scholarly journals Allosteric Regulation of HIV-1 Capsid Structure for Gag Assembly, Virion Production, and Viral Infectivity by a Disordered Interdomain Linker

2019 ◽  
Vol 93 (17) ◽  
Author(s):  
Takaaki Koma ◽  
Osamu Kotani ◽  
Kei Miyakawa ◽  
Akihide Ryo ◽  
Masaru Yokoyama ◽  
...  
Keyword(s):  
In Silico ◽  
Particle Production ◽  
Allosteric Regulation ◽  
Viral Infectivity ◽  
Linker Region ◽  
Virion Production ◽  
Virion Morphogenesis ◽  
Interdomain Linker ◽  
Gag Assembly ◽  
Hiv 1

ABSTRACTThe retroviral Gag capsid (Gag-CA) interdomain linker is an unstructured peptide segment connecting structured N-terminal and C-terminal domains. Although the region is reported to play roles in virion morphogenesis and infectivity, underlying molecular mechanisms remain unexplored. To address this issue, we determined biological and molecular phenotypes of HIV-1 CA linker mutants by experimental andin silicoapproaches. Among the nine linker mutants tested, eight exhibited attenuation of viral particle production to various extents mostly in parallel with a reduction in viral infectivity. Sucrose density gradient, confocal microscopy, and live-cell protein interaction analyses indicated that the defect is accompanied by attenuation of Gag-Gag interactions following Gag plasma membrane targeting in the cells.In silicoanalyses revealed distinct distributions of interaction-prone hydrophobic patches between immature and mature CA proteins. Molecular dynamics simulations predicted that the linker mutations can allosterically alter structural fluctuations, including the interaction surfaces apart from the mutation sites in both the immature and mature CA proteins. These results suggest that the HIV-1 CA interdomain linker is acis-modulator of the CA interaction surfaces to optimize efficiency of Gag assembly, virion production, and viral infectivity.IMPORTANCEHIV-1 particle production and infection are highly ordered processes. Viral Gag proteins play a central role in the assembly and disassembly of viral molecules. Of these, capsid protein (CA) is a major contributor to the Gag-Gag interactions. CA consists of two structured domains, i.e., N-terminal (NTD) and C-terminal (CTD) domains, connected by an unstructured domain named the interdomain linker. While multiple regions in the NTD and CTD are reported to play roles in virion morphogenesis and infectivity, the roles of the linker region in Gag assembly and virus particle formation remain elusive. In this study, we showed by biological and molecular analyses that the linker region functions as an intramolecular modulator to tune Gag assembly, virion production, and viral infectivity. Our study thus illustrates a hitherto-unrecognized mechanism, an allosteric regulation of CA structure by the disordered protein element, for HIV-1 replication.

scholarly journals Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

2010 ◽  
Vol 84 (13) ◽  
pp. 6438-6451 ◽  
Author(s):  
Lorna S. Ehrlich ◽  
Gisselle N. Medina ◽  
Mahfuz B. Khan ◽  
Michael D. Powell ◽  
Katsuhiko Mikoshiba ◽  
...  
Keyword(s):  
Particle Production ◽  
Receptor Interaction ◽  
Particle Release ◽  
Virus Particles ◽  
Intracellular Ca ◽  
Necessary And Sufficient ◽  
Gag Assembly ◽  
Immature Virus ◽  
Interfering Rna ◽  
Hiv 1

ABSTRACT The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca2+ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca2+ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca2+ stores. Following activation by binding of its ligand, IP3, it releases Ca2+ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca2+ signaling as a potential novel cofactor in viral particle release.

scholarly journals The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability

Virology ◽  
2011 ◽  
Vol 421 (2) ◽  
pp. 253-265 ◽  
Author(s):  
Jiyang Jiang ◽  
Sherimay D. Ablan ◽  
Suchitra Derebail ◽  
Kamil Hercík ◽  
Ferri Soheilian ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Critical Determinant ◽  
Linker Region ◽  
Interdomain Linker ◽  
Hiv 1

scholarly journals The Conserved Tyr176/Leu177 Motif in the α-Helix 9 of the Feline Immunodeficiency Virus Capsid Protein Is Critical for Gag Particle Assembly

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 816
Author(s):  
César A. Ovejero ◽  
Silvia A. González ◽  
José L. Affranchino
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Particle Production ◽  
Equine Infectious Anemia Virus ◽  
Particle Assembly ◽  
Immunodeficiency Virus ◽  
Immature Particle ◽  
Gag Assembly ◽  
Hiv 1

