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Infectious cycle of foot-and mouth disease virus
Abstract
Foot-and-mouth disease virus (FMDV) is a single-stranded, positive-sense RNA virus belonging to the genus Aphthovirus in the family Picornaviridae. FMDV enters cells via the mechanism of receptor-mediated endocytosis in which the low pH of the endosomal compartment triggers uncoating of the viral genome. FMDV enters cells by attaching itself to cellular receptor molecules of the integrin family. For FMDV the receptor has been identified as the Arg-Gly-Asp (RGD) binding integrin. The integrin-binding RGD is located in the G-H loop of VP1 and it is highly conserved among all seven serotypes. The FMDV genome organization is similar to that of other picornaviruses. The genome is composed of three parts, the 5′ non-translated region (5′NTR), the coding region and the 3′ non-translated region (3′NTR) containing a heteropolymeric segment and poly(A) tail, which is required for viral replication. The 5'NTR plays important roles in cap-independent translation initiation of the viral polyprotein and in viral genome replication. After translation, the polyprotein is cleaved into four primary cleavage products: the amino terminal L protease; P1-2A, the precursor of the capsid proteins; 2BC and P3 which are cleaved into nonstructural proteins.
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The poliovirus capsid precursor polyprotein, P1, is cotranslationally modified by the addition of myristic acid. We have examined the importance of myristylation of the P1 capsid precursor during the poliovirus assembly process by using a recently described recombinant vaccinia virus expression system which allows the independent production of the poliovirus P1 protein and the poliovirus 3CD proteinase (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). We constructed a site-directed mutation in the poliovirus cDNA encoding an alanine at the second amino acid position of P1 in place of the glycine residue required for the myristic acid addition and isolated a recombinant vaccinia virus (VVP1myr-) that expressed a nonmyristylated form of the P1 capsid precursor. The 3CD proteinase expressed by a coinfecting vaccinia virus, VVP3, proteolytically processed the nonmyristylated precursor P1 expressed by VVP1myr-. However, the processed capsid proteins, VP0, VP3, and VP1, did not assemble into 14S or 75S subviral particles, in contrast to the VP0, VP3, and VP1 proteins derived from the myristylated P1 precursor. When cells were coinfected with VVP1myr- and poliovirus type 1, the nonmyristylated P1 precursor expressed by VVP1myr- was processed by 3CD expressed by poliovirus, and the nonmyristylated VP0-VP3-VP1 (VP0-3-1) protomers were incorporated into capsid particles and virions which sedimented through a 30% sucrose cushion. Thus, the nonmyristylated P1 precursor and VP0-3-1 protomers were not excluded from sites of virion assembly, and the assembly defects observed for the nonmyristylated protomers were overcome in the presence of myristylated capsid protomers expressed by poliovirus. We conclude that myristylation of the poliovirus P1 capsid precursor plays an important role during poliovirus assembly by facilitating the appropriate interactions required between 5S protomer subunits to form stable 14S pentamers. The results of these studies demonstrate that the independent expression of the poliovirus P1 and 3CD proteins by using recombinant vaccinia viruses provides a unique experimental tool for analyzing the dynamics of the poliovirus assembly process.
A cellular 57-kDa protein (p57) that binds specifically to the internal translation initiation site in the 5' untranslated region of foot-and-mouth disease virus RNA was detected in cell extracts of different mammalian species by UV cross-linking. The protein binds to two distinct sites of the translation control region which have as the only common sequence a UUUC motif. The first binding site consists of a conserved hairpin structure, whereas the second binding site contains an essential pyrimidine-rich region without obvious secondary structure. Competition experiments indicate that the complexes with the two binding sites were formed by a single p57 species. The protein binds also to the 5' untranslated region of other picornaviruses. Results from footprint analyses with foot-and-mouth disease RNA suggest the participation of additional cellular factors in the translation initiation complex.
Mutagenesis of the large untranslated sequence at the 5' end of the genome of foot-and-mouth disease virus revealed that a region of approximately 450 nucleotides preceding the open reading frame of the viral polyprotein is involved in the regulation of translation initiation at two internal start sites. Variations in two domains of this region reduced the translation efficiency up to 10-fold, whereas an intermediate segment seemed to be less essential. A pyrimidine-rich sequence preceding the start codon was most sensitive in that conversion of single pyrimidine residues to purines decreased the translation efficiency strongly. The data are in agreement with a recently proposed general structural model for the internal ribosome entry site of the cardiovirusaphthovirus subgroup of picornaviruses (E. V. Pilipenko, V. M. Blinov, B. K. Chernov, T. M. Dmitrieva, and V. I. Agol, Nucleic Acids Res. 17:5701-5711, 1989). They suggest, however, that this model represents only a core structure for the internal entry of ribosomes and that foot-and-mouth disease virus and other members of the picornaviruses need additional regulatory RNA elements for efficient translation initiation.
