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M‐PMV Capsid Transport Is Mediated by Env/Gag Interactions at the Pericentriolar Recycling Endosome
Abstract
Cytoplasmic transport of Gag molecules to the site of budding is an important but poorly understand process in retroviral assembly. Our previous studies of Mason-Pfizer monkey virus showed that, for this retrovirus, Gag is assembled into capsids at a pericentriolar region and that Env is necessary for efficient transport out of the site. An Env requirement for cytoplasmic transport implicates vesicular trafficking in this process even though the capsids remain cytoplasmic and do not bud into intracellular compartments in the cells studied to date. We show here that the secretory pathway of the cell is not directly involved in Gag transport since the latter was not inhibited by BFA, nor did Gag colocalize with markers of the ER, Golgi, or TGN. Instead, colocalization was observed between Gag and endocytosed transferrin and with Rab11, suggesting that pericentriolar recycling endosomes play a critical role in this process. Mutants of Rab11 that inhibit efflux of transferrin from the recycling endosome also inhibited Gag transport. Our studies show that Env colocalizes with Gag at the pericentriolar assembly site, and provide evidence that Env must travel through this compartment in order to initiate export of the capsids from the site of assembly. Thus, for the first time, endocytic trafficking of a retroviral Env glycoprotein is linked to the efficient cytoplasmic transport of Gag.
... M ason-Pfizer monkey virus (M-PMV) is a D-type retrovirus that has been considered a favorable model for the study of immature particle assembly and trafficking because it preassembles in the cytoplasm of infected cells (1,2). Due to both temporally and spatially separated processes of assembly and budding, M-PMV has been utilized to study individual steps of the retroviral life cycle. ...
... However, little is known about the interaction between Env and retroviral structural proteins. It was previously shown that the cytoplasmic tail (CT) of Env plays an important role in the trafficking of immature M-PMV particles to the site of retroviral budding (2,5), and it was well documented that a tyrosine motif in the CT controls the incorporation of M-PMV Env into virions (6). It has also been suggested that Env interacts directly with Gag, which is crucial for the specific incorporation of Env into the virus envelope in human immunodeficiency virus (HIV) (7) and simian immunodeficiency virus (SIV) (8). ...
... As mentioned above, there is evidence that in D-type retroviruses, the matrix domain (MA) of Gag interacts with Env at the membranes of intracytoplasmic vesicles, and this interaction regulates the transport of preassembled immature M-PMV particles to the plasma membrane (2,4,5). The Env-mediated transport of M-PMV Gag is apparently connected with endocytic trafficking of Env (2,4). ...
By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.
... Rab9a is involved in the retrograde transport of cargo from late endosomes to the TGN [27]. Rab11a specifically localizes to recycling endosomes, which recycle cargo from early/sorting endosomes back to the plasma membrane [10,28]. ...
... Several studies focusing on the molecular mechanism of M-PMV Env intracellular trafficking and its interaction with Gag have been published [5,10,11]. The main focus of these studies was on the role of the CT since it is the only part of Env available for potential interactions with viral structural polyproteins and cytoplasmic proteins. ...
... This generally prevents prolonged exposure to the immune system. Sfakianos et al. proposed that M-PMV particles assemble at the membrane of recycling endosomes [10]. This could subsequently result in the efficient targeting of both Env and Gag. ...
The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.
... The recycling pathway is also involved in the transport of assembled capsids or virions towards the plasma membrane of viruses belonging to the Retroviridae [192][193][194]196] and Flaviviridae [195] families ( Figure 6). The Gag-containing capsids of two retroviruses, Mason-Pfizer monkey virus (M-PMV) [192,196] and Jaagsiekte sheep retrovirus (JRSV) [193], are assembled at the MTOC and exit efficiently the cell via the ERC. ...
... The recycling pathway is also involved in the transport of assembled capsids or virions towards the plasma membrane of viruses belonging to the Retroviridae [192][193][194]196] and Flaviviridae [195] families ( Figure 6). The Gag-containing capsids of two retroviruses, Mason-Pfizer monkey virus (M-PMV) [192,196] and Jaagsiekte sheep retrovirus (JRSV) [193], are assembled at the MTOC and exit efficiently the cell via the ERC. In the case of M-PMV, the immature capsids are co-trafficked with Env-recycling endosome vesicles en route to the plasma membrane [192,196]. ...
... A potential role for the ERC in viral egress has been described for both positive-and negative-sense RNA viruses of the Paramyxoviridae [190,191], Orthomyxoviridae [180], Retroviridae [192][193][194] and Flaviviridae [195] families (Table 3). ...
Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral–host interactions occurring on the endocytic recycling compartment (ERC), and mediated by its regulatory Ras-related in brain (Rab) GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition.
... This site was first discovered in the Mason-Pfizer monkey virus (M-PMV) using mutational analyses resulting redistribution of viral assembly from the cytoplasm to the plasma membrane [1,3]. Subsequently, the Gag polyproteins of Jaagsiekte sheep retrovirus (JSRV) and foamy virus (FV) were found to assemble as capsids at the pericentriolar region [12][13][14][15][16], suggesting that this might be a conserved site for retroviral assembly. ...
... Since the CTRS sequences of M-MPV and FV have been shown to direct pericentriolar localization of their cognate Gag proteins [12][13][14][15][16], we investigated whether the same process was relevant with MMTV Gag assembly in multiple cell types. Centrin-1 and γ-tubulin were used as centriolar markers as both proteins are specifically located at the centrosomes [40,41]. ...
... Immature capsids-like structures were mostly confined to areas surrounding membranous tubules and to vacuole-like structures. Collectively, our results show that MMTV Gag exhibits a punctated subcellular distribution, but a substantial fraction localizes to the vicinity of centrioles, as reported for other retroviruses [12][13][14][15][16]. ...
The Gag protein of the mouse mammary tumor virus (MMTV) is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV), assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A) resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.
... In contrast, oBST2B induces Gag accumulation in a perinuclear region. Previous studies have shown that the Gag proteins of betaretroviruses such as Mason-Pfizer monkey retrovirus (M-PMV) and JSRV target the pericentriolar region in a dynein-and microtubule-dependent fashion (34,39,40). The assembled viral parti- cles then traffic to the cell membrane by a mechanism that is influenced by the recycling endosomes (34) and, at least for M-PMV, by the viral Env protein (39). ...
... Previous studies have shown that the Gag proteins of betaretroviruses such as Mason-Pfizer monkey retrovirus (M-PMV) and JSRV target the pericentriolar region in a dynein-and microtubule-dependent fashion (34,39,40). The assembled viral parti- cles then traffic to the cell membrane by a mechanism that is influenced by the recycling endosomes (34) and, at least for M-PMV, by the viral Env protein (39). Therefore, the absence or strong depletion of Env at the plasma membrane and, subsequently, in recycling endosomes could influence the intracellular trafficking of the newly formed virions and lead to the accumulation of Gag proteins in a pericentrosomal area. ...
... We previously demonstrated that oBST2B blocks viral exit less efficiently than oBST2A and only following transient transfections with proportionally more than twice the maximal amount of oBST2B expression plasmid used in this study (26). Betaretroviral particles can still exit from the cells in the absence of viral Env, although it is expected that this would occur at a lower rate than in its presence (39). Therefore, the sequestration of Env within the Golgi could explain the weak effect of oBST2B on JSRV particle release (detected as released Gag in supernatant of transfected cells) that we previously observed in vitro (26). ...
Unlabelled:
Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the "tethering" mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A.
Importance:
BST2 is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. JSRV, the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study, we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles, which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from those of BST2 proteins of other species.
... These previous studies with M-PMV have employed the use of genetic, biochemical and fixed-cell immunofluorescent methodologies for elucidating the role of viral and cellular components in viral assembly, transport, and budding [2,[6][7][8][9]. Prior data has shown that blocking vesicular trafficking by shifting the temperature of the cell to 20°C causes a significant delay in M-PMV Gag release kinetics and maturation, indicating a role for the cellular vesicular transport system in capsid transport [10,11]. It has also been shown that Env glycoprotein expression is necessary for efficient capsid release and maturation [7,10,12]. ...
