric DNA molecules appeared normal. Omission of IPTG did not alter the kinetics of early and late viral protein synthesis, although the absence of the 11-kDa polypeptide was noted by labeling infected cells with [35S]methionine or [32P]phosphate. Pulse-chase experiments revealed that proteolytic processing of the major viral structural proteins, P4a and P4b, was inhibited under nonpermissive conditions, suggesting a block in virus maturation. Without addition of IPTG, the failure of virus particle formation was indicated by sucrose gradient centrifugation of infected cell lysates and by the absence of vaccinia virus-mediated pH-dependent cell fusion. Electron microscopic examination of infected cells revealed that immature virus particles, with aberrant internal structures, accumulated when synthesis of the 11-kDa DNA-binding protein was prevented.
In contrast to its irreversible effect on the Escherichia coliRNA polymerase beta-subunit, the antibiotic rifampicin reversibly inhibits vaccinia virus morphogenesis at a step during the formation of immature viral particles. The protein affected by the presence of rifampicin is L65, a major late vaccinia polypeptide to which mutations that confer rifampicin resistance have been mapped. We now provide evidence using a monospecific anti-L65 serum in concert with immunofluorescence and sucrose gradient analysis that the mechanism of action of rifampicin on vaccinia virus replication involves the inhibition of localization of L65 to the viral factories (virosomes) thereby blocking further development. Studies on the expression and distribution of L65 during the infection cycle reveal that L65 is a stable, nonglycosylated late protein associated with virions. These results are discussed in relationship to the possible in vivo functions of the L65 protein.
We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia
coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture
cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants
could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous
TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue
within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily
identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms
blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative.
Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.
The genetic locus specifying rifampicin-resistance (RifR) in a vaccinia virus mutant has been localized by marker rescue analysis (J. Tartaglia and E. Paoletti (1985) Virology 147, 394-404). The mutation was defined by DNA sequence analysis as an AT to GC transition occurring 56 bp to the left of the unique XhoI site within HindIII D. The point mutation resulted in an asparagine to aspartic acid substitution 60 amino acids from the predicted C-terminus. Specific DNA probes were used to characterize the RifR designated gene at the transcriptional and translational levels. This region is transcriptionally active only after vaccinia virus DNA synthesis, but not in the presence of cytosine arabinoside suggesting that the RifR function is a late gene product. Translation of hybrid selected RNA to DNA surrounding the mutant marker directed the synthesis of a polypeptide with an apparent mol wt of 63 kDa. Transcriptional and translational mapping studies showed that the mRNA encoding this 63-kDa polypeptide was initiated approximately 460 bp to the right of the HindIII D-A junction and was transcribed in a leftward direction into the HindIII D region.
Vaccinia viral polypeptides, labeled in the presence of rifampicin, were incorporated into virus particles after rifampicin was removed. All structural polypeptides, resolved by polyacrylamide gel electrophoresis, were labeled in the presence of rifampicin. The specific effect of rifampicin on the formation of the vaccinia viral envelope was reversed within 10 min even in the presence of NaF or inhibitors of protein or nucleic acid synthesis. Further maturation of some virus particles, which were characterized by their ultrastructure and isolated by sucrose gradient sedimentation, proceeded in the presence of inhibitors of protein synthesis. In contrast, addition of NaF or actinomycin D almost completely blocked the development of mature virus particles. Rifampicin acted specifically at the stage of envelope formation and did not prevent later steps in maturation.
Vaccinia virions were dissociated into their constituent polypeptides which were then analyzed by means of SDS-urea polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue or by autoradiography. Thirty distinct species of polypeptides were identified, which ranged in molecular weight from about 8000 to over 200,000. They could be classified as follows: 1.a. Five polypeptides were shown to be located at or near the virion surface. This conclusion was based on their ability to react in situ with iodine in the presence of lactoperoxidase and hydrogen peroxide, and with fluorescein isothiocyanate; and on the ability of chymotrypsin and of NP40 to release them from virions. They were VP4c, VP7a, VP10a, VP11a, and VP12, the smallest polypeptide.2.b. Seventeen polypeptides were present in cores prepared by treating virions with NP40 and 2-mercaptoethanol followed by iodoacetamide, sonication, and banding in sucrose density gradients. Among these were the two principal virion polypeptides, VP4a and VP4b, which accounted for about 50% of their protein complement. Cores contained about one-half of the virions' mass.3.c. Two polypeptides, VP6a and VP6b, contained glucosamine and are therefore glycopolypeptides. They were located neither on the virion surface as judged by the criteria in (a) above, nor in cores prepared as described in (b) above.4.d. Vaccinia virus which had multiplied in cells suspended in medium containing 32P yielded 6 labeled bands which may represent phosphoproteins. Most of the label (about 75%) comigrated with VP11b, a major virion component (11.4% of total viral protein, MW about 11,000).
