GB Virus Type C E2 Protein Inhibits Human Immunodeficiency Virus Type 1 Assembly Through Interference With HIV-1 Gag Plasma Membrane Targeting

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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Cell-Type-Dependent Targeting of Human Immunodeficiency Virus Type 1 Assembly to the Plasma Membrane and the Multivesicular Body

Journal of Virology ◽

10.1128/jvi.78.3.1552-1563.2004 ◽

2004 ◽

Vol 78 (3) ◽

pp. 1552-1563 ◽

Cited By ~ 196

Author(s):

Akira Ono ◽

Eric O. Freed

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Multivesicular Body ◽

Cell Type ◽

Gag Proteins ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) assembly-and-release pathway begins with the targeting of the Gag precursor to the site of virus assembly. The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood. Based on the analysis of mutant Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane. In this study, we determined that in HeLa and T cells, the MA mutant Gag proteins that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB). Interestingly, we find that in primary human macrophages, both wild-type (WT) and MA mutant Gag proteins are targeted specifically to the MVB. Despite the fact that particle assembly in macrophages occurs at an intracellular site rather than at the plasma membrane, we observe that WT Gag expressed in this cell type is released as extracellular virions with high efficiency. These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages and that particle release in this cell type may follow an exosomal pathway. To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6Gag and the MVB sorting machinery (e.g., TSG101), we examined the targeting and assembly of Gag mutants lacking p6. Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and TSG101 is not required for Gag targeting to the MVB. These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB.

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Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane

Journal of Virology ◽

10.1128/jvi.00308-07 ◽

2007 ◽

Vol 81 (14) ◽

pp. 7476-7490 ◽

Cited By ~ 75

Author(s):

Andrés Finzi ◽

Alexandre Orthwein ◽

Johanne Mercier ◽

Éric A. Cohen

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Multivesicular Body ◽

Subcellular Fractionation ◽

Particle Assembly ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-β-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.

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Human Immunodeficiency Virus Type 1 Matrix Inhibits and Confers Cooperativity on Gag Precursor-Membrane Interactions

Journal of Virology ◽

10.1128/jvi.78.17.9560-9563.2004 ◽

2004 ◽

Vol 78 (17) ◽

pp. 9560-9563 ◽

Cited By ~ 70

Author(s):

David Perez-Caballero ◽

Theodora Hatziioannou ◽

Juan Martin-Serrano ◽

Paul D. Bieniasz

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Specific Targeting ◽

Immunodeficiency Virus ◽

Targeting Signal ◽

Hiv 1 ◽

Globular Head

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag multimerization and membrane binding are required for particle formation. However, it is unclear what constitutes a minimal plasma membrane-specific targeting signal and what role the matrix (MA) globular head and other Gag domains play in membrane targeting. Here, we use membrane flotation and microscopic analysis of Gag deletion mutants to demonstrate that the HIV-1 MA globular head inhibits a plasma membrane-specific targeting signal contained within the six amino-terminal MA residues. MA-mediated inhibition is relieved by concentration-dependent Gag multimerization and imparts a high degree of cooperativity on Gag-membrane association. This cooperativity may confer temporal and spatial regulation on HIV-1 assembly.

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Vpu and Tsg101 Regulate Intracellular Targeting of the Human Immunodeficiency Virus Type 1 Core Protein Precursor Pr55gag

Journal of Virology ◽

10.1128/jvi.80.8.3765-3772.2006 ◽

2006 ◽

Vol 80 (8) ◽

pp. 3765-3772 ◽

Cited By ~ 43

Author(s):

Kirsi Harila ◽

Ian Prior ◽

Mathilda Sjöberg ◽

Antti Salminen ◽

Jorma Hinkula ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Core Protein ◽

Virus Assembly ◽

Host Protein ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55 gag . Depending on the cell type, Pr55 gag accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55 gag determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55 gag are poorly characterized. We have analyzed targeting of newly synthesized Pr55 gag in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55 gag was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55 gag remained at the plasma membrane and virions assembled at this site. In contrast, Pr55 gag expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55 gag required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55 gag and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55 gag .

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Human Immunodeficiency Virus Type 1 Assembly, Budding, and Cell-Cell Spread in T Cells Take Place in Tetraspanin-Enriched Plasma Membrane Domains

Journal of Virology ◽

10.1128/jvi.01845-06 ◽

2007 ◽

Vol 81 (15) ◽

pp. 7873-7884 ◽

Cited By ~ 126

Author(s):

Clare Jolly ◽

Quentin J. Sattentau

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Membrane Domains ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Cell Cell