... Prior data has shown that blocking vesicular trafficking by shifting the temperature of the cell to 20°C causes a significant delay in M-PMV Gag release kinetics and maturation, indicating a role for the cellular vesicular transport system in capsid transport [10,11]. It has also been shown that Env glycoprotein expression is necessary for efficient capsid release and maturation [7,10,12]. Several capsid mutations have provided key insights into the processes involved in M-PMV assembly and transport. Specifically, a single amino acid change from arginine to tryptophan (R55W) in the CTRS causes a switch from B/D type capsid assembly to C-type assembly [3] by preventing nascent Gag molecules from interacting with the dynein light chain Tc-tex and their subsequent transport on microtubules to the pericentriolar region of the cell. ...
... Since previous reports have shown that Env expression is necessary for efficient virion release and capsid maturation [2,7,10,12], and the TEM data showed improper capsid morphology when cells were transfected with pSARM-GagGFP-M100A alone, a pulse-chase experiment was carried out to determine whether cotransfection with WT pSARM-X changed the kinetics and efficiency of Gag-GFP release from cells. Proteins were pulse-labeled with 35 S methionine for 30min and chased with unlabeled media for 2 or 4 hours. ...
Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.
... Our data show that HCMV then sequentially travels from the TGN to Rab11 + recycling endosomes before nuclear translocation. Such viruses as respiratory syncytial virus (33), HIV-1 (34), and Mason-Pfizer monkey virus (35) have been found in Rab11 + recycling endosomes, but in these viruses this is a site for viral assembly, not a part of the entry process. Thus, we suggest that this is the initial documentation of HCMV trafficking to the TGN and then to Rab11 + recycling endosomes before nuclear translocation. ...
... HCMV Culture and Infection. The HCMV strains used included TB40/E (GFP-labeled TB40/E-UL32); Towne/E (passages [35][36][37][38][39][40][41][42][43][44][45]; BADwt (defective in gH/gL/UL128-131) and BADrUL131 (with gH/gL/UL128-131), both of which are AD169-derived BACs (47); and TR5 and TR5ΔUL128-131 (BAC clinical strains with and without the gH/gL/UL128-131). All strains were cultured in human embryonic lung (HEL) fibroblasts (16)(17)(18)(19)(20)(21)(22). ...
Significance
Human cytomegalovirus (HCMV)-infected monocytes play a critical role in the hematogenous dissemination of the virus, establishment of persistence, and progression of HCMV-mediated diseases. Here we identify a unique HCMV nuclear translocation pathway in monocytes. HCMV moves from early endosomes to the trans-Golgi network, then to recycling endosomes before its nuclear translocation. Signaling (via c-Src) induced by viral binding of the gH/gL/UL128-131 complex to integrins triggers this unique pathway. Because HCMV is degraded without this signal, we suggest that this distinct signal-induced nuclear translocation functions as a viral evasion pathway in infected monocytes.
... By contrast, M-PMV gRNAs target the MTOC even prior to Gag synthesis (Fig 8F), perhaps priming the cell for viral capsid biogenesis in the perinuclear region (Fig 9A, bottom). Consistent with this notion, Sfakianos et al. previously suggested that M-PMV capsids are surrounded by an abundance of ribosomes at the MTOC, and sit in close proximity to recycling endosomes bearing viral glycoproteins [102]. Thus, an attractive model is that the MTOC compartmentalizes Gag/Gag-Pol synthesis prior to "loading" nascent capsids onto recycling transport vesicles bound for the cell surface. ...
... To our knowledge, this is the first demonstration of a discrete, transferable RNA element capable of linking mRNAs to the microtubule cytoskeleton, and with remarkable specificity. Unlike for HIV-1, there is strong evidence for microtubules and a role for the MTOC in regulating M-PMV capsid assembly and virion egress [95,96,101,102,131]. Regarding the molecular mechanism by which the CTE and NXF1/NXT1 target mRNAs to the MTOC, we found that 1) the 4xCTE was more active than a single CTE in directing heterologous mRNAs to the centrosome (Fig 6D), 2) NXF1 traffics with 4xCTE-gRNAs to the MTOC (Fig 4D), 3) that heterologous mRNAs can be readily targeted to the centrosome even in the absence of a CTE by using MS2-NXF1 tethering (Fig 7A-7C), and 4) that a similar result is seen when tethering NXF1 to RRE-gRNAs using Rev-NXF1 fusion proteins co-expressed with NXT1 (Fig 7D-7F). ...
Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.
... Assembly and particle production of Mason-Pfizer monkey virus (M-PMV) is perhaps the most distinct from all cases discussed above. M-PMV is thought to catalyze the membrane envelopment of a preassembled spherical capsid shell to release infectious virions (Sfakianos and Hunter, 2003). Assembly of M-PMV capsids occurs in a pericentriolar region of the cytoplasm prior to transport to the PM for budding. ...
... Assembly of M-PMV capsids occurs in a pericentriolar region of the cytoplasm prior to transport to the PM for budding. It is thought that both MA and CA domains of M-PMV Gag interact with the PM (Sfakianos and Hunter, 2003). Initial evidence for a potential role of PI(4,5)P 2 in M-PMV Gag-PM binding has been reported by Hunter and colleagues (Stansell et al., 2007). ...
Targeting of the Gag polyprotein to the plasma membrane (PM) for assembly is a critical event in the late phase of immunodeficiency virus type-1 (HIV-1) infection. Gag binding to the PM is mediated by interactions between the myristoylated matrix (MA) domain and PM lipids. Despite the extensive biochemical and in vitro studies of Gag and MA binding to membranes over the last two decades, the discovery of the role of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in Gag binding to the PM has sparked a string of studies aimed at elucidating the molecular mechanism of retroviral Gag-PM binding. Electrostatic interactions between a highly conserved basic region of MA and acidic phospholipids have long been thought to be the main driving force for Gag-membrane interactions. However, recent studies suggest that the mechanism is rather complex since other factors such as the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. Here we summarize the current understanding of HIV-1 Gag-membrane interactions at the molecular and structural levels and briefly discuss the underlying forces governing interactions of other retroviral MA proteins with the PM.
... 17,182 An early study in MPMV revealed that a single amino acid mutation in MA changed the assembly location from the cytoplasm to the plasma membrane. [183][184] Later on, similar observations were made for other members of the betaretroviruses genus, such as MMTV and Jaagsiekte sheep retrovirus (JSRV) as well as spumaviruses [185][186][187][188][189][190][191] . The MA domain of these viruses contains a "cytoplasmic targeting and retention signal (CTRS)" which is necessary for the intracytoplasmic assembly of virus particles. ...
Members of the family Retroviridae are important animal and human pathogens. Being obligate parasites, their replication involves a series of steps during which the virus hijacks the cellular machinery. Additionally, many of the steps of retrovirus replication are unique among viruses, including reverse transcription, integration, and specific packaging of their genomic RNA (gRNA) as a dimer. Progress in retrovirology has helped identify several molecular mechanisms involved in each of these steps, but many are still unknown or remain controversial. This review summarizes our present understanding of the molecular mechanisms involved in various stages of retrovirus replication. Furthermore, it provides a comprehensive analysis of our current understanding of how different retroviruses package their gRNA into the assembling virions. RNA packaging in retroviruses holds a special interest because of the uniqueness of packaging a dimeric genome. Dimerization and packaging are highly regulated and interlinked events, critical for the virus to decide whether its unspliced RNA will be packaged as a “genome” or translated into proteins. Finally, some of the outstanding areas of exploration in the field of RNA packaging are highlighted, such as the role of epitranscriptomics, heterogeneity of transcript start sites, and the necessity of functional polyA sequences. An in-depth knowledge of mechanisms that interplay between viral and cellular factors during virus replication is critical in understanding not only the virus life cycle, but also its pathogenesis, and development of new anti-retroviral compounds, vaccines, as well retroviral-based vectors for human gene therapy.
... Retroviruses historically classified as C-type viruses, such as MLV, assemble at the plasma membrane to produce immature virions that undergo maturation after release from the cell. In contrast, the D-type retroviruses, including M-PMV and mouse mammary tumor virus (MMTV), assemble at pericentriolar regions of the cell into particles with the so-called A-type morphology and upon maturation are converted into the Dtype morphology (32)(33)(34)(35). Once the particles are formed, they are transported to the cell membrane for budding. ...