Several variants of vaccinia and cowpox were employed to study the problems of virus dissemination involving wrapping in host membranes or integration within A-type inclusions (ATI).All the cowpox variants employed induce ATI and hemagglutinin (HA). The control of synthesis of HA and ATI and its relationship to the process of occlusion was examined by means of a variety of metabolic inhibitors. The experimental data indicated that ATI and HA are virus-directed late functions whose synthesis is under regulation that is distinctive from that related to biogenesis of mature virus. Capability to become occluded within ATI, a heritable character possessed only by some strains, is termed the V factor (Ichihashi and Matsumoto, 1968). Expression of the V trait can occur in the presence of rifampicin, as revealed by integration into ATI of immature V+ strain CP58 particles. The V+ trait can also be transferred to the V− strain CPRC1 by complementation in a double infection with the rifampicin-resistant NR4 vaccinia. These observations suggest that V may be acquired as a component of the virus membrane.To study the fate of progeny not involved in occlusion in ATI, we employed vaccinia IHD-W, which induces polykaryocyte formation (F+) but is HA−, and strain IHD-J, which is HA+ and F−. An orderly progression of events, particularly evident with IHD-W virus, was associated with the movement of progeny particles which were formed earliest. According to the reconstructed sequence, the wrapping of individual virus particles in cell-derived membranes occurred prior to their release at the cell surface. The IHD-J progeny were usually not enclosed by cisternae and migrated to the cell surface as unwrapped or naked particles. Both the cell membranes which formed themselves into membranes of cisternae and certain defined regions of plasma membrane in contact with naked intracellular progeny virus possessed virus-specified antigen(s).
The biogenesis of vaccinia envelopes was examined by combined chemical and electron microscopic procedures. These lipoprotein membranes develop within discrete viroplasmic foci, initially 3–3.5 hours after infection. Sequential appearance of viral envelopes and immature and mature particles could be arrested at defined stages by means of actinomycin D or streptovitacin A. Application of these compounds in advance or following morphogenesis indicated that transcription into the requisite RNA precedes by 60 minutes, and translation into protein by less than 30 minutes, the assembly of membranes. Proteins required for maturation are synthesized within 30 minutes following morphogenesis of immature virus. Experiments with isotopically labeled choline, a specific precursor of lecithin in these membranes, indicated that nascent phospholipid is preferentially integrated into the progeny, and analyses using thin-layer gas chromatography revealed that the fatty acid composition of vaccinia can be distinguished from that of host cell membranes. These combined results imply that unique membranes of vaccinia can condense de novo from precursors, two of which are lecithin and viral protein(s), to become the envelope surrounding immature particles. Further differentiation into mature virus occurs inside this envelope. The applicability of the vaccinia model to biogenesis of cellular membranes in general is discussed.
Previously published investigations with inhibitors and temperature-sensitive (ts) mutants revealed that post-translational cleavage (PTC) of virion core polypeptides is a necessary step for development of infectious, mature vaccinia virus. Present studies focused on the nature of the protease factor(s) required for vaccinia biogenesis. To ascertain whether the proteolytic factor(s) can move freely through the cytoplasm. PTC occurring during complementation between cleavage defective and DNA-ts mutants was compared with that evident following induced syncytiogenesis, involving cells singly inoculated with wild-type and cleavage-defective ts 1095 virus. Since PTC can occur during coinfection but not after cell-cell fusion, the protease factor is presumed to be on diffusible. This notion is supported by the incapacity of extracts from infected cells to bring about in vitro PTC. Data from temperature shift experiments with ts 1085 indicate that the factor(s) for proteolysis is probably a short-lived activity and affinity labeling suggests that if may be a virus-induced, nonvirion polypeptide p 12.5 in molecular weight, possessing the specificity of chymotrypsin for protease inhibitors TPCK and ZPCK. Evidence indicating that the factor has a brief half-life implies that it must be synthesized on a continuous basis to effect viral maturation. A model of vaccina self-assembly, which takes into account previous observations and current data, is proposed according to which induction of core enzymatic activities, internal differentiation, and aquisition of infectiousness are temporally coordinated, closely coupled phenomena requiring PTC.
