Electrostatic Interactions Drive Membrane Association of the Human Immunodeficiency Virus Type 1 Gag MA Domain

ABSTRACT The assembly of most retroviruses occurs at the plasma membrane. Membrane association is directed by MA, the N-terminal domain of the Gag structural protein. For human immunodeficiency virus type 1 (HIV-1), this association is mediated in part by a myristate fatty acid modification. Conflicting evidence has been presented on the relative importance of myristoylation, of ionic interactions between protein and membrane, and of Gag multimerization in membrane association in vivo. We addressed these questions biochemically by determining the affinity of purified myristoylated HIV-1 MA for liposomes of defined composition, both for monomeric and for dimeric forms of the protein. Myristoylation increases the barely detectable intrinsic affinity of the apo-protein for liposomes by only 10-fold, and the resulting affinity is still weak, similar to that of the naturally nonmyristoylated MA of Rous sarcoma virus. Membrane binding of HIV-1 MA is absolutely dependent on the presence of negatively charged lipid and is abrogated at high ionic strength. Forced dimerization of MA increases its membrane affinity by several orders of magnitude. When green fluorescent protein fusions of monomeric or dimeric MA are expressed in cells, the dimeric but not the monomeric protein becomes strongly membrane associated. Computational modeling supports these results and suggests a molecular mechanism for the modest effect of myristoylation on binding, wherein the membrane provides a hydrophobic environment for the myristate that is energetically similar to that provided by the protein. Overall, the results imply that the driving force for membrane association stems largely from ionic interactions between multimerized Gag and negatively charged phospholipids.

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Genetic Recombination between Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2, Two Distinct Human Lentiviruses

Journal of Virology ◽

10.1128/jvi.01937-07 ◽

2007 ◽

Vol 82 (4) ◽

pp. 1923-1933 ◽

Cited By ~ 25

Author(s):

Kazushi Motomura ◽

Jianbo Chen ◽

Wei-Shau Hu

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Fluorescent Protein ◽

Genetic Recombination ◽

Green Fluorescent Protein Gene ◽

Low Frequencies ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are genetically distinct viruses that each can cause AIDS. Approximately 1 million people are infected with both HIV-1 and HIV-2. Additionally, these two viruses use the same receptor and coreceptors and can therefore infect the same target cell populations. To explore potential genetic interactions, we first examined whether RNAs from HIV-1 and HIV-2 can be copackaged into the same virion. We used modified near-full-length viruses that each contained a green fluorescent protein gene (gfp) with a different inactivating mutation. Thus, a functional gfp could be reconstituted via recombination, which was used to detect the copackaging of HIV-1 and HIV-2 RNAs. The GFP-positive (GFP+) phenotype was detected in approximately 0.2% of the infection events, which was 35-fold lower than the intrasubtype HIV-1 rates. We isolated and characterized 54 GFP+ single-cell clones and determined that all of them contained proviruses with reconstituted gfp. We then mapped the general structures of the recombinant viruses and characterized the recombination junctions by DNA sequencing. We observed several different recombination patterns, including those that had crossovers only in gfp. The most common hybrid genomes had heterologous long terminal repeats. Although infrequent, crossovers in the viral sequences were also identified. Taken together, our study demonstrates that HIV-1 and HIV-2 can recombine, albeit at low frequencies. These observations indicate that multiple factors are likely to restrict the generation of viable hybrid HIV-1 and HIV-2 viruses. However, considering the large coinfected human population and the high viral load in patients, these rare events could provide the basis for the generation of novel human immunodeficiency viruses.

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Macrophages in Vaginal but Not Intestinal Mucosa Are Monocyte-Like and Permissive to Human Immunodeficiency Virus Type 1 Infection

Journal of Virology ◽

10.1128/jvi.01796-08 ◽

2009 ◽

Vol 83 (7) ◽

pp. 3258-3267 ◽

Cited By ~ 121

Author(s):

Ruizhong Shen ◽

Holly E. Richter ◽

Ronald H. Clements ◽

Lea Novak ◽

Kayci Huff ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Fluorescent Protein ◽