The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located downstream of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the ‘clasp motif’, important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and Mason-Pfizer Monkey Virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide comparable function. The importance of the sequences spanning the CA-NC cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant protein in vitro , and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant virus in vivo . The mutants revealed major defects in virion assembly and release in 293T and HeLa cells, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection.
Importance
The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short ‘clasp’ motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.
... It has been reported that the dynein complex is used to travel toward the nucleus by some retroviruses, such as bovine immunodeficiency virus (BIV), Mason-Pfizer monkey virus (MPMV), prototype foamy virus (PFV), and human immunodeficiency virus type 1 (HIV-1) (10)(11)(12)(13)(14). In particular, the association of viral proteins with the light chains of the dynein complex was reported to be essential for BIV and PFV viral retrograde transport, suggesting a shared mechanism among older retroviruses (revised in reference 15). ...
Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that Dynein light chain roadblock-type2 (Dynlrb2) knock-down significantly decreases MLV infection compared to non-silenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). Here we aim to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells were the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. On the contrary, an increase in nuclear localization is observed when Dynlrb2 is overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport.
Importance
Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light chain Dynlrb2 for infection, retrograde traffic and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.
... Retroviruses depend upon microtubule associated dynein for their retrograde transport inside the host cell, were each retrovirus have a particular dynein partner to mediate the movement [19,21,28,29,35,36]. In the last 5 years, most research attention has focused on HIV-1, the causative agent of AIDS, and less advance has been made regarding other retroviruses and their association with the cytoskeleton. ...
Following entry into the host cell, retroviruses generate a dsDNA copy of their genomes via reverse transcription, and this viral DNA is subsequently integrated into the chromosomal DNA of the host cell. Before integration can occur, however, retroviral DNA must be transported to the nucleus as part of a ‘preintegration complex’ (PIC). Transporting the PIC through the crowded environment of the cytoplasm is challenging, and retroviruses have evolved different mechanisms to accomplish this feat. Within a eukaryotic cell, microtubules act as the roads, while the microtubule-associated proteins dynein and kinesin are the vehicles that viruses exploit to achieve retrograde and anterograde trafficking. This review will examine the various mechanisms retroviruses have evolved in order to achieve retrograde trafficking, confirming that each retrovirus has its own strategy to functionally subvert microtubule associated proteins.
... This association was demonstrated by co-immunoprecipitation and co-localization assays, but the isoform of DYNLL responsible was not defined. (Pereira et al., 2014;Sfakianos & Hunter, 2003). ...
Retroviruses are obligate intracellular parasites of eukaryotic cells. After reverse transcription, the viral DNA contained in the preintegration complex (PIC) is delivered to the nucleus of the host cell, where it integrates. Before reaching the nucleus, the incoming particle and the PIC must travel throughout the cytoplasm. Likewise the newly synthesized viral proteins and viral particles must transit the cytoplasm during exit. The cytoplasm is a crowded environment and simple diffusion is difficult. Therefore, viruses have evolved to utilize the cellular mechanisms of movement through the cytoplasm, where microtubules are the roads, and the ATP dependent motors dynein and kinesin are the vehicles for retrograde and anterograde trafficking. This review will focus on how different retroviruses (Mazon-pfizer monkey virus, prototype foamy virus, bovine immunodeficiency virus, human immunodeficiency virus type I and murine leukemia virus) have subjugated the microtubule associated motor proteins for viral replication. Although there have been advances in our understanding of how retroviruses move along microtubules, the strategies are different among them. Thus, a better understanding of the mechanisms used by each retrovirus to functionally subvert microtubule motor proteins will provide important clues in the design of new anti-retroviral drugs that can specifically disrupt intracellular viral trafficking.
... The matrix protein (MA), the N-terminal part of the main structural polyprotein Gag, is crucial for membrane targeting of immature virus particles and their interactions with the plasma membrane [2,3]. MAs of most retroviruses are myristoylated and structurally highly similar [4]. ...
... Retroelements-with their (1) higher-order regulatory functions, (2) capability for genetic creativity and (3) innovation competence of new regulatory patterns and combinations-are descended from retroviruses which can be easily identified by their three essential parts gag, pol and env (Rashkova et al. 2002;Weiss 2006;Tang et al. 1999). Most endogenous retroviruses have been degraded into formerly connected domains, but they can still be recognized by one of these three genes (Gao et al. 2003;Sfakianos and Hunter 2003;Ryan 2004;Gabus et al. 2006). The gag gene encodes structural proteins, pol encodes enzymes such as reverse transcriptase and integrase functions, and env encodes envelope proteins. ...
The biocommunicative approach investigates rule-governed, sign-mediated interactions both within and among cells, tissues, organs and organisms. It also investigates genetic sequences as codes/texts that are coherent with the laws of physics and chemistry but, in addition, follow a complementary mix of combinatorial (syntactic), context-sensitive (pragmatic), content-specific (semantic) rules. In this respect, the roles of telomeres and telomerases in evolution, structure and content arrangement of genomes are of particular interest. This involves deciphering the relationships between the ‘molecular syntax’ of telomere repeats and their meaning, i.e. their function in the genomic content. This requires their evolutionary roots to be examined. The telomere replication process by telomerase is the most important feature here because it is processed by a very ancient competence, i.e. reverse transcriptase with a great variety of functions in most key processes of living nature.
... On the other hand, during late infection stage, several viruses, such as HIV, HSV, poliovirus, and norovirus, have been suggested to exploit this perinuclear accumulation to concentrate newly synthesized viral components through MTs in order to facilitate late infection events, including virion replication, assembly, or egress. [23][24][25][26] Classical virology assays usually study replication-competent viruses and measure the production of viral progeny as the readout, which is the sum of the total effects of all events in viral infection with these viruses. Given the potential involvement of MTs and MTOC throughout the entire viral infection, this method is unable to precisely dissect the perinuclear retention of incoming virions during early infection stage. ...
Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After Nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) post virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting the MT-MTOC in brain, liver and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at the MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion.
... The pathogenic human viruses, HIV and human T-cell lymphotropic virus assemble via C-type intermediates, whereas M-PMV is the prototypic D-type retrovirus. The Gag polyprotein of M-PMV is first transported to an intracytoplasmic pericentriolar site, where particle assembly occurs (1)(2)(3). This targeting requires a cytoplasmic targeting/retention signal (CTRS) localized in the MA domain that mediates the interaction of Gag with components of dynein to transport cargo molecules toward the minus ends of the microtubules (4). ...
Importance:
Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles, and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and self-assembly of Gag molecules. Here we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.
... Interestingly, these mutations influenced also transport of assembled particles to PM and resulted in decreased production of extracellular particles. The current model describing the relocation of betaretroviral particles to PM involves anterograde transport mediated by microtubuleassociated Env-containing vesicles [18,[41][42][43]. However, a detailed mechanism of this transport is not yet fully understood. ...
Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated.
Here, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release.
Based on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.
... Rab11 is also essential for trafficking components of respiratory syncytial virus (RSV) 17 , HIV-1 45 , and another retrovirus, Mason-Pfizer monkey virus 46 , to the cell surface where mature viral particles assemble. For example, RSV, a leading cause of lower respiratory illness in children and the elderly, co-opts the Rab11-dependent apical recycling machinery for exit from the host 17 (Fig. 4). ...
Many bacterial and viral pathogens block or subvert host cellular processes to promote successful infection. One host protein that is targeted by invading pathogens is the small GTPase RAB11, which functions in vesicular trafficking. RAB11 functions in conjunction with a protein complex known as the exocyst to mediate terminal steps in cargo transport via the recycling endosome to cell-cell junctions, phagosomes and cellular protrusions. These processes contribute to host innate immunity by promoting epithelial and endothelial barrier integrity, sensing and immobilizing pathogens and repairing pathogen-induced cellular damage. In this Review, we discuss the various mechanisms that pathogens have evolved to disrupt or subvert RAB11-dependent pathways as part of their infection strategy.
... Therefore, viral budding requires the co-expression and interaction of Gag and Env [27,28]. This feature is most closely resembled by Mason Pfizer Monkey Virus (MPMV), where although Env co-expression is not required, it was shown to enhance viral particle release by mediating capsid transport [29]. Similarly, it is thought that FV Env interacts with the preassembled capsids at a pericentriolar region, probably the trans-Golgi network, which then leads to membrane targeting of the fully assembled viral particles [30]. ...