The synthesis of picornavirus polyproteins is initiated cap independently far downstream from the 5' end of the viral RNA at the internal ribosome entry site (IRES). The cellular polypyrimidine tract-binding protein (PTB) binds to the IRES of foot-and-mouth disease virus (FMDV). In this study, we demonstrate that PTB is a component of 48S and 80S ribosomal initiation complexes formed with FMDV IRES RNA. The incorporation of PTB into these initiation complexes is dependent on the entry of the IRES RNA, since PTB and IRES RNA can be enriched in parallel either in 48S or 80S ribosomal complexes by stage-specific inhibitors of translation initiation. The formation of the ribosomal initiation complexes with the IRES occurs slowly, is temperature dependent, and correlates with the incorporation of PTB into these complexes. In a first step, PTB binds to the IRES, and then the small ribosomal subunit encounters this PTB-IRES complex. Mutations in the major PTB-binding site interfere simultaneously with the formation of initiation complexes, translation efficiency, and PTB cross-linking. PTB stimulates translation directed by the FMDV IRES in a rabbit reticulocyte lysate depleted of internal PTB, and the efficiency of translation can be restored to the original level by the addition of PTB. These results indicate that PTB plays an important role in the formation of initiation complexes with FMDV IRES RNA and in stimulation of internal translation initiation with this picornavirus.
Picornavirus positive-strand RNAs are selectively encapsidated despite the coexistence of viral negative-strand RNAs and cellular
RNAs in infected cells. However, the precise mechanism of the RNA encapsidation process in picornaviruses remains unclear.
Here we report the first identification of an RNA element critical for encapsidation in picornaviruses. The 5′ end of the
genome of Aichi virus, a member of the family Picornaviridae, folds into three stem-loop structures (SL-A, SL-B, and SL-C, from the most 5′ end). In the previous study, we constructed
a mutant, termed mut6, by exchanging the seven-nucleotide stretches of the middle part of the stem in SL-A with each other to maintain the base
pairings of the stem. mut6 exhibited efficient RNA replication and translation but formed no plaques. The present study showed that in cells transfected
with mut6 RNA, empty capsids were accumulated, but few virions containing RNA were formed. This means that mut6 has a severe defect in RNA encapsidation. Site-directed mutational analysis indicated that as the mutated region was narrowed,
the encapsidation was improved. As a result, the mutation of the 7 bp of the middle part of the stem in SL-A was required
for abolishing the plaque-forming ability. Thus, the 5′-end sequence of the Aichi virus genome was shown to play an important
role in encapsidation.
Foot-and-mouth disease virus (FMDV) is an important animal pathogen that belongs to the Aphthovirus genus of the Picornaviridae family and infects cattle and other cloven-hoofed animals. Seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3) have been identified serologically, and multiple subtypes occur within each serotype. FMDV enters cells by receptor-mediated endocytosis. By electron microscopy the FMD virion appears to be a round particle with a smooth surface and a diameter of about 25 nm. The FMD viral particle contains a positive-strand RNA genome of about 8500 nucleotides, enclosed within a protein capsid. The virus capsid is made up from 60 copies each of four virus-encoded proteins VP1 to VP4. The FMDV genome is composed of the 5' non-translated region (5'NTR), the coding region, and the 3' non-translated region (3'NTR). The genome encodes a single polyprotein, from which the different viral polypeptides are derived by viral proteases. FMDV populations are genetically and anti-genetically heterogeneous. FMDV have very high mutation rates.
A wealth of experimental data on the mechanism of the picornavirus genome replication has accumulated. Not infrequently, however, conclusions derived from these data appear to contradict each other. On the one hand, initiation of a complementary RNA strand can be demonstrated to occur in a solution containing only the poliovirus RNA polymerase, VPg, uridine triphosphate, poly(A) template and appropriate ions. On the other hand, convincing experiments suggest that efficient initiation of a viral complementary RNA strand requires complex cis-acting signals on the viral RNA template, additional viral and possibly cellular proteins as well as a membrane-containing environment. On the one hand, there is evidence that the viral RNA, in order to be replicated, should first be translated, but on the other hand, the viral RNA polymerase appears to be unable to overcome the ribosome barrier. Possible solutions for these and several other similar paradoxes are discussed, along with less contradictory results on the properties of the picornaviral replicative proteins. Recent results suggesting that recombination and other rearrangements of the viral RNA genomes may be accomplished not only by the replicative template switching but also by nonreplicative mechanisms are also briefly reviewed.
