Interaction between the Human Immunodeficiency Virus Type 1 Gag Matrix Domain and Phosphatidylinositol-(4,5)-Bisphosphate Is Essential for Efficient Gag Membrane Binding

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P2 depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P2 depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P2-depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P2. To examine a putative Gag interaction with PI(4,5)P2, we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P2. Using this assay, we observed that PI(4,5)P2 significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P2 for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P2 binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P2-containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P2 on the membrane and that the MA basic domain mediates this interaction.

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The role of viral coreceptors and enhanced macrophage tropism in human immunodeficiency virus type 1 disease progression

Sexual Health ◽

10.1071/sh03006 ◽

2004 ◽

Vol 1 (1) ◽

pp. 23 ◽

Cited By ~ 14

Author(s):

Paul R. Gorry ◽

Jasminka Sterjovski ◽

Melissa Churchill ◽

Kristie Witlox ◽

Lachlan Gray ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Disease Progression ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Molecular Mechanisms ◽

Coreceptor Usage ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

Despite numerous studies on the impact of viral diversity, human immunodeficiency virus type 1 (HIV-1)-specific immune responses and host factors on disease progression, we still do not have a firm understanding of the long-term pathogenesis of HIV-1 infection. Rapid depletion of CD4+ T-lymphocytes has been associated with a switch in viral coreceptor usage from CCR5 to CXCR4 in ~40 to 50% of infected individuals. However, the majority of infected individuals who progress to AIDS harbour only CCR5-dependent (R5) viral strains. The progression HIV-1 disease is associated with an enhanced tropism of R5 viral strains for monocyte/macrophage lineage cells (enhanced M-tropism). However, the underlying molecular mechanisms contributing to enhanced M-tropism by R5 HIV-1 strains, and how HIV-1 variants with enhanced M-tropism cause CD4+ T-cell depletion in vivo are unknown. This review examines the relationship between viral coreceptor usage, M-tropism, and pathogenicity of HIV-1. We highlight evidence supporting the hypothesis that enhanced M-tropism of R5 HIV-1 results from adaptive viral evolution, resulting in HIV-1 variants that have increased ability to utilise relatively low levels of CCR5 expressed on macrophages, by way of increased CCR5 affinity. The evidence also suggests that these late-emerging, R5 viral strains have reduced sensitivity to entry inhibitors, and increased ability to cause CD4+ T-lymphocyte loss. These variants are likely to impact HIV-1 disease progression, especially in patients who persistently harbour only R5 viral strains.

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Matrix-Induced Inhibition of Membrane Binding Contributes to Human Immunodeficiency Virus Type 1 Particle Assembly Defects in Murine Cells

Journal of Virology ◽

10.1128/jvi.79.24.15586-15589.2005 ◽

2005 ◽

Vol 79 (24) ◽

pp. 15586-15589 ◽

Cited By ~ 25

Author(s):

Theodora Hatziioannou ◽

Juan Martin-Serrano ◽

Trinity Zang ◽

Paul D. Bieniasz

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Membrane Binding ◽

Proteolytic Processing ◽

Particle Assembly ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Globular Head

ABSTRACT Defective human immunodeficiency virus type 1 (HIV-1) assembly in murine cells is accompanied by poor plasma membrane binding and proteolytic processing of the HIV-1 Gag precursor. Here, we show that such defects are induced by the propensity of the HIV-1 MA globular head to inhibit membrane binding and particle assembly, particularly at the low expression levels observed in murine cells. Simple additions to or deletion of the MA globular head can improve the yield of infectious virions from murine cells by >50-fold. Expression level and autoinhibition can be important confounding variables in studies of HIV-1 assembly and contribute to defects encountered in murine cells.

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Analysis of Human Immunodeficiency Virus Type 1 Gag Dimerization-Induced Assembly

Journal of Virology ◽

10.1128/jvi.79.23.14498-14506.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14498-14506 ◽

Cited By ~ 32

Author(s):

Ayna Alfadhli ◽

Tenzin Choesang Dhenub ◽

Amelia Still ◽

Eric Barklis

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Protein Assembly ◽

Particle Assembly ◽

Gag Protein ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Terminal Domains

ABSTRACT The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.

