Specific interaction of CXCR4 with CD4 and CD8α: Functional analysis of the CD4/CXCR4 interaction in the context of HIV-1 envelope glycoprotein-mediated membrane fusion

Abstract

We investigated possible interactions between HIV-1 receptor (CD4) and the main coreceptors CXCR4 and CCR5. We found that CD4 and CXCR4 coexpressed in 293T cells form a complex that can be immunoprecipitated with antibodies directed against the extracellular domain of either protein. Mutagenesis revealed that the CD4/CXCR4 interaction maps to two previously uncharacterized basic motifs in the cytoplasmic domain of CD4. HIV-1 envelope glycoprotein-mediated membrane fusion was found to be independent of the ability of CD4 and CXCR4 to interact, whether fusion was studied in a virus-cell or a cell-cell model. However, this interaction might explain the adaptation of HIV-1 to CXCR4 as an alternative to CCR5. We found that CXCR4 also interacts with the cytoplasmic domain of CD8alpha in a way that is similar to the CD4/CXCR4 interaction. The CD4/CXCR4 and CD8alpha/CXCR4 interactions may thus be involved in cellular signaling pathways shared by the CD4 and CD8alpha molecules.

Nef-Induced CD4 Endocytosis in Human Immunodeficiency Virus Type 1 Host Cells: Role of p56lckKinase

“…Recombinant viruses were obtained as described previously (4). Briefly, 293T cells were cotransfected by the calcium phosphate precipitation technique, with pVPack-GP, pVPack-VSV-G, and pMSCVhyg containing the appropriate CD4 mutant.…”

“…293T cells were cotransfected by the calcium phosphate precipitation technique, with pBR-NL43-env*nefϩvpu*-IRES-EGFP or pBR-NL43-env*nef-vpu*-IRES-EGFP and pHCMV-G to allow for vesicular stomatitis virus G glycoprotein (VSV-G) expression. Transfected cells were treated as described above, and supernatant were assayed for reverse transcriptase activity, as described elsewhere (4). A total of ϳ5 ϫ 10 5 A2.01 cells were transduced, with 200,000 reverse transcriptase counts at a final volume of 250 l, and cell surface CD4 expression was analyzed by FACS 48 h posttransduction.…”

“…Cell lysates were prepared as described above, and 20 g of proteins was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% acrylamide NuPAGE Novex bis-Tris precast gels (Invitrogen). When MG132 and NH 4 Cl treatments were performed, cells were transduced with the indicated viruses and treated 36 h later for a 6-hour period of time. Nef, CD4, p56 lck , GFP, and p24 were visualized by Western blotting, followed by immunodetection.…”

“…We next investigated the mechanisms responsible for Nef-induced CD4 degradation. Cells were treated with 100 mM NH 4 Cl in order to buffer lysosome acidification and to inhibit pH-dependent proteases or with 10 M MG132 in order to inhibit the proteasome-dependent degradation pathway. To check the efficiency of such treatments, cells were incubated with VSV-G-pseudotyped GFP reporter viruses in the presence of NH 4 Cl or MG132.…”

CD4 and CCR5 Constitutively Interact at the Plasma Membrane of Living Cells

“…Such interactions are likely to favor the association rate of gp120 with CCR5, recently proposed as being strictly dependent upon a close vicinity of CD4 and CCR5 in living cells (20). Furthermore, the observation that CD4 poorly co-immunoprecipitated with CXCR4 (19,21,22) supports the attractive hypothesis that preferential interactions between CCR5 and CD4 may be relevant to the predominance of R5-tropic HIV isolates in the course of viral infection. Nevertheless, this possibility remains debated, as it was recently inferred using coprecipitation, high-resolution deconvolution microscopy and resonance energy transfer techniques, that CD4 and CCR5 do not exist in a stable complex at the cell membrane (23,24).…”