Gag proteins play an important role in many stages of the retroviral replication cycle. They orchestrate viral assembly, interact with numerous host cell proteins, engage in regulation of viral gene expression, and provide the main driving force for virus intracellular trafficking and budding. Foamy Viruses (FV), also known as spumaviruses, display a number of unique features among retroviruses. Many of these features can be attributed to their Gag proteins. FV Gag proteins lack characteristic orthoretroviral domains like membrane-binding domains (M domains), the major homology region (MHR), and the hallmark Cys-His motifs. In contrast, they contain several distinct domains such as the essential Gag-Env interaction domain and the glycine and arginine rich boxes (GR boxes). Furthermore, FV Gag only undergoes limited maturation and follows an unusual pathway for nuclear translocation. This review summarizes the known FV Gag domains and motifs and their functions. In particular, it provides an overview of the unique structural and functional properties that distinguish FV Gag proteins from orthoretroviral Gag proteins.
... M-PMV MA may also interact with other PIPs in vivo because it has been shown that M-PMV Gag protein interacts with Env in the endosomal membrane during transport to the PM. 31 However, interaction between the MA and PI(4,5)P 2 is essential for virus contact with the PM because depletion of PI(4,5)P 2 from the membrane or mutation of the lysine or arginine residues in the PIP binding site blocks virus budding. 20 These data show that despite some structural differences between M-PMV and HIV MA, as well as other retroviruses, M-PMV myrMA interacts with PI(4,5)P 2 similarly to the other retroviral MAs. ...
We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein.
... Retroelements-with their (1) higher-order regulatory functions, (2) capability for genetic creativity and (3) innovation competence of new regulatory patterns and combinations-are descended from retroviruses which can be easily identified by their three essential parts gag, pol and env (Rashkova et al. 2002;Weiss 2006;Tang et al. 1999). Most endogenous retroviruses have been degraded into formerly connected domains, but they can still be recognized by one of these three genes (Gao et al. 2003;Sfakianos and Hunter 2003;Ryan 2004;Gabus et al. 2006). The gag gene encodes structural proteins, pol encodes enzymes such as reverse transcriptase and integrase functions, and env encodes envelope proteins. ...
Whereas telomeres protect terminal ends of linear chromosomes, telomerases identify natural chromosome ends, which differ
from broken DNA and replicate telomeres. Although telomeres play a crucial role in the linear chromosome organization of eukaryotic
cells, their molecular syntax most probably descended from an ancient retroviral competence. This indicates an early retroviral
colonization of large double-stranded DNA viruses, which are putative ancestors of the eukaryotic nucleus. This contribution
demonstrates an advantage of the biosemiotic approach towards our evolutionary understanding of telomeres, telomerases, other
reverse transcriptases and mobile elements. Their role in genetic/genomic content organization and maintenance is no longer
viewed as an object of randomly derived alterations (mutations) but as a highly sophisticated hierarchy of regulatory networks
organized and coordinated by natural genome-editing competences of viruses.
... Depletion of FIP3 inhibits cell abscission (Wilson et al., 2005 ) and impairs the release of filamentous virus particles (Bruce et al., 2010). The ability of M2 to induce negative curvature in a cholesterol-rich environment (Rossman et al., 2010a,b) might be essential for the rapid internalization of partially assembled IAV particles to an intracellular compartment (Jo et al., 2010 ), as suggested for some retroviruses (Basyuk et al., 2003; Sfakianos and Hunter, 2003; Spearman, 2006). Thus, M2 might be indirectly involved in the pinching-off of infectious IAV particles. ...
The mechanism of budding of influenza A virus revealed important deviation from the consensus mechanism of budding of retroviruses and of a growing number of negative-strand RNA viruses. This study is focused on the role of the influenza A virus matrix protein M1 in virus release. We found that a mutation of the proline residue at position 16 of the matrix protein induces inhibition of virus detachment from cells. Depletion of the M1-binding protein RACK1 also impairs virus release and RACK1 binding requires the proline residue at position 16 of M1. The impaired M1-RACK1 interaction does not affect the plasma membrane binding of M1; in contrast, RACK1 is recruited to detergent-resistant membranes in a M1-proline-16-dependent manner. The proline-16 mutation in M1 and depletion of RACK1 impairs the pinching-off of the budding virus particles. These findings reveal the active role of the viral matrix protein in the release of influenza A virus particles that involves a cross-talk with a RACK1-mediated pathway.
... At this stage, the viral protease, in association with cellular proteases, cleave the Gag structural protein, leading to core disassembly (15). Interestingly, during the late stages of infection, nascent Gag is also targeted to the centrosome for intracellular capsid assembly (16), a pathway reminiscent of what is known for the Mason-Pfizer monkey virus (17,18), another type B/D retrovirus. Similar to other retroviruses, the prototypic foamy virus (PFV) Gag harbors L domain (PT/SAP), which binds to the Tsg101 component of endosomal sorting complex required for transport-I (ESCRT-I) to induce budding (19)(20)(21). ...
Retroviruses hijack cellular machineries to productively infect their hosts. During the early stages of viral replication, proviral integration relies on specific interactions between components of the preintegration complex and host chromatin-bound proteins. Here, analyzing the fate of incoming primate foamy virus, we identify a short domain within the C-terminus of the structural Gag protein that efficiently binds host chromosomes, by interacting with H2A/H2B core histones. While viral particle production, virus entry and intracellular trafficking are not affected by mutation of this domain, chromosomal attachment of incoming subviral complexes is abolished, precluding proviral integration. We thus highlight a new function of the structural foamy Gag protein as the main tether between incoming subviral complexes and host chromatin prior to integration.
... Congregation of viral components at the budding membrane may be facilitated by their cosorting into the same membrane microdomain. A specific interaction of transmembrane proteins and viral capsid proteins or preassembled nucleocapsids leading to their recruitment to the respective budding regions has been observed, for example, herpesviruses, HBV, and some retroviruses (Wilk et al. 2001;Sfakianos and Hunter 2003;Mettenleiter et al. 2009;Patient et al. 2009). In these cases, virus envelopment and release are completely dependent on the viral glycoproteins, whereas assembly of the viral nucleocapsid is not. ...
Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.
... The subcellular location at which Gag incorporates the viral RNA, either en route to or at the plasma membrane, remains unknown, however, with the TIR-FMbased work as well as a large corpus of earlier studies compatible so far with either scenario. A pericentriolar region was reported to be the assembly site for type D retroviruses (50,51). Subsequently, HIV-1 genomic RNA and Gag were also reported to colocalize in a region at or adjacent to the centriole (45). ...
Human immunodeficiency virus type 1 (HIV-1) Gag and genomic RNA determinants required for encapsidation are well established, but where and when encapsidation occurs in the cell is unknown. We constructed MS2 phage coat protein labeling systems to track spatial dynamics of primate and nonprimate lentiviral genomic RNAs (HIV-1 and feline immunodeficiency virus [FIV]) vis-à-vis their Gag proteins in live cells. Genomic RNAs of both lentiviral genera were observed to traffic into the cytoplasm, and this was Rev dependent. In transit, FIV Gag and genomic RNA accumulated independently of each other at the nuclear envelope, and focal colocalizations of genomic RNA with an intact packaging signal (psi) and Gag were observed to extend outward from the cytoplasmic face. In contrast, although HIV-1 genomic RNA was detected at the nuclear envelope, HIV-1 Gag was not. For both lentiviruses, genomic RNAs were seen at the plasma membrane if and only if Gag was present and psi was intact. In addition, HIV-1 and FIV genomes accumulated with Gag in late endosomal foci, again, only psi dependently. Thus, lentiviral genomic RNAs require specific Gag binding to accumulate at the plasma membrane, packaged genomes cointernalize with Gag into the endosomal pathway, and plasma membrane RNA incorporation by Gag does not trigger committed lentiviral particle egress from the cell. Based on the FIV results, we hypothesize that the Gag-genome association may initiate at the nuclear envelope.
