Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion.
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We have examined mutations in the ectodomain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using the recently developed three-color assay (I. Munoz-Barroso, S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal, J. Cell Biol. 140:315-323, 1998), we have assessed the ability of the mutant gp41s to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. The results were compared with the syncytium-inducing capabilities of these gp41 mutants. Two mutant proteins were incapable of mediating both dye transfer and syncytium formation. Two mutant proteins mediated dye transfer but were less effective at inducing syncytium formation than was wild-type gp41. The most interesting mutant proteins were those that were not capable of inducing syncytium formation but still mediated dye transfer, indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. Fusion mediated by the mutant gp41s was inhibited by the peptides DP178 and C34. (+info)
Reappearance of founder virus sequence in human immunodeficiency virus type 1-infected patients.
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Different patterns of temporal evolution in human immunodeficiency virus type 1 V3 and p17 regions are described for eight patients studied during the first years following primary infection. In samples from three patients, a rapid replacement of the major sequence occurred but the original sequence reappeared later simultaneously with clinical deterioration and increased plasma viral load. (+info)
A mechanism of resistance to HIV-1 entry: inefficient interactions of CXCR4 with CD4 and gp120 in macrophages.
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To test the hypothesis that inefficient interactions of CXCR4 with CD4 and gp120 could affect HIV-1 entry, we incubated macrophages, monocytes, and lymphocytes with gp120 and coimmunoprecipitated CD4 by using anti-CXCR4 antibodies. CD4 was efficiently coimmunoprecipitated in lymphocytes and monocytes but not in macrophages. Overexpression of CD4 in macrophages resulted in detection of CD4-CXCR4 and gp120-CD4-CXCR4 complexes in parallel with the restoration of macrophage fusion susceptibility. These results suggest a mechanism of resistance to entry of some X4 HIV-1 strains into macrophages and a method for dissection of the initial stages of HIV entry. (+info)
High diversity of HIV-1 subtype F strains in Central Africa.
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In this paper, we studied the variability of HIV-1 subtype F strains in Africa. For 11 viruses, mainly of Central African origin, different parts of the genome were genetically characterized. For all strains the V3-V5 region of the envelope gene was sequenced, and for 7 strains, the entire envelope gene was studied. For 10 strains, the p24 region of the gag gene was also sequenced. For each region studied, three subgroups in the F subtype were identified, F1, F2, and F3. These three subgroups were supported by high bootstrap values and the intra- and inter-subgroup F distances were comparable to those obtained for the known subtypes A, B, C, D, E, G, and H. In subgroup F1, some African strains clustered with previously described strains from Brazil and Romania, suggesting an African origin of the HIV-1 epidemic in these countries. A more detailed analysis of the gag and the envelope sequences allowed the identification of four recombinant viruses. Our data show a high diversity among subtype F strains, suggesting the presence of new subtypes in the regions studied. If biological differences exist among subtypes, it is important that these subtypes be well defined. The data from our study show that there is a need to clearly identify the different subgroups within the F subtype. (+info)
Constitutive cell surface association between CD4 and CCR5.
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HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor. For most strains of HIV, this coreceptor is CCR5. Here, we provide evidence that CD4 is specifically associated with CCR5 in the absence of gp120 or any other receptor-specific ligand. The amount of CD4 coimmunoprecipitated with CCR5 was significantly higher than that with the other major HIV coreceptor, CXCR4, and in contrast to CXCR4 the CD4-CCR5 coimmunoprecipitation was not significantly increased by gp120. The CD4-CCR5 interaction probably takes place via the second extracellular loop of CCR5 and the first two domains of CD4. It can be inhibited by CCR5- and CD4-specific antibodies that interfere with HIV-1 infection, indicating a possible role in virus entry. These findings suggest a possible pathway of HIV-1 evolution and development of immunopathogenicity, a potential new target for antiretroviral drugs and a tool for development of vaccines based on Env-CD4-CCR5 complexes. The constitutive association of a seven-transmembrane-domain G protein-coupled receptor with another receptor also indicates new possibilities for cross-talk between cell surface receptors. (+info)
HIV and SIV gp120 binding does not predict coreceptor function.
