scholarly journals Human immunodeficiency virus type 1 membrane fusion mediated by a laboratory-adapted strain and a primary isolate analyzed by resonance energy transfer.

1996 ◽  
Vol 70 (9) ◽  
pp. 6437-6441 ◽  
Author(s):  
V Litwin ◽  
K A Nagashima ◽  
A M Ryder ◽  
C H Chang ◽  
J M Carver ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Energy Transfer ◽  
Membrane Fusion ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Primary Isolate ◽  
Immunodeficiency Virus

scholarly journals Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

2009 ◽  
Vol 83 (14) ◽  
pp. 7322-7336 ◽  
Author(s):  
Ian B. Hogue ◽  
Adam Hoppe ◽  
Akira Ono
Keyword(s):  
Human Immunodeficiency Virus ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Membrane Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 structural polyprotein Pr55Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.

scholarly journals Detection of Human Immunodeficiency Virus Type 1 Nef and CD4 Physical Interaction in Living Human Cells by Using Bioluminescence Resonance Energy Transfer

2005 ◽  
Vol 79 (13) ◽  
pp. 8629-8636 ◽  
Author(s):  
David Cluet ◽  
Christophe Bertsch ◽  
Christian Beyer ◽  
Liliane Gloeckler ◽  
Mathieu Erhardt ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Energy Transfer ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Human Cells ◽  
Bioluminescence Resonance Energy Transfer ◽  
Immunodeficiency Virus

ABSTRACT CD4 down-regulation by human immunodeficiency virus type 1 (HIV-1) Nef protein is a key function for virus virulence. This activity may be mediated by a direct Nef-CD4 interaction. We investigated the formation, in situ, of such a complex between proteins using bioluminescence resonance energy transfer technology and coimmunoprecipitations. Our data clearly demonstrate that Nef and CD4 interact in intact human cells. Moreover, our results clearly indicate that the dileucine motif of the CD4 cytoplasmic domain, critical for the Nef-induced CD4 down-regulation, is not implicated in the Nef/CD4 complex formation in the cellular context.

scholarly journals Contrasting Use of CCR5 Structural Determinants by R5 and R5X4 Variants within a Human Immunodeficiency Virus Type 1 Primary Isolate Quasispecies

2003 ◽  
Vol 77 (22) ◽  
pp. 12057-12066 ◽  
Author(s):  
Yanjie Yi ◽  
Anjali Singh ◽  
Farida Shaheen ◽  
Andrew Louden ◽  
ChuHee Lee ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Terminal Region ◽  
Primary Isolate ◽  
Structural Basis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Macrophagetropic R5 human immunodeficiency virus type 1 (HIV-1) isolates often evolve into dualtropic R5X4 variants during disease progression. The structural basis for CCR5 coreceptor function has been studied in a limited number of prototype strains and suggests that R5 and R5X4 Envs interact differently with CCR5. However, differences between unrelated viruses may reflect strain-specific factors and do not necessarily represent changes resulting from R5 to R5X4 evolution of a virus in vivo. Here we addressed CCR5 domains involved in fusion for a large set of closely related yet functionally distinct variants within a primary isolate swarm, employing R5 and R5X4 Envs derived from the HIV-1 89.6PI quasispecies. R5 variants of 89.6PI could fuse using either N-terminal or extracellular loop CCR5 sequences in the context of CCR5/CXCR2 chimeras, similar to the unrelated R5 strain JRFL, but R5X4 variants of 89.6PI were highly dependent on the CCR5 N terminus. Similarly, R5 89.6PI variants and isolate JRFL tolerated N-terminal CCR5 deletions, but fusion by most R5X4 variants was markedly impaired. R5 89.6PI Envs also tolerated multiple extracellular domain substitutions, while R5X4 variants did not. In contrast to CCR5 use, fusion by R5X4 variants of 89.6PI was largely independent of the CXCR4 N-terminal region. Thus, R5 and R5X4 species from a single swarm differ in how they interact with CCR5. These results suggest that R5 Envs possess a highly plastic capacity to interact with multiple CCR5 regions and support the concept that viral evolution in vivo results from the emergence of R5X4 variants with the capacity to use the CXCR4 extracellular loops but demonstrate less-flexible interactions with CCR5 that are strongly dependent on the N-terminal region.