Microtubules (MTs) form rapidly adaptable, complex intracellular networks of filaments that not only provide structural support, but also form the tracks along which motors traffic macromolecular cargos to specific sub-cellular sites. These dynamic arrays play a central role in regulating various cellular processes including cell shape and motility as well as cell division and polarization. Given their complex organization and functional importance, MT arrays are carefully controlled by many highly specialized proteins that regulate the nucleation of MT filaments at distinct sites, their dynamic growth and stability, and their engagement with other subcellular structures and cargoes destined for transport. This review focuses on recent advances in our understanding of how MTs and their regulatory proteins function, including their active targeting and exploitation, during infection by viruses that utilize a wide variety of replication strategies that occur within different cellular sub-compartments or regions of the cell.
The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub1), here we show that it can also bind several diubiquitins (K48-Ub2, N-Ub2, and K63-Ub2), with a preference for K63-linked Ub2. The NMR structure of the Tsg101:K63-Ub2 complex showed that while the Ub1-binding site accommodates the distal domain of Ub2, the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site results in distinct phenotypes regarding the recruitment of Tsg101 partners. Mutation in the vestigial active site abrogates interaction between Tsg101 and the HIV-1 protein Gag but not Hrs, a cellular protein. Mutation at the N-terminal helix alters Gag but not Hrs-Tsg101 localization. Given the broad involvement of Tsg101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo.
Human immunodeficiency virus (HIV) is thought to assemble and bud at the plasma membrane of infected cells. However, published electron micrographs have indicated that virus can also be found in intracellular compartments in some infected cells. Intracellular assembly is particular prominent in macrophages, although the nature of the virus-containing compartment and the relevance of intracellular virus in HIV infection are unclear. This thesis shows that in human monocyte derived macrophages (MDM) infectious HIV-1 is assembled on intracellular membranes of multivesicular late endosomes. The nature of the compartment into which virus particles bud was identified by immunolabelling techniques with organelle-specific antibodies. This showed a strong co-localisation of viral antigen with late endosomal markers such as CD63. Immuno-electron microscopy detected CD63 on the viral membrane and internal vesicles of multivesicular bodies, suggesting that cellular proteins resident in late endosomes are incorporated in intracellular virus. To determine whether intracellular virus is released and is infectious, virus derived from MDM supernatants was precipitated with organelle-specific antibodies. This revealed the presence of late endosomal proteins, such as CD63 and LAMP-1, on the viral membrane of infectious particles. Plasma membrane proteins, in contrast, were not incorporated, suggesting that infectious virus released from MDM had assembled intracellularly. The detection of infectivity in MDM-homogenates further supports the finding that intracellularly assembled virus is infectious. Virus release seems to be an active process, as indicated by temperature sensitivity, and probably occurs through fusion of internal compartments with the plasma membrane. Immunoprecipitation of T-cell derived HIV-1 indicated that T-cells may also assemble virus intracellularly, alongside plasma membrane assembly. This was suggested by the detection of late endosomal and plasma membrane proteins on the viral membrane and supported by preliminary morphological analysis. These findings may have broad implications for our understanding of the biology of HIV infection and its cell-cell transmission.
La domestication des bétails représente une étape importante dans l'histoire de l'humanité. Le mouton était l'un des premiers animaux à être domestiqués dans le croissant fertile. Ces événements de domestication, probablement initiés au début du Néolithique, ont génétiquement construit les races contemporaines du Moyen-Orient mais aussi du monde entier. L'élevage de moutons, principalement mouton de la race Awassi, représente une activité économique essentielle du Liban ; cependant, jusqu'à présent, il n'existe que très peu de données génétiques sur cette race. De nos jours, les outils moléculaires disponibles nous permettent de définir en détail la diversité génétique des populations de moutons et de retracer leur histoire évolutive. Par conséquent, l'objectif principal de mon projet de thèse était de caractériser génétiquement la race Awassi du Liban. Pour cette étude, 277 échantillons d'ADN génomique prélevés des moutons Awassi du Liban (n = 254) et de la Syrie (n = 23) ont été analysés. Au début, nous avons utilisé cinq rétrovirus endogènes (rétrovirus endogène de moutons de Jaagsiekte-enJSRV) qui sont polymorphiques par insertion dans les génomes du mouton domestique (enJSRV-18, -7, -15, -16 et -22) et ont été précédemment considérés comme très informatifs principalement pour distinguer génétiquement les moutons primitifs des races plus modernes (c.-à-d. le dernier issu de l'épisode migratoire impliquant des moutons avec des traits de production améliorés). En utilisant cette approche, nos résultats montrent une prédominance du type R2 (enjSRV-18 seulement) confirmant que le mouton Awassi du Liban est une race moderne. Comme prévu, le rétrotype R4 (à la fois enJSRV-18 et enJSRV-7), une caractéristique commune des populations de moutons du bassin méditerranéen, se trouve également dans le génome des moutons d'Awassi du Liban et plus accentué dans les troupeaux Syriens. Il est intéressant de noter que les populations de moutons d'Awassi situés dans le nord-est du Liban et ayant ainsi un accès plus restreint à la mer Méditerranée que les autres populations (c'est-à-dire en raison de la chaîne de montagne centrale qui coupe le pays sur deux), présentent une faible fréquence de R4. Bien que l'origine des animaux utilisés pour établir les troupeaux analysés au cours de cette étude soit inconnue, nos résultats fournissent également certaines preuves que le mode d'élevage (ouvert ou fermé) peut influencer les rétrotypes observés et en particulier le R4. De manière surprenante, au cours de cette étude, nous avons également dévoilé la présence de soi-disant "Solo-LTR" (c'est-à-dire généré par une recombinaison homologue) pour un autre enJSRV (enJSRV-6) qui prédomine dans deux troupeaux d'une région particulière du Liban (Nabatieh). Et comme approche complémentaire, deux marqueurs mitochondriaux ont été utilisés, le cytochrome b (Cyt-b) et D-Loop, pour étudier l'origine maternelle de cette race et sa relation phylogénétique au sein de la famille Ovis aries. Dans notre étude, le Cyt-b se révèle plus discriminant que le D-Loop. Des mouton d'Awassi analysé, quatre haplogroupes (HPG) du Moyen-Orient ont été trouvés avec l'analyse du Cyt-b : HPG A, B, C et E, ce dernier étant peu fréquent. De même, l’analyse de la super-séquence, alignement Cyt-b_D-Loop, a permis l’identification de l’HPG D, un HPG extrêmement rare et limité jusqu’à présent aux moutons à queue grasse tel que l’Awassi. Enfin, une expansion passée de la population est observée pour les HPG A, B et C (mais pas pour HPG E) avec les distributions incompatibles et des tests de neutralité négatifs significatifs. Dans l'ensemble, les résultats obtenus au cours de cette étude fournissent une caractérisation génétique complète ainsi que quelques idées sur la structure phylogéographique des populations de moutons de la race Awassi au Liban.
In the last decade it was found that the number of genes of some nematodes and humans was similar but their regulation was completely different. Today we know that the higher-order regulation of protein-coding datasets depends on complex interconnected networks of a great variety of non-coding RNAs that are read and transcribed in the developmental and growth processes of every cell within multicellular organisms. The evolutionary origins of these non-coding RNAs are not randomly-derived mixtures of nucleotide acids but formerly intact viral agents which infected all cellular host genomes in a non-lytic but persistent way. Although some of these viral agents still fulfill vital functions, e.g., endogenous retroviruses which are active in placentation of mammals, in most cases they split up (‘defectives’) into several functional parts which now serve as ‘effectives’, i.e. symbiogenetic integrated functional tools for cellular needs of host organisms.
This is the first uniform description of all key levels of communication in the organismic kingdoms of plants, fungi, animals and bacteria based on the most recent empirical data. Biocommunication occurs on three levels (A) intraorganismic, i.e. intra- and intercellular, (B) interorganismic, between the same or related species and (C) transorganismic, between organisms which are not related. The biocommunicative approach demonstrates both that cells, tissues, organs and organisms coordinate and organize by communication processes and genetic nucleotide sequence order in cellular and non-cellular genomes is structured language-like, i.e. follow combinatorial (syntactic), context-sensitive (pragmatic) and content-specific (semantic) rules. Without sign-mediated interactions no vital functions within and between organisms can be coordinated. Exactly this feature is absent in non-living matter.
