scholarly journals Virion Incorporation of Human Immunodeficiency Virus Type 1 Nef Is Mediated by a Bipartite Membrane-Targeting Signal: Analysis of Its Role in Enhancement of Viral Infectivity

1998 ◽  
Vol 72 (11) ◽  
pp. 8833-8840 ◽  
Author(s):  
Reinhold Welker ◽  
Mark Harris ◽  
Bettina Cardel ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
High Titer ◽  
Membrane Targeting ◽  
Viral Infectivity ◽  
Virion Incorporation ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The nef gene of primate immunodeficiency viruses is essential for high-titer virus replication and AIDS pathogenesis in vivo. In tissue culture, Nef is not required for human immunodeficiency virus (HIV) infection but enhances viral infectivity. We and others have shown that Nef is incorporated into HIV-1 particles and cleaved by the viral proteinase. To determine the signal for Nef incorporation and to analyze whether virion-associated Nef is responsible for enhancement of infectivity, we generated a panel of nef mutants and analyzed them for virion incorporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV particles. Incorporation was reconstituted by targeting the respective proteins to the plasma membrane by using a heterologous signal. Mutational analysis revealed that both myristoylation and an N-terminal cluster of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domain of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These results indicate that Nef incorporation into HIV-1 particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef correlated with enhanced infectivity of the respective viruses in a single-round replication assay. However, the phenotypes of HIV mutants with reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or different mechanisms may be involved in this system.

scholarly journals Role of the Gag Matrix Domain in Targeting Human Immunodeficiency Virus Type 1 Assembly

2000 ◽  
Vol 74 (6) ◽  
pp. 2855-2866 ◽  
Author(s):  
Akira Ono ◽  
Jan M. Orenstein ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Virus Particle ◽  
Particle Formation ◽  
Membrane Targeting ◽  
Basic Domain ◽  
Golgi Vesicles ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6Gag or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.

scholarly journals Sequence of Human Immunodeficiency Virus Type 1 (HIV-1) Gag Localization and Oligomerization Monitored with Live Confocal Imaging of a Replication-Competent, Fluorescently Tagged HIV-1

2007 ◽  
Vol 81 (22) ◽  
pp. 12596-12607 ◽  
Author(s):  
Wolfgang Hübner ◽  
Ping Chen ◽  
Armando Del Portillo ◽  
Yuxin Liu ◽  
Ronald E. Gordon ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Confocal Imaging ◽  
Hiv Protease ◽  
Viral Gene ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The assembly of infectious human immunodeficiency virus (HIV) requires that Gag transport and oligomerization be coordinated with its association with other viral proteins, viral RNAs, and cellular membranes. We have developed a replication-competent HIV type 1 molecular clone that carries a Gag-internal or interdomain green fluorescent protein (iGFP) fusion to reveal a physiologically accurate temporal sequence of Gag localization and oligomerization during the formation of infectious HIV. This recombinant HIV is as infectious as native HIV in single-round infectivity assays, validating its use for trafficking studies. It replicates robustly in permissive MT4 cells and is infectious, yet it spreads poorly in other T-cell lines. Immunofluorescence of Gag-iGFP showed a pattern very similar to that of native Gag. However, the intense plasma membrane Gag-iGFP fluorescence contrasts markedly with its immunofluorescence at this site, indicating that many Gag epitopes can be masked by oligomerization. Consistent with this, fluorescence resonance energy transfer studies visualized intense Gag oligomerization at the plasma membrane and weaker oligomerization at cytoplasmic sites. Four-dimensional, time-lapse confocal imaging reveals a temporal progression of Gag distribution over hours in which Gag is initially diffusely localized within the cytoplasm. Plasma membrane signals then accumulate as Gag levels increase and vesicular association appears late, only after plasma membrane site signals have reached high intensity. Lastly, the cell rounds up and HIV protease activation induces diffuse fluorescence throughout the cell. These distinct phases reveal a natural progression of Gag trafficking during the viral gene expression program. HIV Gag-iGFP is a useful tool for dissecting mechanisms of viral assembly and transmission.

scholarly journals Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1

2007 ◽  
Vol 81 (11) ◽  
pp. 5547-5560 ◽  
Author(s):  
Clare Jolly ◽  
Ivonne Mitar ◽  
Quentin J. Sattentau
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
Immunodeficiency Virus ◽  
Fixed Cell ◽  
Cell Spread ◽  
Hiv 1 ◽  
Cell Cell

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection of CD4+ T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

scholarly journals Immunoglobulin G3 from Polyclonal Human Immunodeficiency Virus (HIV) Immune Globulin Is More Potent than Other Subclasses in Neutralizing HIV Type 1

2001 ◽  
Vol 75 (14) ◽  
pp. 6558-6565 ◽  
Author(s):  
Orit Scharf ◽  
Hana Golding ◽  
Lisa R. King ◽  
Nancy Eller ◽  
Doug Frazier ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Immune Globulin ◽  
High Titer ◽  
Passive Immunization ◽  
Hinge Region ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fab Fragments ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab′)2 fragments or with papain to generate Fab fragments. IgG3 F(ab′)2 fragments were still more efficient in neutralization than F(ab′)2 of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.