The 5'-terminal, RNase T1-resistant oligonucleotide of poliovirus mRNA has been isolated. Its sequence is pU-U-A-A-A-A-C-A-Gp, which is identical to that of virion RNA except that the genome-linked protein VPg is absent [Nomoto, A., Detjen, B., Pozzatti, R. & Wimmer, E. (1977) Nature 268, 208-213]. Because all newly synthesized viral RNAs are VPg-linked, we propose that VPg is cleaved from progeny RNA at the linkage between protein and nucleic acid prior to polyribosome formation. This may represent a new mode of processing of viral macromolecules. Virion RNA from which VPg has been cleaved proteolytically retains its specific infectivity, an observation suggesting that VPg is not involved in early steps (penetration and translation) of the infectious cycle initiated by RNA.
The role of procapsids during foot-and-mouth disease virus multiplication was studied on infected BHK-21 cells. Purified virus and procapsids were obtained by treating the infected cytoplasmic extracts with RNase and EDTA. The synthesis of virus, procapsids, and total particles was determined in pulse-chase experiments. A precursor-product relationship between procapsids and virions was obtained. The results show that the rate of synthesis of total particles (virus + procapsids) was linear from the addition of the label and was identical to that corresponding to virions. Therefore, the speed of the morphogenetic process as well as the existence of a precursor pool of structural proteins was established. Furthermore, the rate of virus synthesis from procapsids was identical to the rate of synthesis of procapsids from their structural precursors. A quantitative recovery of label from procapsids into virions was obtained by the use of cycloheximide or tosyl-lysine chloromethyl ketone. Under these conditions, virus synthesis proceeds, indicating that these drugs do not affect the morphogenetic step studied in this paper.
RNA template- and primer-dependent preparations of RNA polymerase were purified from encephalomyocarditis virus-infected Krebs-2 cells, using a three-step chromatographic procedure. The RNA duplex-unwinding activity of these preparations was investigated by two assays, using a partially double-stranded RNA template (encephalomyocarditis virus RNA annealed with a long segment of antisense transcript). Less purified preparations of the polymerase appeared to be able to efficiently displace, in an ATP-dependent and RNA elongation-dependent reaction, the antisense segment from the template. However, upon a more extensive purification, the unwinding activity of the RNA polymerase preparations was lost.
The effect of three lysosomotropic compounds, chloroquine, monensin and NH4Cl, on the replication of foot-and-mouth disease virus (FMDV) type A12 was studied. Viral replication was almost totally inhibited by 0.5 mM chloroquine, 50 microM monensin, or 25 mM NH4Cl. Monensin and NH4Cl affected replication when added either before or within the first hour of infection. Chloroquine, however, still inhibited viral replication when added up to 2.5 h after infection. Assays of binding of radiolabeled virus to cells showed that these compounds had no effect on viral adsorption. Neither monensin nor NH4Cl had any significant effect on cellular protein synthesis, but there was no evidence of viral protein synthesis in cells infected in the presence of these compounds. In contrast, chloroquine inhibited both cellular and viral protein synthesis. Eclipse assays, performed in the presence of the compounds, showed that while chloroquine and NH4Cl had little effect on cell-induced degradation of incoming virions to 12 S protein subunits, monensin inhibited this reaction. The replication of representative members of all seven serotypes of FMDV was inhibited by monensin although some types were less sensitive to the compound than others. These results are consistent with a model which postulates that viral eclipse is the result of acidification of endocytic vesicles which degrade entrapped virions to 12 S protein subunits resulting in the release of genome RNA.
Foot-and-mouth disease virus (FMDV) and bovine enterovirus (BEV) were used for simultaneous dual infection of calf kidney tissue cultures. In the harvest fluid a viral agent was found that had the FMDV genome, but (1) was neutralized by BEV-antiserum and not by FMDV-antiserum; (2) had the acid-stability of BEV and not FMDV; (3) had the buoyant density of BEV (1.35 class) and not FMDV (1.45 class); and (4) had the s-rate class (140–150 S) of both parental viruses. Such a particle meets criteria for simple genomic masking. Its ultracentrifugal properties show that the protein coat influenced the buoyant density in CsCl more than nucleic acid, presumably because of permeability to salt.
