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Brett D Lindenbach - One of the best experts on this subject based on the ideXlab platform.

  • the ins and outs of hepatitis c virus entry and Assembly
    Nature Reviews Microbiology, 2013
    Co-Authors: Brett D Lindenbach, Charles M Rice
    Abstract:

    Hepatitis C virus, a major human pathogen, produces infectious virus Particles with several unique features, such as an ability to interact with serum lipoproteins, a dizzyingly complicated process of virus entry, and a pathway of virus Assembly and release that is closely linked to lipoprotein secretion. Here, we review these unique features, with an emphasis on recent discoveries concerning virus Particle structure, virus entry and virus Particle Assembly and release.

  • virion Assembly and release
    Current Topics in Microbiology and Immunology, 2013
    Co-Authors: Brett D Lindenbach
    Abstract:

    Hepatitis C Virus (HCV) Particles exhibit several unusual properties that are not found in other enveloped RNA viruses, most notably their low buoyant density and interaction with serum lipoproteins. With the advent of systems to grow HCV in cell culture, the molecular basis of HCV Particle Assembly and release can now be addressed. The process of virus Assembly involves protein–protein interactions between viral structural and nonstructural proteins and the coordinated action of host factors. This chapter reviews our current understanding of these interactions and factors.

  • Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly
    PLOS Pathogens, 2011
    Co-Authors: Natalie A. Counihan, Stephen M. Rawlinson, Brett D Lindenbach
    Abstract:

    Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus Particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus Assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of Assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus Particle Assembly.

  • hepatitis c virus ns2 coordinates virus Particle Assembly through physical interactions with the e1 e2 glycoprotein and ns3 ns4a enzyme complexes
    Journal of Virology, 2011
    Co-Authors: Kenneth A Stapleford, Brett D Lindenbach
    Abstract:

    The hepatitis C virus (HCV) NS2 protein is essential for Particle Assembly, but its function in this process is unknown. We previously identified critical genetic interactions between NS2 and the viral E1-E2 glycoprotein and NS3-NS4A enzyme complexes. Based on these data, we hypothesized that interactions between these viral proteins are essential for HCV Particle Assembly. To identify interaction partners of NS2, we developed methods to site-specifically biotinylate NS2 in vivo and affinity capture NS2-containing protein complexes from virus-producing cells with streptavidin magnetic beads. By using these methods, we confirmed that NS2 physically interacts with E1, E2, and NS3 but did not stably interact with viral core or NS5A proteins. We further characterized these protein complexes by blue native polyacrylamide gel electrophoresis and identified ≈520-kDa and ≈680-kDa complexes containing E2, NS2, and NS3. The formation of NS2 protein complexes was dependent on coexpression of the viral p7 protein and enhanced by cotranslation of viral proteins as a polyprotein. Further characterization indicated that the glycoprotein complex interacts with NS2 via E2, and the pattern of N-linked glycosylation on E1 and E2 suggested that these interactions occur in the early secretory pathway. Importantly, several mutations that inhibited virus Assembly were shown to inhibit NS2 protein complex formation, and NS2 was essential for mediating the interaction between E2 and NS3. These studies demonstrate that NS2 plays a central organizing role in HCV Particle Assembly by bringing together viral structural and nonstructural proteins.

  • hepatitis c virus ns2 protein contributes to virus Particle Assembly via opposing epistatic interactions with the e1 e2 glycoprotein and ns3 ns4a enzyme complexes
    Journal of Virology, 2009
    Co-Authors: Tung Phan, Ivo C Lorenz, Rudolf K. F. Beran, Christopher Peters, Brett D Lindenbach
    Abstract:

    The hepatitis C virus NS2 protein has been recently implicated in virus Particle Assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus Assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in Assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus Assembly.

Alan Rein - One of the best experts on this subject based on the ideXlab platform.

  • hiv 1 gag protein with or without p6 specifically dimerizes on the viral rna packaging signal
    Journal of Biological Chemistry, 2020
    Co-Authors: Samantha Sarni, Alan Rein, Banhi Biswas, Shuohui Liu, Erik D Olson, Jonathan P Kitzrow, Vicki H Wysocki, Karin Musierforsyth
    Abstract:

    The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral Particle Assembly. Although the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form Particles resembling gRNA-containing Particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral Assembly. However, the heterogeneity of Gag–RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native MS to investigate RNA binding of wild-type (WT) Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in Particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically promotes nucleation of Gag–Gag interactions at the early stages of immature viral Particle Assembly in a p6-independent manner.