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Interaction of HIV and SIV Envelope (Env) proteins with viral coreceptors is a critical step in viral entry. By using a sensitive and specific gp120 binding assay, we have identified a discordance between the ability of a coreceptor to support Env-mediated membrane fusion and high-affinity binding of gp120. Direct binding of gp120 from the dual-tropic HIV-1 strain 89.6 was not detectable for any coreceptor that it uses for fusion, while detectable binding of gp120s from the R5 HIV-1 strains JRFL and CM235 and the SIV strain 239 was not measurable for many CCR5 chimeras and mutants that function efficiently as viral coreceptors. In comparison, binding of chemokines to these same mutants was highly predictive of their ability to signal. Thus, gp120 is more sensitive than chemokines to perturbations of CCR5 structure. We conclude that while chemokine binding to CCR5 is a good predictor of chemokine receptor function, gp120 binding does not always predict coreceptor function. (+info)
Expression and detection of macrophage-tropic HIV-1 gp120 in the brain using conformation-dependent antibodies.
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HIV-1 envelope proteins gp120 and gp41 are likely to play a role in the pathogenesis of HIV-associated neurocognitive disorders. While detection of gp120 in HIV-infected cell cultures is easy, it has not yet been possible to identify gp120 in human or animal brains in situ. The difficulty in detecting gp120 could be due to low expression levels of the protein, to the shedding of gp120 from infected macrophages/microglia, or to the use of inappropriate gp-specific antibodies. We addressed these questions by analyzing the subcellular localization, oligomeric structure, and shedding behavior of gp120 from a macrophage-tropic, CCR5-dependent primary isolate, BX08, expressed by a Semliki Forest virus replicon (SFVenvBX08) in vitro. We used the same SFV system injected in vivo into the rat brain in an attempt to detect gp120 in situ. Our results show that gp120/41 is expressed as monomers, dimers, and trimers in cell culture. Immunocytochemical analysis revealed that intracytoplasmic gp120 can be recognized by an anti-V3 antibody, whereas gp120 at the plasma membrane is detected exclusively by a conformation-dependent antibody. In the rat brain, the SFV vector allows gene expression in neurons from day 3 to day 9 after injection without any apparent brain damage nor reactive astrogliosis. In SFVenvBX08-infected neurons only conformation-dependent antibodies allowed gp120 labeling. These results suggest that previous difficulties in detecting gp120 in brain tissues may be due to the use of antibodies which were unable to recognize gp120 at the plasma membrane. (+info)
Changes in and discrepancies between cell tropisms and coreceptor uses of human immunodeficiency virus type 1 induced by single point mutations at the V3 tip of the env protein.
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We examined the effect of amino acid substitutions of the GPGR (glycine-proline-glycine-arginine) tip sequence at the V3 domain of the Env protein of human immunodeficiency virus type 1 (HIV-1) on its cell tropism and coreceptor use. We changed the GPGR sequence of a T-cell line (T)- and macrophage (M)-tropic (R5-R3-X4) HIV-1 strain, GUN-1wt, to GA(alanine)GR (the resulting mutant was designated GUN-1/A), GL(leucine)GR (GUN-1/L), GP(proline)GR (GUN-1/P), GR(arginine)GR (GUN-1/R), GS(serine)GR (GUN-1/S), or GT(threonine)GR (GUN-1/T). GUN-1/A, GUN-1/S, and GUN-1/T mutants infected brain-derived cells such as a CD4-transduced glioma cell line, U87/CD4, and a brain-derived primary cell strain, BT-20/N, as well as T-cell lines in a CD4-dependent manner, although the plating of these mutants onto macrophages was inhibited. GUN-1/L, GUN-1/P, and GUN-1/R mutants showed both T- and M-tropism, but did not plate onto the brain-derived cells. A CCR3, CCR5, CCR8, or CXCR4 gene was introduced into a CD4-positive glioma cell line, NP-2/CD4, which demonstrated complete resistance to various HIV-1 strains. Not only HIV-1 strains, which were infectious to macrophages, such as GUN-1wt, GUN-1v, GUN-1/L, and GUN-1/P, but also an HIV-1 strain, GUN-1v, which was hardly infectious to macrophages, grew well in NP-2/CD4 cells expressing CCR3 or CCR5. However, the M-tropic GUN-1/R mutant could not efficiently use CCR5 nor CCR3. No point mutants, except GUN-1/L, grew well in NP-2/CD4 cells expressing CCR8. These findings indicate that the cell tropism of HIV-1 to macrophages and brain-derived cells and their use of the coreceptors were markedly, though not always concomitantly, affected by the tip sequence of the V3 domain. (+info)