The adsorption of foot-and-mouth disease virus (FMDV) types A12119, O1B, and C3Res were studied in baby hamster kidney (BHK-21) cells by measuring the amount of radioactively labeled purified virus which remained attached to cells at various times after infection. At 4°, over half of the maximum adsorption of type A virus occurred within 15 min, and approximately 65% of the radioactivity bound by 45 min was removed by brief treatment with EDTA, indicating the assay measures primarily adsorption and not penetration. Upon shifting the temperature from 4 to 37°, about 60 to 70% of the bound virus eluted by 1 hr in an unmodified form. Only 140 S virus particles and 12 S subunits could be found associated with the infected cell 20 min after shifting the temperature from 4 to 37°. By 50 min, the number of 140 S particles decreased slightly. The number of viral receptors per BHK-21 cell was determined to be 1–2.5 × 104 for virus types A12 and O1B. Competition experiments between FMDV types A12, O1B, and C3Res indicated that they can utilize at least some common receptor sites on cells.
Foot-and-mouth disease virus (FMDV) was examined for its ability to adsorb specifically to plasma membranes isolated from BHK-21 cells. The membranes were prepared by the polyethylene glycol-dextran method, and characterized by increases in specific activity of ouabain-sensitive Na+K+-ATPase and 5′-nucleotidase, and by enrichment in 3H-fucose over unfractionated homogenates. The membranes adsorbed purified radiolabeled FMDV type A12119 with kinetics characteristic of intact cells. Plasma membranes prepared from cells pretreated with trypsin were unable to adsorb virus. The adsorption of labeled FMDV was inhibited by unlabeled virus. Treatment of virus with trypsin, which cleaves capsid protein 3, greatly reduced its ability to adsorb to both plasma membranes and intact cells. After adsorption of virus to membranes at 4°, subsequent incubation at 37° under physiological conditions resulted in a rapid elution of bound virus in an unmodified form which reached approximately 80% by 1 hr. Incubation of the membrane-virus complex at 33° under low-salt conditions degraded the virus particles to intact and fragmented viral RNA and 12 S protein subunits. Membrane-induced viral degradation did not occur at 4° but was observed within 5 min after shifting to 33°. Thus, isolated plasma membranes from BHK-21 cells retain receptors for FMDV possessing uncleaved capsid protein 3. In addition, the eclipse and uncoating of FMDV in intact cells probably occurs at the plasma membrane, and in confirmation of previously reported results, the postadsorptive degradation, unlike that of other picornaviruses, occurs in a single step without the production of intermediate subviral particles.
The surfaces of primary and continuous line cell cultures displayed the same sequence of morphological changes during the course of infection with foot-and-mouth disease virus. These changes could be classified into four broad stages: I) cells were flattened, closely attached to one another and microvilli appeared, II) cells rounded, microvilli began to disappear and the cells started to separate from one another by cytoplasmic strands, III) cells were discrete, rounded structures and IV) cells were rounded and had numerous attached buds, some of which contained virus. The internal changes included the appearance of increasing amounts of smooth membranous vacuoles lined with the viral induced RNA polymerase and the presence of buds, some with viral particles inside. While the different cell cultures showed similar internal and external changes as a result of infection, they responded to infection at different rates and contained subpopulations of resistant cells.
Infection of BHK cells with foot-and-mouth disease virus (FMDV) causes a thorough change in the electrophoretic profile of whole nuclear histones. It consists in the disappearance of histone H3 and the appearance of a new polypeptide (Pi) which migrates between histones H2A and H4 on SDS-polyacrylamide gels. Protein Pi is detected at 2 hr postinfection (pi), the time in which viral RNA synthesis begins to increase, and reaches equimolecular amounts with the remaining core histones 1 hr later, when the disappearance of histone H3 is almost complete. Labeling of cells prior to infection demonstrates that Pi is not a novo product but the result of a viral-induced processing of a host precursor synthetized beforehand. Protein Pi comigrates with histone H2A/B in acetic acid/urea polyacrylamide gels and it shares common major peptides with histone H3 under controlled proteolysis with protease V8 or trypsin. The mononucleosomal and nucleosomal DNA pattern analysis after micrococcal nuclease treatment of nuclei from infected and mock-infected cells did not show any significant differences even though after 3 hr (p.i.), protein Pi replaces histone H3 in the nucleosomal structure. It was concluded that FMDV infection is responsible for a specific modification in the nucleus of infected cells which leads, after 3 hr (p.i.), to a complete histone H3 protein Pi transition in the nucleosomes.