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Mutation of Dileucine-Like Motifs in the Human Immunodeficiency Virus Type 1 Capsid Disrupts Virus Assembly, Gag-Gag Interactions, Gag-Membrane Binding, and Virion Maturation

Journal of Virology ◽

10.1128/jvi.00355-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 7939-7951 ◽

Cited By ~ 47

Author(s):

Anjali Joshi ◽

Kunio Nagashima ◽

Eric O. Freed

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Particle Production ◽

Virus Assembly ◽

Membrane Binding ◽

Single Amino Acid ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55Gag drives the assembly and release of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role in these processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CA contains two dileucine-like motifs (L189/L190 and I201/L202) implicated in regulating the localization of Gag to multivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimer interface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactions during core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1 replication, we introduced a series of mutations into these motifs in the context of a full-length, infectious HIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virus particle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization, and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting, the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages. Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-like motifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported here provide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CA dimerization, particle maturation, and virion infectivity.

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Kinesin KIF4 Regulates Intracellular Trafficking and Stability of the Human Immunodeficiency Virus Type 1 Gag Polyprotein

Journal of Virology ◽

10.1128/jvi.00819-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 9937-9950 ◽

Cited By ~ 54

Author(s):

Nathaniel W. Martinez ◽

Xiaoxiao Xue ◽

Reem G. Berro ◽

Geri Kreitzer ◽

Marilyn D. Resh

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Particle Production ◽

Particle Assembly ◽

Gag Protein ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.

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Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness

Journal of General Virology ◽

10.1099/vir.0.045492-0 ◽

2012 ◽

Vol 93 (12) ◽

pp. 2625-2634 ◽

Cited By ~ 6

Author(s):

Elena Capel ◽

Glòria Martrus ◽

Mariona Parera ◽

Bonaventura Clotet ◽

Miguel Angel Martínez

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Replication Capacity ◽

Recombinant Viruses ◽

Subtype B ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naïve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naïve isolates (P = 0.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P<0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P<0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naïve proteases from early or recent sample isolates (P = 0.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P = 0.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naïve proteases.

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Incorporation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase into Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.01796-06 ◽

2007 ◽

Vol 81 (10) ◽

pp. 5155-5165 ◽

Cited By ~ 9

Author(s):

Wei-Hao Liao ◽

Kuo-Jung Huang ◽

Yu-Fen Chang ◽

Shiu-Mei Wang ◽

Ying-Tzu Tseng ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Binding Capacity ◽

Membrane Binding ◽

Virus Like Particles ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We demonstrate that a genetically engineered human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) composed mainly of p66 or p51 subunits can be incorporated into virus-like particles (VLPs) when coexpressed with HIV-1 Pr55 gag . VLP-associated RT exhibited a detergent-resistant association with immature cores during sucrose gradient equilibrium centrifugation, suggesting that RT is incorporated into VLPs. However, RT that retains downstream integrase (IN) is severely inhibited in terms of incorporation into VLPs. Results from immunofluorescence tests reveal that RT-IN is primarily localized at the perinuclear area and exhibits poor colocalization with Gag. IN removal leads to a redistribution of RT throughout the cytoplasm and improved RT incorporation into VLPs. Similar results were observed for RT-IN in which alanine was substituted for 186-Lys-Arg-Lys-188 residues of the IN putative nuclear localization signal, suggesting that IN karyophilic properties may partly account for the inhibitory effect of IN on RT incorporation. Although the membrane-binding capacity of RT was markedly reduced compared to that of wild-type Gag or Gag-Pol, the correlation of membrane-binding ability with particle incorporation efficiency was incomplete. Furthermore, we observed that membrane-binding-defective myristylation-minus RT can be packaged into VLPs at the same level as its normal myristylated counterpart. This suggests that the incorporation of RT into VLPs is independent of membrane affinity but very dependent on RT-Gag interaction. Results from a genetic analysis suggest that the Gag-interacting regions of RT mainly reside in the thumb subdomain and that the RT-binding domains of Gag are located in the matrix (MA) and p6 regions.