Vaccinia virus encodes two protein kinases; the B1 kinase is expressed early and appears to play a role during DNA replication, whereas the F10 kinase is expressed late and is encapsidated in virions. Here we report that the F10 kinase gene is the locus affected in a complementation group of temperature-sensitive mutants composed of ts15, ts28, ts54, and ts61. Although these mutants have a biochemically normal phenotype at the nonpermissive temperature, directing the full program of viral gene expression, they fail to form mature virions. Electron microscopic analysis indicates that morphogenesis undergoes arrest at a very early stage, prior to the formation of membrane crescents or immature virions. An essential role for the F10 protein kinase in orchestrating the onset of virion assembly is implied.
Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts28, ts54, ts61, and ts15) composing a single complementation group have been mapped by marker rescue to the F10 open reading frame located within the genomic HindIII F DNA fragment. Sequencing of the F10 gene from wild-type and mutant viruses revealed single-amino-acid substitutions in the F10 polypeptide responsible for thermolabile growth. Although the ts mutants displayed normal patterns of viral protein synthesis, electron microscopy revealed a profound morphogenetic defect at the nonpermissive temperature (40 degrees C). Virion assembly was arrested at an early stage, with scant formation of membrane crescents and no progression to normal spherical immature particles. The F10 gene encodes a 52-kDa serine/threonine protein kinase (S. Lin and S. S. Broyles, Proc. Natl. Acad. Sci. USA 91:7653-7657, 1994). We expressed a His-tagged version of the wild-type, ts54, and ts61 F10 polypeptides in bacteria and purified these proteins by sequential nickel affinity and phosphocellulose chromatography steps. The wild-type F10 protein kinase activity was characterized in detail by using casein as a phosphate acceptor. Whereas the wild-type and ts61 kinases displayed similar thermal inactivation profiles, the ts54 kinase was thermosensitive in vitro. These findings suggest that protein phosphorylation plays an essential role at an early stage of virion assembly.
Within vaccinia virus-infected cells, the product of the L1R open reading frame is covalently modified by myristic acid at the penultimate NH2-terminal glycine residue. Previously we have shown that while the L1R protein is a constituent of both intracellular mature virus particles and extracellular enveloped virions which are released from the infected cell, it is associated exclusively with the primary membranes surrounding the virion core. Given this rather specific localization, it was of interest to study the potential role of this essential gene in virus replication and morphogenesis. To this end, we have constructed a recombinant vaccinia virus in which expression of the L1R gene can be transcriptionally repressed. Without the inducer isopropylthiogalactopyranoside (IPTG), synthesis of the L1R protein was blocked, resulting in a total inhibition of plaque formation. Velocity sedimentation of viral particles labeled in the presence of [3H]thymidine, grown in the absence of IPTG, revealed a substantial reduction in viral DNA incorporation into virions. Likewise, proteolysis of the major core proteins p4a, p4b, and p25K, believed to occur during the final stages of virion maturation, was severely impaired. In the absence of L1R expression, only immature virions could be detected by electron microscopy. Transient expression of a plasmid containing the full-length L1R gene driven by its own promoter was able to complement and rescue the defective phenotype. However, a plasmid bearing a mutation in the myristyl acceptor glycine residue was unable to biologically rescue the recombinant, and the protein was not detected in purified virions.trans complementation using a truncated, myristylated form of the L1R protein partially rescued the defective mutant. Collectively, these data suggest that myristic acid mediates essential interactions of the L1R protein with viral membranes and/or other virion components that lead to the productive assembly, maturation, and release of particles.