  • hiv 1 gag protein with or without p6 specifically dimerizes on the viral rna packaging signal
    bioRxiv, 2020
    Co-Authors: Samantha Sarni, Alan Rein, Banhi Biswas, Shuohui Liu, Erik D Olson, Jonathan P Kitzrow, Vicki H Wysocki, Karin Musierforsyth
    Abstract:

    The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral Particle Assembly. While the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form Particles resembling gRNA-containing Particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral Assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native mass spectrometry to investigate RNA binding of wild-type Gag and Gag lacking the p6 domain (Gagp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. Gagp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in Particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically nucleates Gag-Gag interactions at the early stages of immature viral Particle Assembly in a p6-independent manner.

  • on the role of the sp1 domain in hiv 1 Particle Assembly a molecular switch
    Journal of Virology, 2011
    Co-Authors: Siddhartha A K Datta, Ferri Soheilian, Kunio Nagashima, Lakew G Temeselew, Rachael M Crist, Anne Kamata, Jane Mirro, Demetria Harvin, Raul E Cachau, Alan Rein
    Abstract:

    Expression of a retroviral protein, Gag, in mammalian cells is sufficient for Assembly of immature virus-like Particles (VLPs). VLP Assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP Assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP Assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.

  • interactions between hiv 1 gag molecules in solution an inositol phosphate mediated switch
    Journal of Molecular Biology, 2007
    Co-Authors: Siddhartha A K Datta, Stephen Campbell, Zhuojun Zhao, Patrick K Clark, Sergey Tarasov, Jerry Alexandratos, Mamuka Kvaratskhelia, Jacob Lebowitz, Alan Rein
    Abstract:

    Retrovirus Particle Assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like Particles (VLPs) in a fully defined system in vitro. However, this Assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the Assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during Particle Assembly.

  • modulation of hiv like Particle Assembly in vitro by inositol phosphates
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Stephen Campbell, Robert J Fisher, Eric M Towler, Stephen B Fox, Haleem J Issaq, Tracy L Wolfe, Lawrence R Phillips, Alan Rein
    Abstract:

    HIV-1 Gag protein assembles into 100- to 120-nm diameter Particles in mammalian cells. Recombinant HIV-1 Gag protein assembles in a fully defined system in vitro into Particles that are only 25–30 nm in diameter and that differ significantly in other respects from authentic Particles. However, Particles with the size and other properties of authentic virions were obtained in vitro by addition of inositol phosphates or phosphatidylinsitol phosphates to the Assembly system. Thus, the interactions between HIV-1 Gag protein molecules are altered by binding of inositol derivatives; this binding is apparently essential for normal HIV-1 Particle Assembly. This requirement is not seen in a deleted Gag protein lacking residues 16–99 within the matrix domain.

Ian M Jones - One of the best experts on this subject based on the ideXlab platform.

  • roles of matrix p2 and n terminal myristoylation in human immunodeficiency virus type 1 gag Assembly
    Journal of Virology, 2000
    Co-Authors: Yuko Morikawa, David J Hockley, Milan V Nermut, Ian M Jones
    Abstract:

    Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus Particle Assembly occurs. Particle Assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like Particles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of the Assembly-competent Gag; (ii) the p2 domain, located at the capsid/nucleocapsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag Assembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimerization stages but plays a key role in conversion of these multimers to Gag VLP. We suggest that the Gag trimer and the >1,000-kDa multimer are intermediates in the Assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VLP development and suggest that each stage involves a separate domain, MA, p2, or N-terminal myristoylation, each of which contributes to HIV Particle Assembly.

  • gag gag interactions in the c terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for gag Particle Assembly
    Journal of General Virology, 1996
    Co-Authors: Weihong Zhang, David J Hockley, Milan V Nermut, Yuko Morikawa, Ian M Jones
    Abstract:

    Seven internal deletions within the p24 domain of the human immunodeficiency virus type 1 Gag precursor have been assessed for their effect on Gag Particle formation following their expression using recombinant baculoviruses. In addition, each deleted molecule was assessed for its ability to bind soluble p24 antigen in vitro. The mutants fell into three different phenotypic groups: (i) three mutants that had no effect on either p24 binding or Gag Particle Assembly, (ii) three mutants that abolished both features and (iii) one mutant that bound p24 in vitro but failed to assemble Particles. Mutations that abolished both in vitro p24 binding and Particle Assembly mapped to the C terminus of p24 confirming this region as critical for virion Assembly. We suggest the division of virion Assembly into at least two distinct phases and suggest a model in which the critical sequences mapped to date are combined with available structural information.

Paul D Bieniasz - One of the best experts on this subject based on the ideXlab platform.