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Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5555 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5555-5562 ◽

Cited By ~ 15

Author(s):

J D DeZazzo ◽

J M Scott ◽

M J Imperiale

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dependent Manner ◽

Immunodeficiency Virus ◽

A Site ◽

The Impact ◽

Hiv 1 ◽

Rna Levels

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.

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Selected amino acid substitutions in the C-terminal region of human immunodeficiency virus type 1 capsid protein affect virus assembly and release

Journal of General Virology ◽

10.1099/vir.0.80137-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 2903-2913 ◽

Cited By ~ 21

Author(s):

Samir Abdurahman ◽

Stefan Höglund ◽

Laura Goobar-Larsson ◽

Anders Vahlne

Keyword(s):

Human Immunodeficiency Virus ◽

Capsid Protein ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Infectious Clone ◽

Particle Assembly ◽

Virus Particles ◽

Immunodeficiency Virus ◽

Hiv 1

The capsid protein (CA or p24) of human immunodeficiency virus type 1 (HIV-1) plays a major role both early and late in the virus replication cycle. Many studies have suggested that the C-terminal domain of this protein is involved in dimerization and proper assembly of the viral core. Point mutations were introduced in two conserved sites of this region and their effects on viral protein expression, particle assembly and infectivity were studied. Eight different mutants (L205A+P207A, L205A, P207A, 223GPG225AAA, G223A, P224A, G225A and V221G) of the infectious clone pNL4-3 were constructed. Most substitutions had no substantial effect on HIV-1 protein synthesis, yet they impaired viral infectivity and particle production. The two mutants P207A and V221G also had a profound effect on Gag–Pol protein processing in HeLa–tat cells. However, these results were cell line-specific and Gag–Pol processing of P207A was not affected in 293T cells. In HeLa–tat cells, no virus particles were detected with the P207A mutation, whereas the other mutant virus particles were heterogeneous in size and morphology. None of the mutants showed normal, mature, conical core structures in HeLa–tat cells. These results indicate that the two conserved sequences in the C-terminal CA domain are essential for proper morphogenesis and infectivity of HIV-1 particles.

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c-Myc and Sp1 Contribute to Proviral Latency by Recruiting Histone Deacetylase 1 to the Human Immunodeficiency Virus Type 1 Promoter

Journal of Virology ◽

10.1128/jvi.01208-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 10914-10923 ◽

Cited By ~ 151

Author(s):

Guochun Jiang ◽

Amy Espeseth ◽

Daria J. Hazuda ◽

David M. Margolis

Keyword(s):

Human Immunodeficiency Virus ◽

Rna Polymerase Ii ◽

Histone Deacetylase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Molecular Mechanisms ◽

Histone Deacetylase 1 ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Histone deacetylase (HDAC) inhibitors such as valproic acid (VPA) induce the expression of quiescent proviral human immunodeficiency virus type 1 (HIV-1) and may deplete proviral infection in vivo. To uncover novel molecular mechanisms that maintain HIV latency, we sought cellular mRNAs whose expression was diminished in resting CD4+ T cells of HIV-1-infected patients exposed to VPA. c-Myc was prominent among genes markedly downregulated upon exposure to VPA. c-Myc expression repressed HIV-1 expression in chronically infected cell lines. Chromatin immunoprecipitation (ChIP) assays revealed that c-Myc and HDAC1 are coordinately resident at the HIV-1 long terminal repeat (LTR) promoter and absent from the promoter after VPA treatment in concert with histone acetylation, RNA polymerase II recruitment, and LTR expression. Sequential ChIP assays demonstrated that c-Myc, Sp1, and HDAC1 coexist in the same DNA-protein complex at the HIV promoter. Short hairpin RNA inhibition of c-Myc reduces both c-Myc and HDAC1 occupancy, blocks c-Myc repression of Tat activation, and increases LTR expression. These results expand the understanding of mechanisms that recruit HDAC and maintain the latency of HIV-1, suggesting novel therapeutic approaches against latent proviral HIV infection.

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