The capsid domain (CA) of the lentiviral Gag polyproteins has two distinct roles during virion morphogenesis. As a domain of Gag, it mediates the Gag–Gag interactions that drive immature particle assembly, whereas as a mature protein, it self-assembles into the conical core of the mature virion. Lentiviral CA proteins are composed of an N-terminal region with seven α-helices and a C-terminal domain (CA-CTD) formed by four α-helices. Structural studies performed in HIV-1 indicate that the CA-CTD helix 9 establishes homodimeric interactions that contribute to the formation of the hexameric Gag lattice in immature virions. Interestingly, the mature CA core also shows inter-hexameric associations involving helix 9 residues W184 and M185. The CA proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) exhibit, at equivalent positions in helix 9, the motifs Y176/L177 and L169/F170, respectively. In this paper, we investigated the relevance of the Y176/L177 motif for FIV assembly by introducing a series of amino acid substitutions into this sequence and studying their effect on in vivo and in vitro Gag assembly, CA oligomerization, mature virion production, and viral infectivity. Our results demonstrate that the Y176/L177 motif in FIV CA helix 9 is essential for Gag assembly and CA oligomerization. Notably, mutations converting the FIV CA Y176/L177 motif into the HIV-1 WM and EIAV FL sequences allow substantial particle production and viral replication in feline cells.

scholarly journals The Nucleocapsid Region of Human Immunodeficiency Virus Type 1 Gag Assists in the Coordination of Assembly and Gag Processing: Role for RNA-Gag Binding in the Early Stages of Assembly

2009 ◽  
Vol 83 (15) ◽  
pp. 7718-7727 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Teresa Shatzer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Protease Activity ◽  
Particle Production ◽  
Rna Binding ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Gag Assembly ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag-RNA interactions are required for virus assembly. However, our prior study found that a defect in particle production exhibited by an HIV-1 proviral mutant with a severe deletion in the RNA-binding nucleocapsid (NC) region of Gag, NX, could be reversed by eliminating its protease activity. While our follow-up study indicated that a secondary RNA-binding site in Gag can also provide the required RNA-binding function, how protease activity inhibits NX virion production is still unclear. Therefore, we tested three possible mechanisms: NX virions are unstable and fall apart after budding; NX Gag assembly is slowed, allowing protease processing to start before particle formation; or the protease region within NX Gag-Pol becomes activated prematurely and processes the assembling Gag. We found that NX particles were as stable as wild-type virions. Furthermore, even a modest slowing of protease activity could rescue NX. Pulse-chase analysis revealed that the initial particle production by NC-deleted Gag was delayed compared to that of wild type Gag, but once started, the rate of production was similar, revealing a defect in the initiation of assembly. Wild-type Gag particle production was not eliminated or decreased in the presence of excess NX Gag-Pol, inconsistent with a premature activation of protease. Overall, these results indicate that the particle formation defect of NX is due to delayed initiation of assembly caused by the absence of NC in Gag, making it vulnerable to protease processing before budding can occur. Therefore, NC plays an important initiating role in Gag assembly.

scholarly journals Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Jordan T. Becker ◽  
Nathan M. Sherer
Keyword(s):  
Particle Production ◽  
Full Length ◽  
Viral Mrnas ◽  
Viral Rnas ◽  
Virion Assembly ◽  
Virion Production ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Virus Particle Production

ABSTRACT Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5′ packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy.

An In silico Approach for Identification of Novel Inhibitors as a Potential Therapeutics Targeting HIV-1 Viral Infectivity Factor

2015 ◽  
Vol 15 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Chanda Sinha ◽  
Anuradha Nischal ◽  
Srinivas Bandaru ◽  
Priyadarshani Kasera ◽  
Ashish Rajput ◽  
...  
Keyword(s):  
In Silico ◽  
Viral Infectivity ◽  
Viral Infectivity Factor ◽  
Hiv 1 ◽  
Potential Therapeutics

scholarly journals Defect of Human Immunodeficiency Virus Type 2 Gag Assembly in Saccharomyces cerevisiae

2007 ◽  
Vol 81 (18) ◽  
pp. 9911-9921 ◽  
Author(s):  
Yuko Morikawa ◽  
Toshiyuki Goto ◽  
Daisuke Yasuoka ◽  
Fumitaka Momose ◽  
Tetsuro Matano
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Membrane Binding ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Gag Assembly ◽  
Hiv 1

ABSTRACT We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.

scholarly journals Association of Human Immunodeficiency Virus Type 1 Gag with Membrane Does Not Require Highly Basic Sequences in the Nucleocapsid: Use of a Novel Gag Multimerization Assay

2005 ◽  
Vol 79 (22) ◽  
pp. 14131-14140 ◽  
Author(s):  
Akira Ono ◽  
Abdul A. Waheed ◽  
Anjali Joshi ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Immunodeficiency Virus ◽  
Gag Assembly ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterol-enriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.

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