A characterization of vaccinia virus strain Western Reserve (WR) open reading frame (ORF) A36R is described. This ORF is predicted to encode a 221-amino-acid protein (M(r) 25.1 K) with an amino-terminal hydrophobic sequence, seven potential sites for attachment of N-linked carbohydrate, but no carboxy-terminal transmembrane anchor. It is identical in vaccinia strain Copenhagen and shares 94.6% amino acid identity with the corresponding ORF in variola virus strains Harvey, India-1967, and Bangladesh-1975. RNA analyses detected a 600-nucleotide, early transcript that initiated 10-13 nucleotides upstream of the A36R ORF, and heterogeneously sized late transcripts running across the ORF. A rabbit antiserum raised against an Escherichia coli glutathione S-transferase fusion protein identified M(r) 43-50 K proteins that accumulated late during vaccinia virus infection and fractionated as integral membrane proteins during Triton X-114 partitioning. Similar polypeptides were expressed by vaccinia virus strains Tian Tan, Tashkent, Lister, Wyeth, Copenhagen, and IHD-J and by rabbitpox virus and cowpox virus (strain Brighton Red). Immunoblot analysis of purified and protease-digested intracellular mature virus (IMV) and extracellular enveloped virus (EEV) showed that the A36R proteins were present on the surface of EEV with type II membrane topology, but were absent from IMV. A WR deletion mutant lacking the A36R ORF (delta A36R) had a small plaque phenotype on all cell lines tested. IMV formation by delta A36R was unaltered but EEV formation was reduced approximately fivefold compared to wild-type (WT) when measured either by density gradient analysis of isotopically labeled virions or by infectivity assays. Thus the loss of the A36R protein from the EEV surface did not reduce EEV specific infectivity in vitro. Despite this, delta A36R showed striking attenuation compared with WT in a murine intranasal model. Finally, a revertant virus in which the A36R ORF was restored showed WT plaque size, EEV formation, and virulence, demonstrating that all the phenotypic differences of delta A36R were attributable to loss of the A36R gene and not to other mutations acquired during its construction.
We analyzed the 186,102 base pairs (bp) that constitute the entire DNA genome of a highly virulent variola virus isolated from Bangladesh in 1975. The linear, double-stranded molecule has relatively small (725 bp) inverted terminal repeat (ITR) sequences containing three 69-bp direct repeat elements, a 54-bp partial repeat element, and a 105-base telomeric end-loop that can be maximally base-paired to contain 17 mismatches. Proximal to the right-end ITR sequences are another seven 69-bp elements and a 53- and a 27-bp partial element. Sequence analysis showed 187 closely spaced open reading frames specifying putative major proteins containing > or = 65 amino acids. Most of the virus proteins correspond to proteins in current databases, including 150 proteins that have > 90% identity to major gene products encoded by vaccinia virus, the smallpox vaccine. Variola virus has a group of proteins that are truncated compared with vaccinia virus counterparts and a smaller group of proteins that are elongated. The terminal regions encode several novel proteins and variants of other poxvirus proteins that potentially augment variola virus transmissibility and virulence for its only natural host, humans.
Proteolytic processing of vaccinia virus core proteins is an essential step in the formation of mature virions and occurs during the process of virion morphogenesis. In order to investigate how the vaccinia virus (VV) structural proteins become integrated into virus particles during normal maturation, immunological reagents were generated against the three major VV core proteins 4a, 4b, and 25K and their precursor molecules P4a, P4b, and P25K. These sera were used in conjunction with immunofluorescent and immunogold labeling of VV-infected tissue culture cells. The immunofluorescent results indicated that all three core precursors and their cleavage products were localized to virosomes. As the infection progressed, punctate staining with these sera became spread throughout the cytoplasm which suggested that individual virion particles were being recognized. Immunoelectron microscopy showed that the core proteins were localized to the center of both immature and mature virus particles. This result was in contrast to the situation observed using antisera directed against L65, a protein previously implicated in the assembly of the viral membrane. Immunogold staining of L65 showed that it was initially located along the inner side of the immature virion membrane and remained with the membrane even as the viroplasm began to condense toward the center of the virus particle. In order to determine whether the core protein localization observed was the result of precursors, products, or both, a synthetic peptide strategy was used to generate an antiserum that recognized only P4a in immunoprecipitation reactions. Immunogold labeling with this reagent indicated that P4a was found in the viroplasm of immature particles and in low levels in the mature virion. Intracellular localization of core and L65 proteins during virion morphogenesis is discussed.
The ts16 mutation of vaccinia virus WR (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) has been mapped by marker rescue to the I7L open reading frame located within the genomic HindIII I DNA fragment. The I7 gene encodes a 423-amino-acid polypeptide. Thermolabile growth was attributed to an amino acid substitution, Pro-344-->Leu, in the predicted I7 protein. A normal temporal pattern of viral protein synthesis was elicited in cells infected with ts16 at the nonpermissive temperature (40 degrees C). Electron microscopy revealed a defect in virion assembly at 40 degrees C. Morphogenesis was arrested at a stage subsequent to formation of spherical immature particles. Western immunoblot analysis with antiserum directed against the I7 polypeptide demonstrated an immunoreactive 47-kDa polypeptide accumulating during the late phase of synchronous vaccinia virus infection. Immunoblotting of extracts of wild-type virions showed that the I7 protein is encapsidated within the virus core. The I7 polypeptide displays amino acid sequence similarity to the type II DNA topoisomerase of Saccharomyces cerevisiae.