scholarly journals Alpha-enolase in viral target cells suppresses the human immunodeficiency virus type 1 integration

Retrovirology ◽  
2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Naoki Kishimoto ◽  
Kengo Yamamoto ◽  
Nozomi Iga ◽  
Chie Kirihara ◽  
Towa Abe ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Early Phase ◽  
Viral Integration ◽  
Target Cells ◽  
Viral Infectivity ◽  
Immunodeficiency Virus ◽  
Viral Target ◽  
Hiv 1 ◽  
Viral Producer

Abstract Background A protein exhibiting more than one biochemical function is termed a moonlighting protein. Glycolytic enzymes are typical moonlighting proteins, and these enzymes control the infection of various viruses. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and alpha-enolase (ENO1) are incorporated into human immunodeficiency virus type 1 (HIV-1) particles from viral producer cells and suppress viral reverse transcription independently each other. However, it remains unclear whether these proteins expressed in viral target cells affect the early phase of HIV-1 replication. Results Here we show that the GAPDH expression level in viral target cells does not affect the early phase of HIV-1 replication, but ENO1 has a capacity to suppress viral integration in viral target cells. In contrast to GAPDH, suppression of ENO1 expression by RNA interference in the target cells increased viral infectivity, but had no effect on the expression levels of the HIV-1 receptors CD4, CCR5 and CXCR4 and on the level of HIV-1 entry. Quantitative analysis of HIV-1 reverse transcription products showed that the number of copies of the late products (R/gag) and two-long-terminal-repeat circular forms of viral cDNAs did not change but that of the integrated (Alu-gag) form increased. In contrast, overexpression of ENO1 in viral target cells decreased viral infectivity owing to the low viral integration efficiency. Results of subcellular fractionation experiments suggest that the HIV integration at the nucleus was negatively regulated by ENO1 localized in the nucleus. In addition, the overexpression of ENO1 in both viral producer cells and target cells most markedly suppressed the viral replication. Conclusions These results indicate that ENO1 in the viral target cells prevents HIV-1 integration. Importantly, ENO1, but not GAPDH, has the bifunctional inhibitory activity against HIV-1 replication. The results provide and new insights into the function of ENO1 as a moonlighting protein in HIV-1 infection.

scholarly journals Human Immunodeficiency Virus Env-Independent Infection of Human CD4− Cells

2000 ◽  
Vol 74 (23) ◽  
pp. 10994-11000 ◽  
Author(s):  
Shen Pang ◽  
Duan Yu ◽  
Dong Sung An ◽  
Gayle C. Baldwin ◽  
Yiming Xie ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Alternative Pathway ◽  
Neutralizing Antibody ◽  
Green Fluorescent Protein Gene ◽  
Immunodeficiency Virus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT CD4− epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4− human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4− cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development.

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

2008 ◽  
Vol 82 (20) ◽  
pp. 9937-9950 ◽  
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.

scholarly journals Genome-Wide Analysis of Chromosomal Features Repressing Human Immunodeficiency Virus Transcription

2005 ◽  
Vol 79 (11) ◽  
pp. 6610-6619 ◽  
Author(s):  
M. K. Lewinski ◽  
D. Bisgrove ◽  
P. Shinn ◽  
H. Chen ◽  
C. Hoffmann ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Fluorescent Protein ◽  
Centromeric Heterochromatin ◽  
Regulatory Sequences ◽  
Hiv Transcription ◽  
Genome Wide ◽  
A Genome ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
In Cells

ABSTRACT We have investigated regulatory sequences in noncoding human DNA that are associated with repression of an integrated human immunodeficiency virus type 1 (HIV-1) promoter. HIV-1 integration results in the formation of precise and homogeneous junctions between viral and host DNA, but integration takes place at many locations. Thus, the variation in HIV-1 gene expression at different integration sites reports the activity of regulatory sequences at nearby chromosomal positions. Negative regulation of HIV transcription is of particular interest because of its association with maintaining HIV in a latent state in cells from infected patients. To identify chromosomal regulators of HIV transcription, we infected Jurkat T cells with an HIV-based vector transducing green fluorescent protein (GFP) and separated cells into populations containing well-expressed (GFP-positive) or poorly expressed (GFP-negative) proviruses. We then determined the chromosomal locations of the two classes by sequencing 971 junctions between viral and cellular DNA. Possible effects of endogenous cellular transcription were characterized by transcriptional profiling. Low-level GFP expression correlated with integration in (i) gene deserts, (ii) centromeric heterochromatin, and (iii) very highly expressed cellular genes. These data provide a genome-wide picture of chromosomal features that repress transcription and suggest models for transcriptional latency in cells from HIV-infected patients.

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