Additionally the biocommunicative approach investigates natural genome editing competences of viruses. Natural genome editing from a biocommunicative perspective is competent agent-driven generation and integration of meaningful nucleotide sequences into pre-existing genomic content arrangements and the ability to (re)combine and (re)regulate them according to context-dependent (i.e. adaptational) purposes of the host organism. The biocommunicative approach is an original scientific field of investigations. Readers must be competent in basic knowledge of biology and genetics.
The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4 h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles.
Productive viral infection is dependent upon post-entry migration of viruses/viral components to sites within a host cell that complement viral deficiencies. Delivery of virions or component proteins to appropriate sites within an infected cell is critical for completing successive stages in viral replication, including release into the cytoplasm, uncoating, genome replication, viral gene expression, assembly and budding. Vesicular transport is essential for steady-state cellular trafficking of membrane-associated proteins. Rob GTPases and their associated effectors are key regulators of vesicular transport pathways. In recent years, Rob proteins have been implicated in the endocytic or exocytic sorting of component viral proteins or intact viruses, most of which are known to be membrane-encapsulated and enveloped. This review will discuss the current understanding of how Rob GTPases and their effectors may regulate individual vesicular transport. steps, and detail emerging discoveries examining how specific Rabs and effectors support viral replication.
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HIV-1 assembly occurs at the plasma membrane of cells or on intracellular membranes as a result of the complex interplay of viral structural proteins and cellular vesicular transport systems. A number of recent discoveries have revealed direct interactions between the viral Gag and Env proteins and cellular molecules involved in trafficking. These direct interactions are often mediated by discrete motifs that may be suitable as targets for antiretroviral therapy in the future. Discrete motifs within the p6 region of Gag bind and recruit members of the endosomal sorting complex required for transport complex of proteins for regulation of particle budding. Gag interacts directly with the δ subunit of the cellular adaptin AP-3, and this interaction mediates movement of Gag to the multivesicular body (MVB). TIP47 has been identified as a linker between Gag and Env that directs vesicular movement of this complex within the cell, leading to Env incorporation into virions. Additional cellular determinants of assembly have been identified that are consistent with a model for assembly in which regulated vesicular transport plays a dominant role. Future discoveries should clarify the pathways utilized for the sequential movement of Gag and Env in the cell, including the differential movement of Gag and Env to the MVB or plasma membrane observed in T cells and macrophages. Research into the assembly pathway of HIV-1 is poised to reveal a number of additional targets for antiviral drug discovery in the coming years.
Endosomes play key roles in the cellular infection cycles of many viruses. Initially implicated in virus entry, recent research
has demonstrated that endosomes can also be required at other stages in viral replication. Endosomes can provide platforms
for viral nucleic acid replication and virus assembly, or play roles in modulating anti-viral immune responses. To these ends
viruses exploit various attributes of endosomes such as the low luminal pH, unique trafficking properties, cellular location
and composition. In turn, viruses have become remarkable tools for analysing endosome function.
Cardiac patients often are obese and have hypertension, but in most studies these conditions are investigated separately. Here, we aimed at 1) elucidating the interaction of metabolic and mechanophysical stress in the development of cardiac dysfunction in mice and 2) preventing this interaction by ablation of the fatty acid transporter CD36. Male wild-type (WT) C57Bl/6 mice and CD36(-/-) mice received chow or Western-type diet (WTD) for 10 wk and then underwent a sham surgery or transverse aortic constriction (TAC) under anesthesia. After a 6-wk continuation of the diet, cardiac function, morphology, lipid profiles, and molecular parameters were assessed. WTD administration affected body and organ weights of WT and CD36(-/-) mice, but it affected only plasma glucose and insulin concentrations in WT mice. Cardiac lipid concentrations increased in WT mice receiving WTD, decreased in CD36(-/-) on chow, and remained unchanged in CD36(-/-) receiving WTD. TAC induced cardiac hypertrophy in WT mice on chow but did not affect cardiac function and cardiac lipid concentrations. WTD or CD36 ablation worsened the outcome of TAC. Ablation of CD36 protected against the WTD-related aggravation of cardiac functional and structural changes induced by TAC. In conclusion, cardiac dysfunction and remodeling worsen when the heart is exposed to two stresses, metabolic and mechanophysical, at the same time. CD36 ablation prevents the metabolic stress resulting from a WTD. Thus, metabolic conditions are a critical factor for the compromised heart and provide new targets for metabolic manipulation in cardioprotection.
Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason–Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.
• capsid assembly
• dynein motor
• matrix protein structure
• retrovirus
• transport
Generation of infectious retroviral particles rely on the targeting of all structural components to the correct cellular sites at the correct time. Gag, the main structural protein, orchestrates the assembly process and the mechanisms that trigger its targeting to assembly sites are well described. Gag is also responsible for the packaging of the viral genome and the molecular details of the Gag/RNA interaction are well characterized. Until recently, much less was understood about the cell biology of retrovirus RNA packaging. However, novel biochemical and live-cell microscopic approaches have identified where in the cell the initial events of genome recognition by Gag occur. These recent developments have shed light on the role played by the viral genome during virion assembly. Other central issues of the cell biology of RNA packaging, such as how the Gag-RNA complex traffics through the cytoplasm towards assembly sites, await characterization.
The plasma membrane is the final barrier that enveloped viruses must cross during their egress from the infected cell. Here, we review recent insights into the cell biology of retroviral assembly and release; these insights have driven a new understanding of the host proteins, such as the ESCRT machinery, that are used by retroviruses to promote their final separation from the host cell. We also review antiviral host factors such as tetherin, which can directly inhibit the release of retroviral particles. These studies have illuminated the role of the lipid bilayer as the unexpected target for virus restriction by the innate immune response.
The exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with highly related and biologically active endogenous
retroviruses (enJSRVs). The endogenous enJS56A1 locus possesses a defective Gag polyprotein which blocks the late replication
steps of related exogenous and endogenous retroviruses by a mechanism known as JSRV late restriction (JLR). Conversely, enJSRV-26, which most likely integrated into the sheep genome less than 200 years ago, is able
to escape JLR. In this study, we demonstrate that the ability of enJSRV-26 to escape JLR is due to a single-amino-acid substitution
in the signal peptide (SP) of its envelope glycoprotein. We show that enJSRV-26 SP does not localize to the nucleolus, unlike
the functional SPs of related exogenous and endogenous sheep betaretroviruses. In addition, enJSRV-26 SP function as a posttranscriptional
regulator of viral gene expression is impaired. enJSRV-26 JLR escape relies on the presence of the functional enJS56A1 SP.
Moreover, we show that the ratio between enJSRV-26 and enJS56A1 Gag is critical to elude JLR. Interestingly, we found that
the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus. These data further reinforce
the notion that transdominant enJSRV proviruses have been positively selected in domestic sheep, and that the coevolution
between endogenous and exogenous sheep betaretroviruses and their host is still occurring.
The first human retrovirus, human T-lymphotropic virus 1 (HTLV-1), was discovered 30 years ago. Despite intensive study, the cell surface molecules involved in virus entry have only been identified over the past few years. Three molecules form the receptor complex for HTLV-1: glucose transporter 1, neuropilin 1 and heparan sulfate proteoglycans. Another molecule on the surface of dendritic cells, DC-SIGN, may play a role in dendritic cell-mediated infection of cells. In addition to the cell surface molecules used for entry, the HTLV-1 envelope interacts with cellular proteins, enabling the virus to traffic by exploiting cellular delivery pathways. To facilitate both these steps, HTLV-1 encodes motifs that mimic cellular binding partners for the trafficking system and ligands for the receptors. Here we review the interactions between the HTLV-1 envelope and cellular proteins.
A mandatory step in the formation of an infectious retroviral particle is the acquisition of its envelope glycoprotein (Env). This step invariably occurs by Env positioning itself in the host membrane at the location of viral budding and being incorporated along with the host membrane into the viral particle. In some ways, this step of the viral life cycle would appear to be imprecise. There is no specific sequence in Env or in the retroviral structural protein, Gag, that is inherently required for the production of an infectious Env-containing particle. Additionally, Env-defective proviruses can efficiently produce infectious particles with any of a number of foreign retroviral Env glycoproteins or even glycoproteins from unrelated viral families, a process termed pseudotyping. However, mounting evidence suggests that Env incorporation is neither passive nor random. Rather, several redundant mechanisms appear to contribute to the carefully controlled process of Env acquisition, many of which are apparently used by a wide variety of enveloped viruses. This review presents and discusses the evidence for these different mechanisms contributing to incorporation.