Vaccinia virus gene B5R encodes a M(r) 42K glycoprotein that is expressed throughout infection and forms part of the envelope of extracellular virus. In this paper deletion mutants (delta B5R) lacking the B5R open reading frame (ORF) from the Western Reserve (WR) and IHD-J strains of vaccinia virus have been constructed and shown to form very small plaques compared with the wild-type viruses. This phenotype was directly attributable to loss of the B5R gene since re-insertion of this gene from WR or IHD-J into the WR mutant lacking B5R (W-delta B5R) restored a normal plaque phenotype. In the latter case the failure of the revertant to form comets indicated that the nine amino acid differences in the B5R ORF between the IHD-J and WR strains of virus are not responsible for comet formation by IHD-J virus. Furthermore, the B5R deletion mutant of IHD-J (I-delta B5R) still formed small comets. Despite the small plaque phenotype of the deletion mutants, normal yields of intracellular naked virus (INV) were produced. In contrast, deletion of B5R had a profound affect on the formation of the extracellular enveloped virus (EEV). Transmission electron microscopy indicated that INV particles were not wrapped by a double layer of Golgi-derived membrane and enveloped particles were not detected within the cell or on the cell surface without expression of the B5R protein. Biochemical measurement of EEV formation, by labeling infected cells with [3H]thymidine followed by cesium chloride density gradient centrifugation of particles released from the cells 24 hr postinfection, showed that only 10% of WT levels of EEV were produced by I-delta B5R. The loss of the B5R ORF caused severe attenuation in intranasally infected mice. At doses between 10(4) and 3 x 10(7) plaque-forming units there were no signs of disease in animals infected with W-delta B5R, whereas at comparable doses the WR parent virus caused significant mortalities. Finally, an ORF with 93.4% amino acid identity to vaccinia WR B5R is present in variola major virus strain Harvey and the B5R protein was shown by Western blotting to be expressed by all orthopoxviruses tested.
We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.
Vaccinia virus early transcription factor (VETF) is a heterodimeric protein that is packaged in virus particles for expression of early genes during the next round of infection. To investigate additional roles of VETF, we constructed a conditionally lethal recombinant vaccinia virus in which the D6R gene, encoding the 70-kDa subunit of VETF, is under stringent Escherichia coli lac operator control. When cells were infected with the recombinant virus in the absence of an inducer, synthesis of the 70-kDa protein was undetectable and the yield of infectious virus was severely reduced. Under these nonpermissive conditions, DNA replication and synthesis of viral proteins other than the one encoded by D6R occurred, suggesting that de novo synthesis of VETF is not required for expression of early or late genes during the virus growth cycle. Electron microscopy, however, revealed that immature virus particles and masses of electron-dense material accumulated in the absence of an inducer. We concluded that VETF has a direct role in virion morphogenesis or is required for expression of a novel subset of genes that have such a role.
The product of the vaccinia virus I1 gene was characterized biochemically and genetically. This 35-kDa protein is conserved in diverse members of the poxvirus family but shows no homology to nonviral proteins. We show that recombinant I1 binds to both single-stranded and double-stranded DNA in a sequence-nonspecific manner in an electrophoretic mobility shift assay. The protein is expressed at late times during infection, and approximately 700 copies are encapsidated within the virion core. To determine the role of the I1 protein during the viral life cycle, a inducible viral recombinant in which the I1 gene was placed under the regulation of the Escherichia coli lac operator/repressor was constructed. In the absence of isopropyl-beta-D-thiogalactopyranoside, plaque formation was abolished and yields of infectious, intracellular virus were dramatically reduced. Although all phases of gene expression and DNA replication proceeded normally during nonpermissive infections, no mature virions were produced. Electron microscopic analysis confirmed the absence of mature virion assembly but revealed that apparently normal immature virions accumulated. Thus, I1 is an encapsidated DNA-binding protein required for the latest stages of vaccinia virion morphogenesis.