ABSTRACT Human immunodeficiency virus type-1 (HIV-1) egress from infected CD4+ T cells is thought to be via assembly and budding at the plasma membrane and may involve components of the T-cell secretory apparatus, including tetraspanins. However, many studies on HIV-1 assembly have examined the trafficking of viral proteins in isolation, and most have used immortalized epithelial, fibroblastic, or hematopoietic cell lines that may not necessarily reflect natural infection of susceptible T cells. Here we have used immunofluorescence and cryoimmunoelectron microscopy (CEM) to examine protein transport during HIV-1 assembly in productively infected Jurkat CD4+ T cells and primary CD4+ T cells. The HIV-1 envelope glycoprotein (Env) and the core protein (Gag) colocalize strongly with CD63 and CD81 and less strongly with CD9, whereas no colocalization was seen between Env or Gag and the late endosome/lysosomal marker Lamp2. CEM revealed incorporation of CD63 and CD81 but not Lamp2 into virions budding at the plasma membrane, and this was supported by immunoprecipitation studies, confirming that HIV-1 egress in T cells is trafficked via tetraspanin-enriched membrane domains (TEMs) that are distinct from lysosomal compartments. CD63, CD81, and, to a lesser extent, CD9 were recruited to the virological synapse (VS), and antibodies against these tetraspanins reduced VS formation. We propose that HIV-1 promotes virus assembly and cell-cell transfer in T cells by targeting plasma membrane TEMs.

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Chimpanzee GB virus C and GB virus A E2 envelope glycoproteins contain a peptide motif that inhibits human immunodeficiency virus type 1 replication in human CD4+ T-cells

Journal of General Virology ◽

10.1099/vir.0.047126-0 ◽

2013 ◽

Vol 94 (4) ◽

pp. 774-782 ◽

Cited By ~ 2

Author(s):

James H. McLinden ◽

Jack T. Stapleton ◽

Donna Klinzman ◽

Krishna K. Murthy ◽

Qing Chang ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Persistent Infection ◽

Amino Acid Level ◽

E2 Protein ◽

Immunodeficiency Virus ◽

Hiv 1

GB virus type C (GBV-C) is a lymphotropic virus that can cause persistent infection in humans. GBV-C is not associated with any disease, but is associated with reduced mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Related viruses have been isolated from chimpanzees (GBV-Ccpz) and from New World primates (GB virus type A, GBV-A). These viruses are also capable of establishing persistent infection. We determined the nucleotide sequence encoding the envelope glycoprotein (E2) of two GBV-Ccpz isolates obtained from the sera of captive chimpanzees. The deduced GBV-Ccpz E2 protein differed from human GBV-C by 31 % at the amino acid level. Similar to human GBV-C E2, expression of GBV-Ccpz E2 in a tet-off human CD4+ Jurkat T-cell line significantly inhibited the replication of diverse HIV-1 isolates. This anti-HIV-replication effect of GBV-Ccpz E2 protein was reversed by maintaining cells in doxycycline to reduce E2 expression. Previously, we found a 17 aa region within human GBV-C E2 that was sufficient to inhibit HIV-1. Although GBV-Ccpz E2 differed by 3 aa differences in this region, the chimpanzee GBV-C 17mer E2 peptide inhibited HIV-1 replication. Similarly, the GBV-A peptide that aligns with this GBV-C E2 region inhibited HIV-1 replication despite sharing only 5 aa with the human GBV-C E2 sequence. Thus, despite amino acid differences, the peptide region on both the GBV-Ccpz and the GBV-A E2 protein inhibit HIV-1 replication similar to human GBV-C. Consequently, GBV-Ccpz or GBV-A infection of non-human primates may provide an animal model to study GB virus–HIV interactions.

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Deficiency of Niemann-Pick Type C-1 Protein Impairs Release of Human Immunodeficiency Virus Type 1 and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments

Journal of Virology ◽

10.1128/jvi.00259-09 ◽

2009 ◽

Vol 83 (16) ◽

pp. 7982-7995 ◽

Cited By ~ 45

Author(s):

Yuyang Tang ◽

Ihid Carneiro Leao ◽

Ebony M. Coleman ◽

Robin Shepard Broughton ◽

James E. K. Hildreth

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Cholesterol Trafficking ◽

Immunodeficiency Virus ◽

Type C ◽

Intracellular Cholesterol ◽

Niemann Pick ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study, we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells, wherein these pathways are severely compromised, causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further, NPCD Epstein-Barr virus-transformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone, respectively, replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results, suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally, normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein, and overexpression of NPC1 increased HIV-1 release. Taken together, our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.

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Human Immunodeficiency Virus Type 1 Protease Triggers a Myristoyl Switch That Modulates Membrane Binding of Pr55gag and p17MA

Journal of Virology ◽

10.1128/jvi.73.3.1902-1908.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 1902-1908 ◽

Cited By ~ 69

Author(s):

Luz Hermida-Matsumoto ◽

Marilyn D. Resh

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Membrane Binding ◽

Immunodeficiency Virus ◽

Membrane Bound ◽

Myristoyl Switch ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Pr55 gag gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55 gag is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55 gag maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540–8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55 gag in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55 gag was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55 gag was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the “trigger” for a myristoyl switch mechanism that modulates the membrane associations of Pr55 gag and p17MA in virions and membranes.

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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

Journal of Virology ◽

10.1128/jvi.79.22.14131-14140.2005 ◽

2005 ◽

Vol 79 (22) ◽

pp. 14131-14140 ◽

Cited By ~ 70

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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