Coreceptor usage of HIV-1 after deletion of Gly317–Pro318–Gly319 motif in the gp120 V3 loop

The V3 loop of HIV-1 gp120 is required for viral entry into the cell via membrane fusion, and is believed to interact with cell surface chemokine receptors on T cells and macrophages, although other regions in gp120 also play roles in this process [1–3] In spite of the hypervariability of the V3 loop, the three amino acids Gly317–Pro318–Gly319 (GPG) at the crown of the loop are highly conserved [4] The reason for the conservation of this GPG motif is not fully understood. It has been suggested that deletion of the GPG motif might be lethal for viral infectivity because the expression of recombinant gp160 having such a deletion did not induce syncytia in CEM cells [5] We constructed a GPG-deleted provirus based on the infectious clone pBRu-2. Successful infection and replication of GPG-deleted virus (clone mp8 and mp10) was detected in MT-2 cells, although the mutant virus showed lower infectivity. Infection could also be observed in the C8166, C91-PL, Molt-3 and THP-1 CD4 cell lines, and peripheral blood mononuclear cell (PBMC)-derived dendritic cells, but not in CEM-SS, HUT78, H9, Jurkat, U937 CD4 cell lines or in PBMC. The mutant virus could also induce syncytia and apoptosis to a similar level as did the wild-type (WT) virus (to be published elsewhere). Here we extend the studies of the biology of this GPG-deleted virus as to its coreceptor usage. In the present study, the coreceptor usage analysis was performed on two coreceptor indicator cell lines, the human glioma cell line U87 and the human osteosarcoma cell line HOS, which both co-express CD4 cells and one of the HIV coreceptors. Virus production was determined by measuring HIV-1 p24 antigen in the culture supernatants collected at days 4, 7, 11 and 14 post-infection by an in-house HIV-1 p24 enzyme-linked immunosorbent assay [6] Viral infection of both WT and the mutants was observed in U87–CD4–CXCR4 cells, together with the formation of syncytia in the infected cells. This indicated that entry of the mutant virus was CXCR4-dependent, as was the WT virus. No other chemokine receptors (CCR1, CCR2b, CCR3 or CCR5) could replace the function of CXCR4 for this entry (Table 1). In support of this finding, infection of the mutant virus was efficiently inhibited by 500 ng of the CXC chemokine SDF-1α (R&D Systems, UK), whereas 100 μg of the CC chemokine regulated upon activation: normal T cell expressed/secreted (RANTES) had no inhibitory effects at all in MT-2 cells (Table 2). These observations confirmed the usage of CXCR4 by the GPG-deleted virus for entry. However, none of the coreceptor-expressing HOS cell lines could support productive infection of the mutant virus, including the HOS-CD4-CXCR4 (Table 1). These results implied that the CD4 and CXCR4 molecules were not enough to support the mutant virus entry. Additional cellular surface molecule(s) would thus seem to be involved in this step.Table 1: Infection of the coreceptor indicator cell lines by wild type and the mutant viruses. Table 2: Chemokines inhibition assay on MT-2 cells. The post-CD4 cell binding events of HIV-1 entry remain elusive. It is not known whether the conserved sequence or structure in the V3 loop interacts directly with chemokine receptors or if it is conserved for other interactions between gp120 and cellular molecules. It has been implied that multiple interactions may occur along a diffuse surface between envelope glycoprotein and coreceptors [7,8] Post-CD4 cell interactions are driven both by conformational changes induced within the envelope protein complex upon CD4 cell binding and by proximity on the membrane, but ultimately involve multiple weak contacts over a variable surface of envelope-coreceptor interaction [8] Our observations that the GPG-motif may not be necessary for a successful entry suggest that the relationship between the conserved structural requirements of the V3 loop and the coreceptor CXCR4 might be relatively relaxed. If the envelope–coreceptor interaction is diffuse, other cellular factor(s) might get into contact with gp120. Therefore, a successful entry might be determined by the sum of multiple interactions between envelope glycoprotein and the host cell surface, and the effects of specific changes in gp120 on overall binding may not always be readily predictable. The ability of a coreceptor, in conjugation with CD4 cells, to support HIV-1 entry may vary depending on the cellular context in which it is expressed. Therefore, here the entry by the mutant virus appeared to be cell-dependent irrespective of the expression of both CD4 and CXCR4 molecules. One other possible interpretation of the results presented here is that the GPG motif might be involved in the interaction with additional cell surface molecule(s) of MT-2 cells, which may affect CXCR4, and therefore the deletion of the GPG motif increased the inhibitory effects of SDF-1α. Jin Su Anders Vahlne

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