  • Clathrin facilitates the morphogenesis of retrovirus Particles
    2011
    Co-Authors: Fengwen Zhang, Trinity Zang, Sam J Wilson, Marc C Johnson, Paul D Bieniasz
    Abstract:

    The morphogenesis of retroviral Particles is driven by Gag and GagPol proteins that provide the major structural component and enzymatic activities required for Particle Assembly and maturation. In addition, a number of cellular proteins are found in retrovirus Particles; some of these are important for viral replication, but many lack a known functional role. One such protein is clathrin, which is assumed to be passively incorporated into virions due to its abundance at the plasma membrane. We found that clathrin is not only exceptionally abundant in highly purified HIV-1 Particles but is recruited with high specificity. In particular, the HIV-1 Pol protein was absolutely required for clathrin incorporation and point mutations in reverse transcriptase or integrase domains of Pol could abolish incorporation. Clathrin was also specifically incorporated into other retrovirus Particles, including members of the lentivirus (simian immunodeficiency virus, SIVmac), gammaretrovirus (murine leukemia virus, MLV) and betaretrovirus (Mason-Pfizer monkey virus, M-PMV) genera. However, unlike HIV-1, these other retroviruses recruited clathrin primarily using peptide motifs in their respective Gag proteins that mimicked motifs found in cellular clathrin adaptors. Perturbation of clathrin incorporation into these retroviruses, via mutagenesis of viral proteins, siRNA based clathrin depletion or adaptor protein (AP180) induced clathrin sequestration, had a range of effects on the accuracy of Particle morphogenesis. These effects varied according to which retrovirus was examined, and included Gag and/or Pol protein destabilization, inhibition of Particle Assembly and reduction in virion infectivity. For each retroviru

  • analysis of the initiating events in hiv 1 Particle Assembly and genome packaging
    PLOS Pathogens, 2010
    Co-Authors: Sebla B Kutluay, Paul D Bieniasz
    Abstract:

    HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the Assembly of virus-like Particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 Assembly has accrued, events that occur at the initiation of Assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete Particle Assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 Particle Assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent Particle Assembly in cells.

  • the cell biology of hiv 1 virion genesis
    Cell Host & Microbe, 2009
    Co-Authors: Paul D Bieniasz
    Abstract:

    Recent work has illuminated three critical aspects of the cell biology of HIV-1 Particle genesis. First, we have come to understand which cellular membranes are selected as platforms for virus Particle Assembly and how this occurs. Second, an understanding of how the host ESCRT pathway enables virion budding is accruing. Third, it has become apparent that a host inhibitor can block HIV-1 Particle release and that antagonism of this inhibitor underlies the ability of HIV and SIV accessory genes to facilitate Particle release. Here, I review recent progress in these three areas.

  • plasma membrane is the site of productive hiv 1 Particle Assembly
    PLOS Biology, 2006
    Co-Authors: Nolwenn Jouvenet, Stuart J D Neil, Cameron Bess, Marc C Johnson, Cesar A Virgen, Sanford M Simon, Paul D Bieniasz
    Abstract:

    Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag) to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM) and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when Assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 Assembly is initiated and completed at the PM, and not at endosomal membranes.

  • hiv 1 and ebola virus encode small peptide motifs that recruit tsg101 to sites of Particle Assembly to facilitate egress
    Nature Medicine, 2001
    Co-Authors: Juan Martinserrano, Trinity Zang, Paul D Bieniasz
    Abstract:

    Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of Particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like Particles. EbVp40 recruits Tsg101 to sites of Particle Assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain–defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus–host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens.

Mudathir Alim - One of the best experts on this subject based on the ideXlab platform.

  • inhibition of hiv 1 Particle Assembly by 2 3 cyclic nucleotide 3 phosphodiesterase
    Cell Host & Microbe, 2012
    Co-Authors: Sam J Wilson, Trinity Zang, Sebla B Kutluay, John W Schoggins, Nolwenn Jouvenet, Mudathir Alim
    Abstract:

    The expression of hundreds of interferon-stimulated genes (ISGs) causes the cellular “antiviral state” in which the replication of many viruses, including HIV-1, is attenuated. We conducted a screen for ISGs that inhibit HIV-1 virion production and found that 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP), a membrane-associated protein with unknown function in mammals has this property. CNP binds to the structural protein Gag and blocks HIV-1 Particle Assembly after Gag and viral RNA have associated with the plasma membrane. Several primate lentiviruses are CNP-sensitive, and CNP sensitivity/resistance is determined by a single, naturally dimorphic, codon (E/K40) in the matrix domain of Gag. Like other antiretroviral proteins, CNP displays interspecies variation in antiviral activity. Mice encode an inactive CNP variant and a single amino acid difference in murine versus human CNP determines Gag binding and antiviral activity. Some cell types express high levels of CNP and we speculate that CNP evolved to restrict lentivirus replication therein.