研究では､HIV-1 Nef蛋白質由来で､同一のMHC分子に提示され且つ構造が非常によく類似した２つの抗原ペプチドに対するCTL応答を基に､どのような抗原ペプチドの性質がCTLの抗ウイルス機能制御に影響を与えるかを明らかにすることを目的とした。
Baculoviruses produce two progeny phenotypes during their replication cycles. The occlusion-derived virus (ODV) is responsible for initiating primary infection in the larval midgut, and the budded virus (BV) phenotype is responsible for the secondary infection. The proteomics of several baculovirus ODVs have been revealed, but so far, no extensive analysis of BV-associated proteins has been conducted. In this study, the protein composition of the BV of Autographa californica nucleopolyhedrovirus (AcMNPV), the type species of baculoviruses, was analyzed by various mass spectrometry (MS) techniques, including liquid chromatography-triple quadrupole linear ion trap (LC-Qtrap), liquid chromatography-quadrupole time of flight (LC-Q-TOF), and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). SDS-PAGE and MALDI-TOF analyses showed that the three most abundant proteins of the AcMNPV BV were GP64, VP39, and P6.9. A total of 34 viral proteins associated with the AcMNPV BV were identified by the indicated methods. Thirteen of these proteins, PP31, AC58/59, AC66, IAP-2, AC73, AC74, AC114, AC124, chitinase, polyhedron envelope protein (PEP), AC132, ODV-E18, and ODV-E56, were identified for the first time to be BV-associated proteins. Western blot analyses showed that ODV-E18 and ODV-E25, which were previously thought to be ODV-specific proteins, were also present in the envelop fraction of BV. In addition, 11 cellular proteins were found to be associated with the AcMNPV BV by both LC-Qtrap and LC-Q-TOF analyses. Interestingly, seven of these proteins were also identified in other enveloped viruses, suggesting that many enveloped viruses may commonly utilize certain conserved cellular pathways.
Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.
The role of myristylation, a fatty acid modification of nascent polypeptides, in the assembly and intracellular transport of D-type retroviral capsids was investigated through the use of oligonucleotide-directed mutagenesis. Myristic acid is normally esterified through an amide linkage to a glycine residue at the amino terminus of the Mason-Pfizer monkey virus gag gene products. Mutant pA-1, which has a codon for valine substituted for that of the normally myristylated glycine, is completely noninfectious. While the mutant gag polyprotein precursors are synthesized at normal levels, they are not myristylated and are not cleaved to the mature virion proteins. No extracellular virus particles are released from mutant pA-1-infected cells, but intracytoplasmic A-type particles (capsids) accumulate in the cytoplasm. Since none of the intracellular capsids can be found associated with the plasma membrane, these results strongly suggest that myristylation is a critical signal for intracytoplasmic transport of completed viral capsids to their normal site of budding and release.
The functional roles of the matrix (MA) protein in the assembly and maturation of retroviruses was investigated with a series of MA mutants of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus. The mutants we describe here were generated by the introduction of random point mutations within the MA coding domain by use of sodium bisulphite mutagenesis. Studies of these mutants show that the MA protein plays a critical role in three different, sequential events in the final stages of type D retrovirus replication: (i) folding of the gag gene-encoded precursor poly-proteins into a stable conformation for capsid assembly in the cytoplasm of infected cells; (ii) capsid transport from the site of assembly to the plasma membrane; and (iii) capsid association with, and extrusion of the membrane during virus budding. The mutants described here interfere with or block M-PMV replication at each of these stages. Large numbers of preassembled capsids accumulate within the cytoplasm of transport-defective mutant-infected cells, suggesting that transport of M-PMV capsids to the plasma membrane is an active and specific intracellular targeting process. The initial association of the capsid with the membrane may depend upon this intracytoplasmic transport process but additional protein-lipid interactions that involve the MA protein are required for membrane extrusion around the preformed capsids; in cells infected with the budding-defective mutant, assembled capsids accumulate under the inner surface of the cell plasma membrane, and are retarded in their release from the infected cell.
The capsids of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus, are preassembled in the infected cell cytoplasm and are then transported to the plasma membrane, where they are enveloped in a virus glycoprotein-containing lipid bilayer. The role of viral glycoprotein in intracellular transport of M-PMV capsids was investigated with a spontaneous mutant (5A) of M-PMV, which we show here to be defective in envelope glycoprotein biosynthesis. DNA sequence analysis of the env gene of mutant 5A reveals a single nucleotide deletion in the middle of the gene, which results in the synthesis of a truncated form of the envelope glycoprotein. Evidence is presented showing that the mutant glycoprotein is not expressed at the cell surface but is retained in the endoplasmic reticulum. Normal levels of gag-pro-pol precursor polyproteins are made and processed in mutant genome-transfected cells, and high levels of noninfectious particles lacking viral glycoprotein are released with normal kinetics into the culture medium. No intracisternal budding of capsids is observed. We conclude that viral glycoprotein is required neither for targeting preassembled capsids of M-PMV to the plasma membrane for final maturation nor for the budding process. Since the presence or absence of M-PMV glycoprotein at the site of budding does not affect the efficiency or kinetics of the targeting process, the preassembled capsid of M-PMV, in contrast to those of intracisternal type A particles, appears to have an intrinsic signal for intracellular transport to the plasma membrane.
SUMMARY The present studies were undertaken to investigate the ultrastructure and development of virus particles detected in a spontaneous mammary tumor of a rhesus monkey. Thin-section electron microscopy of the tumor tissue has revealed 2 types of particles, viz., an intracytoplasmic, electron-dense, ring-shaped particle measuring about 70 m/u in diameter, and an extracellular particle with an outer unit membrane and a central dense nucleoid measuring about 110 m/n in diameter. From the electron micrographs, the intracytoplasmic development and virus maturation by a process of budding at the level of the cell membrane are reconstructed. The observations are discussed in view of the known oncogenic RNA-type virus particles.
The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly.
Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.
Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883-1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481-490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end-directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins.
Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.
The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.
We report here that a broad spectrum of phospholipase A(2) (PLA(2)) antagonists produce a concentration-dependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (approximately 1 microm), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (approximately 10 microm), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA(2) antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA(2) antagonists. And third, endosome tubule formation in a cell-free, cytosol-dependent reconstitution system was equally sensitive PLA(2) antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA(2) and that PLA(2)-dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA(2) activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.
In this study, we have identified a novel Nedd4-like ubiquitin ligase, BUL1, as the host factor involved in budding of type D retrovirus Mason-Pfizer monkey virus (M-PMV). Overexpression of BUL1 enhanced virus particle release, while a BUL1 mutant in which a W to G substitution was introduced into a WW domain, W791G, lost the ability to bind to the viral Gag protein and abolished its ability to mediate virus budding. In addition, a fragment of BUL1 containing only the WW domains inhibited virus budding in a dominant negative manner. These results, together with previous findings, indicate that the M-PMV Gag L domain interacts with the BUL1 WW domain and that this interaction is essential for virus budding. Our observations provide new insights into the mechanism of virus budding, and could be useful in establishing new antiviral strategies targeted at progeny virus release from a host cell.
Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.
The Nef protein from the human immunodeficiency virus (HIV) induces down-regulation of the CD4 and major histocompatibility complex class I molecules from the cell surface by interfering with the endocytic machinery. This work focuses on the interaction of HIV-1 Nef with the μ1 chain of adaptor protein type 1 (AP1) complex and its contribution to the Nef-induced alterations of membrane trafficking. Two independent regions surrounding a disordered loop located in the C-terminal part of Nef are involved in μ1 binding. Each region can separately interact with μ1, and simultaneous point mutations within both regions are needed to abolish binding. We used CD8 chimeras in which the cytoplasmic tail was replaced by Nef mutants to show that these μ1-binding sites contain determinants required to induce CD4 down-regulation and to target the chimera to the endocytic pathway by promoting AP1 complex recruitment. Ultrastructural analysis revealed that the CD8-Nef chimera provokes morphological alterations of the endosomal compartments and co-localizes with AP1 complexes. These data indicate that the recruitment by Nef of AP1 via binding to μ1 participates in the connection of Nef with the endocytic pathway.