scholarly journals Role of Cholesterol in Human Immunodeficiency Virus Type 1 Envelope Protein-Mediated Fusion with Host Cells

2002 ◽  
Vol 76 (22) ◽  
pp. 11584-11595 ◽  
Author(s):  
Mathias Viard ◽  
Isabella Parolini ◽  
Massimo Sargiacomo ◽  
Katia Fecchi ◽  
Carlo Ramoni ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Membrane Fusion ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Cholesterol Depletion ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-β-cyclodextrin (MβCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1α- and macrophage inflammatory protein 1β-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MβCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.

scholarly journals Antibodies That Are Cross-Reactive for Human Immunodeficiency Virus Type 1 Clade A and Clade B V3 Domains Are Common in Patient Sera from Cameroon, but Their Neutralization Activity Is Usually Restricted by Epitope Masking

2005 ◽  
Vol 79 (2) ◽  
pp. 780-790 ◽  
Author(s):  
Chavdar Krachmarov ◽  
Abraham Pinter ◽  
William J. Honnen ◽  
Miroslaw K. Gorny ◽  
Phillipe N. Nyambi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fusion Proteins ◽  
Primary Isolate ◽  
Neutralization Activity ◽  
Neutralizing Activity ◽  
Clade B ◽  
Immunodeficiency Virus

ABSTRACT Sera from human immunodeficiency virus type 1 (HIV-1)-infected North American patients recognized a fusion protein expressing a V3 loop from a clade B primary isolate virus (JR-CSF) but not from a clade A primary isolate virus (92UG037.8), while most sera from Cameroonian patients recognized both fusion proteins. Competition studies of consensus V3 peptides demonstrated that the majority of the cross-reactive Cameroonian sera contained cross-reactive antibodies that reacted strongly with both V3 sequences. V3-specific antibodies purified from all six cross-reactive sera examined had potent neutralizing activity for virus pseudotyped with envelope proteins (Env) from SF162, a neutralization-sensitive clade B primary isolate. For four of these samples, neutralization of SF162 pseudotypes was blocked by both the clade A and clade B V3 fusion proteins, indicating that this activity was mediated by cross-reactive antibodies. In contrast, the V3-reactive antibodies from only one of these six sera had significant neutralizing activity against viruses pseudotyped with Envs from typically resistant clade B (JR-FL) or clade A (92UG037.8) primary isolates. However, the V3-reactive antibodies from these cross-reactive Cameroonian sera did neutralize virus pseudotyped with chimeric Envs containing the 92UG037.8 or JR-FL V3 sequence in Env backbones that did not express V1/V2 domain masking of V3 epitopes. These data indicated that Cameroonian sera frequently contain cross-clade reactive V3-directed antibodies and indicated that the typical inability of such antibodies to neutralize typical, resistant primary isolate Env pseudotypes was primarily due to indirect masking effects rather than to the absence of the target epitopes.

scholarly journals Regulation of Human Immunodeficiency Virus Type 1 Env-Mediated Membrane Fusion by Viral Protease Activity

2004 ◽  
Vol 78 (2) ◽  
pp. 1026-1031 ◽  
Author(s):  
Tsutomu Murakami ◽  
Sherimay Ablan ◽  
Eric O. Freed ◽  
Yuetsu Tanaka
Keyword(s):  
Human Immunodeficiency Virus ◽  
Membrane Fusion ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytoplasmic Tail ◽  
Target Cells ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR+) or inactive (PR−) viral PR. We observed that PR− virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.

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