Female Genital Tract ◽

Vaginal Mucosa ◽

Innate Response ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Mucosal surfaces play a major role in human immunodeficiency virus type 1 (HIV-1) transmission and pathogenesis, and yet the role of lamina propria macrophages in mucosal HIV-1 infection has received little investigative attention. We report here that vaginal and intestinal macrophages display distinct phenotype and HIV-1 permissiveness profiles. Vaginal macrophages expressed the innate response receptors CD14, CD89, CD16, CD32, and CD64 and the HIV-1 receptor/coreceptors CD4, CCR5, and CXCR4, similar to monocytes. Consistent with this phenotype, green fluorescent protein-tagged R5 HIV-1 entered macrophages in explanted vaginal mucosa as early as 30 min after inoculation of virus onto the epithelium, and purified vaginal macrophages supported substantial levels of HIV-1 replication by a panel of highly macrophage-tropic R5 viruses. In sharp contrast, intestinal macrophages expressed no detectable, or very low levels of, innate response receptors and HIV-1 receptor/coreceptors and did not support HIV-1 replication, although virus occasionally entered macrophages in intestinal tissue explants. Thus, vaginal, but not intestinal, macrophages are monocyte-like and permissive to R5 HIV-1 after the virus has translocated across the epithelium. These findings suggest that genital and gut macrophages have different roles in mucosal HIV-1 pathogenesis and that vaginal macrophages play a previously underappreciated but potentially important role in mucosal HIV-1 infection in the female genital tract.

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Virological Consequences of Early Events following Cell-Cell Contact between Human Immunodeficiency Virus Type 1-Infected and Uninfected CD4+ Cells

Journal of Virology ◽

10.1128/jvi.00695-08 ◽

2008 ◽

Vol 82 (16) ◽

pp. 7773-7789 ◽

Cited By ~ 26

Author(s):

Eliana Ruggiero ◽

Roberta Bona ◽

Claudia Muratori ◽

Maurizio Federico

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Fluorescent Protein ◽

Cell Contact ◽

Target Cells ◽

Immunodeficiency Virus ◽

Virus Expression ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected cells transmit viral products to uninfected CD4+ cells very rapidly. However, the natures of the transmitted viral products and the mechanism of transmission, as well as the relative virological consequences, have not yet been fully clarified. We studied the virological events occurring a few hours after contact between HIV-1-infected and uninfected CD4+ cells using a coculture cell system in which the virus expression in target cells could be monitored through the induction of a green fluorescent protein reporter gene driven by HIV-1 long terminal repeats. Within 16 h of coculture, we observed two phenomena not related to the cell-free virus infection, i.e., the formation of donor-target cell fusions and a fusion-independent internalization of viral particles likely occurring at least in part through intercellular connections. Both events depended on the expression of Env and CD4 in donor and target cells, respectively, whereas the HIV-1 internalization required clathrin activity in target cells. Importantly, both phenomena were also observed in cocultures of primary CD4+ lymphocytes, while primary macrophages supported only HIV-1 endocytosis. By investigating the virological consequences of these events, we noticed that while fused cells released infectious HIV-1 particles, albeit with reduced efficiency compared with donor cells, no virus expression was detectable upon HIV-1 endocytosis in target cells. In sum, the HIV-1 transmission following contact between an HIV-1-infected and an uninfected CD4+ cell can occur through different mechanisms, leading to distinguishable virological outcomes.

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Cell-Based Fluorescence Assay for Human Immunodeficiency Virus Type 1 Protease Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.9.2616-2622.2001 ◽

2001 ◽

Vol 45 (9) ◽

pp. 2616-2622 ◽

Cited By ~ 27

Author(s):

Kristina Lindsten ◽

Tat'ána Uhlı́ková ◽

Jan Konvalinka ◽

Maria G. Masucci ◽

Nico P. Dantuma

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Fluorescent Protein ◽

Autocatalytic Cleavage ◽

Immunodeficiency Virus ◽

Green Fluorescent ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells.

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Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein

Journal of General Virology ◽

10.1099/vir.0.81200-0 ◽

2006 ◽

Vol 87 (3) ◽

pp. 563-571 ◽

Cited By ~ 73

Author(s):

Matthew Bentham ◽

Sabine Mazaleyrat ◽

Mark Harris

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Electrostatic Interactions ◽

Membrane Association ◽

Subcellular Targeting ◽

Immunodeficiency Virus ◽

And Function

Human immunodeficiency virus type 1 Nef protein is N-terminally myristoylated, a modification reported to be required for the association of Nef with cytoplasmic membranes. As myristate alone is not sufficient to anchor a protein stably into a membrane, it has been suggested that N-terminal basic residues contribute to Nef membrane association via electrostatic interactions with acidic phospholipids. Here, data are presented pertaining to the role of the myristate and basic residues in Nef membrane association, subcellular localization and function. Firstly, by using a biochemical assay for membrane association it was shown that, whereas myristoylation of Nef was not essential, mutation of a cluster of four arginines between residues 17 and 22 reduced membrane association dramatically. Mutation of two lysines at residues 4 and 7 had negligible effect alone, but when combined with the arginine substitutions, abrogated membrane association completely. By using indirect immunofluorescence, it was demonstrated that mutation of either of the two basic clusters altered the subcellular distribution of Nef dramatically. Thirdly, the requirement of the arginine and lysine clusters for Nef-mediated CD4 downmodulation was shown to correlate precisely with membrane association. These data suggest that membrane localization and subcellular targeting of Nef are controlled by a complex interplay of signals at the N terminus of the protein.

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