A critical phase in the infection cycle of HIV and other retroviruses is the assembly of new infectious virus particles. This process requires complex but coordinated targeting of capsid precursor proteins, virus genomic RNA and viral glycoproteins to a common assembly site on the plasma membrane. Domains within the capsid precursor proteins define the route taken to the plasma membrane and direct the process of virus budding. However, in order for the assembled virus to be infectious, viral glycoproteins, replicative enzymes and genomic RNA must also be included. The mechanisms by which this complex of interactions occur are discussed in this chapter.
Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.
Two different morphogenic processes of retroviral capsid assembly have been observed: the capsid is either assembled at the plasma membrane during the budding process (type C), or preassembled within the cytoplasm (types B and D). We describe here a gag mutant of Mason-Pfizer monkey virus, a type D retrovirus, in which a tryptophan substituted for an arginine in the matrix protein results in efficient assembly of capsids at the plasma membrane through a morphogenic process similar to that of type C retroviruses. We conclude that a type D retrovirus Gag polyprotein contains an additional, dominant signal that prevents immediate transport of precursors from the site of biosynthesis to the plasma membrane. Instead, they are directed to and retained at a cytoplasmic site where a concentration sufficient for self-assembly into capsids occurs. Thus, capsid assembly processes for different retroviruses appear to differ only in the intracellular site to which capsid precursors are directed.
Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, encodes two glycosylated virion proteins, gp20 and gp70. The polyprotein precursor to these proteins was identified by immunoprecipitation of pulse labeled M-PMV-infected cells with an antiserum raised against gp70, the major glycoprotein of the virus. The relationship of this precursor to the two viral glycoproteins was verified by tryptic peptide mapping, which demonstrated that gp20 and gp70 were independent products of the env gene. The types and degree of glycosylation of the precursor and its products was investigated by tunicamycin inhibition of glycosylation, endo-beta-N-acetyl glucosaminidase H (Endo-H) and endo-beta-N-acetylglucosaminidase F (Endo-F) catalyzed removal of glycosylated residues. The results suggest that the precursor, a molecule with a mol wt of 86,000, is composed of approximately 55,000 Da of protein to which 14-15 oligosaccharide chains are attached. The precursor is cleaved post-translationally to yield the two glycoproteins of M-PMV, gp70 and gp20. Most, if not all, of the glycan units associated with the gp70 molecule are of the complex variety, as shown by their resistance to Endo-H cleavage. The gp20 molecule, on the other hand, appears to contain a single glycan unit predominantly of the high mannose type since this side chain is sensitive to digestion by Endo-H.
The transport kinetics of the influenza virus hemagglutinin from its site of synthesis to the apical plasma membrane of Madin-Darby canine kidney cells, a polarized epithelial cell line, were studied by a sensitive tryptic assay. Hemagglutinin acquired terminal sugars, as judged by sensitivity to endo-beta-N-acetylglucosaminidase H, 10-15 min after synthesis, and first appeared on the apical domain 15 min later. None of the pulse-labeled hemagglutinin accumulated on the basolateral domain. At 20 degrees C, terminal glycosylation continued, but no hemagglutinin was detected on the cell surface within 2 hr. If the incubation temperature was raised from 20 degrees C to 37 degrees C, hemagglutinin was quickly externalized, demonstrating that the inhibition at low temperature was reversible.
The tubular structures of endosomes are thought to mediate the sorting and recycling of endocytosed macromolecules. These structures have been reported to show considerable morphological variety. However, it is not clear whether they are functionally identical. To address this question, we applied quantitative imaging analysis to characterize tubular organelles loaded with a recycling protein marker, fluorescent transferrin, in living human carcinoma HEp2 cells, using laser scanning confocal microscopy. High-resolution images of the cells demonstrated two types of tubular structures with a distinct morphology and showing a time dependency in their appearance: the fine tubular element and the extensive tubular element. Fine tubular elements 2-10 microns long were distributed throughout the cytoplasm after 10 min of loading with the tracer. Extensive tubular elements 5-20 microns long radiated from the cytocenter after 2 h of loading, but not after 10 min. Time-lapse imaging analysis demonstrated that the half-life of transferrin in the fine and extensive tubular elements was 12 min and approximately 50 min, respectively, at 33 degrees C. Double labeling experiments using fluorescent transferrin and epidermal growth factor indicated that the extensive tubular element was neither a late endosome nor a lysosome. From these results, we conclude that the fine tubular and extensive tubular elements are distinct organelles: the former comprising the sorting endosome and recycling compartment which mediate the rapid recycling of transferrin, and the latter being part of a novel pathway of slower transferrin processing.
Temperatures around 20 degrees C are known to block degradation of endocytosed material by preventing its transport to lysosomes, accordingly reduced temperature has been widely used to define endosomes. Newer studies have revealed that the low temperature block is proximal to perinuclear late endosomes, but it is not clear whether the block is already in early endosomes, or whether the traffic proceeds to multivesicular carrier endosomes which mediate transport from early to late compartments. We have now focused on this problem using rat cardiac myocytes. First, cell fractionation on Percoll gradients showed that at reduced temperatures (22 degrees C and 26 degrees C), with prolonged chase periods, endocytosed horseradish peroxidase was able to proceed from early endosomes to later compartments but not up to lysosomes. Further, microscopic experiments with fluorescent endocytic marker FITC-dextran showed that the marker did not accumulate in the perinuclear area, as was the case at 37 degrees C, but stayed in peripheral cytoplasm at reduced temperatures, even after 16-h chase. Second, electron microscopic pulse labeling showed that, at 22 degrees C, endocytosed gold particles (BSA-gold) are transported to compartments not accessible to HRP internalised later to early endosomes. Thus, these gold particles had reached a later compartment. Morphologically these vesicles were multivesicular bodies of 0.5-1 microm in diameter. Third, we used fluorescence microscopy to study the effect of reduced temperature on transferrin uptake and recycling. At 17 degrees C and 22 degrees C, transferrin was internalized normally to peripheral (sorting) and perinuclear (recycling) vesicles. If transferrin was first taken up at 37 degrees C, and the cells were then chased at various temperatures from 37 degrees C to 17 degrees C, the recycling was slowed down but not entirely blocked at the reduced temperatures. From these results we can conclude that (1) endocytic traffic is blocked in multivesicular carrier endosomes at and below 26 degrees C, and that (2) reduced temperature slows down transport in the recycling pathway, without a complete block.
Although initially recognised as essential for the entry of certain viruses, endocytosis is now known to also play important roles in the replication of, and adaptation to, the host cell of a number of viruses. Here we consider several aspects of this association and recent results that have emerged to support this view.
Macrophages are important targets for HIV-1 infection and harbor the virions in an as yet unidentified organelle. To determine the location of HIV-1 in these cells, an extensive analysis of primary human macrophages infected in vitro with HIV-1 was carried out by immuno-electron microscopy. Virus particles were found to accumulate in intracellular multivesicular compartments which were enriched in major histocompatibility complex class II molecules and CD63. These features are characteristics of major histocompatibility complex class II compartments where maturing class II molecules acquire their peptide cargo. The membrane-delimited, electron-dense virus particles of 100-110 nm diameter labeled strongly for HIV-1 p24 antigen, major histocompatibility complex class II molecules, CD63 and, to a lesser extent for HIV-1 gp120 envelope protein and Lamp 1. Our data suggest that virus particles may access the lumen of the major histocompatibility complex class II compartment by budding from the limiting membrane, thus acquiring proteins of this membrane such as class II and CD63. Viral assembly and budding would therefore occur in macrophages by a process similar to the formation of the internal vesicles in multivesicular bodies and at the same location. This could account for the particular content in lipids and proteins previously found in the membrane wrapping HIV particles. Our observations also suggest direct fusion of the virus containing major histocompatibility complex class II compartment with the plasma membrane, leading to massive release of viral particles into the extracellular medium.
Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason-Pfizer monkey virus specifically targets Gag precursor proteins to the pericentriolar region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting-retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N-terminal cytoplasmic targeting-retention signal functions in a cotranslational manner. Transport of the capsids out of the pericentriolar assembly site requires the env-gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting-retention signal defective abrogates pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting-retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.