Abstract:Like all other viruses, a successful egress of functional particles from infected cells is a prerequisite for foamy virus (FV) spread within the host. The budding process of FVs involves steps, which are shared by other retroviruses, such as interaction of the capsid protein with components of cellular vacuolar protein sorting (Vps) machinery via late domains identified in some FV capsid proteins. Additionally, there are features of the FV budding strategy quite unique to the spumaretroviruses. This includes s… Show more
“…2, 3), the presence of conserved residues in the N-terminus of FV Gag (Fig. 4a) and published data for PFV [9], individual amino acid replacements in Gag were engineered, generating single amino acid proviral Gag mutants pCF-7-L10A, Q11A, Q12A, L13A, Y14A, H25A, G26A, D27A, I28A, I29A, R32A, G36A, W38A, G39A, R43A, L51A and D53A (Fig. 4a).Fig.…”
BackgroundFoamy viruses (FVs) of the Spumaretrovirinae subfamily are distinct retroviruses, with many features of their molecular biology and replication strategy clearly different from those of the Orthoretroviruses, such as human immunodeficiency, murine leukemia, and human T cell lymphotropic viruses. The FV Gag N-terminal region is responsible for capsid formation and particle budding via interaction with Env. However, the critical residues or motifs in this region and their functional interaction are currently ill-defined, especially in non-primate FVs.ResultsMutagenesis of N-terminal Gag residues of feline FV (FFV) reveals key residues essential for either capsid assembly and/or viral budding via interaction with the FFV Env leader protein (Elp). In an in vitro Gag–Elp interaction screen, Gag mutations abolishing particle assembly also interfered with Elp binding, indicating that Gag assembly is a prerequisite for this highly specific interaction. Gradient sedimentation analyses of cytosolic proteins indicate that wild-type Gag is mostly assembled into virus capsids. Moreover, proteolytic processing of Gag correlates with capsid assembly and is mostly, if not completely, independent from particle budding. In addition, Gag processing correlates with the presence of packaging-competent FFV genomic RNA suggesting that Pol encapsidation via genomic RNA is a prerequisite for Gag processing. Though an appended heterogeneous myristoylation signal rescues Gag particle budding of mutants unable to form capsids or defective in interacting with Elp, it fails to generate infectious particles that co-package Pol, as evidenced by a lack of Gag processing.ConclusionsChanges in proteolytic Gag processing, intracellular capsid assembly, particle budding and infectivity of defined N-terminal Gag mutants highlight their essential, distinct and only partially overlapping roles during viral assembly and budding. Discussion of these findings will be based on a recent model developed for Gag–Elp interactions in prototype FV.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0291-8) contains supplementary material, which is available to authorized users.
“…2, 3), the presence of conserved residues in the N-terminus of FV Gag (Fig. 4a) and published data for PFV [9], individual amino acid replacements in Gag were engineered, generating single amino acid proviral Gag mutants pCF-7-L10A, Q11A, Q12A, L13A, Y14A, H25A, G26A, D27A, I28A, I29A, R32A, G36A, W38A, G39A, R43A, L51A and D53A (Fig. 4a).Fig.…”
BackgroundFoamy viruses (FVs) of the Spumaretrovirinae subfamily are distinct retroviruses, with many features of their molecular biology and replication strategy clearly different from those of the Orthoretroviruses, such as human immunodeficiency, murine leukemia, and human T cell lymphotropic viruses. The FV Gag N-terminal region is responsible for capsid formation and particle budding via interaction with Env. However, the critical residues or motifs in this region and their functional interaction are currently ill-defined, especially in non-primate FVs.ResultsMutagenesis of N-terminal Gag residues of feline FV (FFV) reveals key residues essential for either capsid assembly and/or viral budding via interaction with the FFV Env leader protein (Elp). In an in vitro Gag–Elp interaction screen, Gag mutations abolishing particle assembly also interfered with Elp binding, indicating that Gag assembly is a prerequisite for this highly specific interaction. Gradient sedimentation analyses of cytosolic proteins indicate that wild-type Gag is mostly assembled into virus capsids. Moreover, proteolytic processing of Gag correlates with capsid assembly and is mostly, if not completely, independent from particle budding. In addition, Gag processing correlates with the presence of packaging-competent FFV genomic RNA suggesting that Pol encapsidation via genomic RNA is a prerequisite for Gag processing. Though an appended heterogeneous myristoylation signal rescues Gag particle budding of mutants unable to form capsids or defective in interacting with Elp, it fails to generate infectious particles that co-package Pol, as evidenced by a lack of Gag processing.ConclusionsChanges in proteolytic Gag processing, intracellular capsid assembly, particle budding and infectivity of defined N-terminal Gag mutants highlight their essential, distinct and only partially overlapping roles during viral assembly and budding. Discussion of these findings will be based on a recent model developed for Gag–Elp interactions in prototype FV.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0291-8) contains supplementary material, which is available to authorized users.
“…6). Unlike other Retroviridae , foamy viruses recruit the ESCRT machinery to facilitate budding at the ER and the trans-Golgi network (TGN), in addition to the plasma membrane (46). Nevertheless, with the exception of foamy viruses, to the best of our knowledge, the ESCRT machinery has not been previously implicated in envelopment of other RNA viruses that preferentially bud intracellularly.…”
Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses.
“…The spumaretroviral envelope protein (Env) harbors essential features that allow for absorption, uptake and fusogenic release of capsids into the cytoplasm. In comparison to other retroviral envelope proteins FV glycoproteins undergo a highly unusual biosynthesis as the precursor (gp130 Env for prototype FV, PFV) is only posttranslationally cleaved by cellular furin-like proteases (reviewed in [10]). Env precursor processing then culminates into an N-terminal signal or leader peptide (LP, gp18 LP for PFV), a central surface (SU, gp80 SU for PFV) and a C-terminal transmembrane subunit (TM, gp48 TM for PFV) [4].…”
Section: Introductionmentioning
confidence: 99%
“…Upon crossing the first physical barrier imposed by the cell membrane and escape of capsids into the cytosol FVs face some daunting challenges. Unique among retroviruses is entry of different kinds of FV particles, containing either a DNA or RNA genome, as a consequence of the reverse transcription initiation in a significant fraction of virions already in the virus producing cell (reviewed in [9,10]). To eventually deliver the viral genome to its final destination—the nucleus—the capsids first migrate through the cytoplasm of the host cell using cytoskeletal structures and motor protein complexes [11,12,13,14].…”
Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.
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