HIV-1 entry is a multistep process that involves sequential interactions of HIV-1 gp120-gp41 with CD4 and chemokine receptors. The process is facilitated by receptor movement, and is influenced by plasma membrane organization and/or composition. In addition to these key players, plasma membrane lipids (GSLs/cholesterol) have been implicated in modulating HIV-1 entry. However, an absolute requirement of target membrane rafts and their components in HIV-1 infection has been challenged.
Our original hypothesis that GSLs per se play a role in HIV-1 entry/fusion [14
] was based on our studies with PPMP, which inhibits the synthesis of glucosyl ceramide, the precursor for GSL biosynthesis. We argued that a reduction of GSL levels by blocking their synthesis would adversely affect HIV-1 fusion. PPMP has a number of different effects on sphingolipid metabolism, which include an increase of ceramide levels. However, in our follow up studies, we reported that GSL-deficient cells (GM95), that were engineered to express HIV-1 receptors, were highly susceptible to HIV-1 Env-mediated fusion [29
]. These results raised questions about a direct involvement of GSLs in fusion of cells expressing high levels of HIV-1 receptors. We have also reported that enhancing plasma membrane ceramide levels by other means (fenretinide, sphingomyelinase) reduces the susceptibility of certain target cells to HIV-1 fusion [9
]. Therefore, it appears that the receptor disposition with it’s surrounding microenvironment (and not the specific lipid components) within the plane of the plasma membrane of a target cell is likely to dictate and/or facilitate fusion process.
Surprisingly, treatment of B16 cells with PPMP enhanced their fusion susceptibility to about the level of fusion susceptibility of NIH3T3 or GM95 cells expressing CD4 and coreceptors. We also noted that the levels of sphingomyelin in B16 cells were significantly increased following PPMP treatment [30
]. Since the relative amount of ceramide to sphingomyelin appears to be important for HIV receptor disposition and fusion susceptibility [9
], we conclude that the high levels of sphingomyelin produced in B16 cells following PPMP treatment will override the inhibitory effect (on fusion) of possible ceramide accumulation.
In this study we have probed the mechanism(s) underlying the block of fusion in B16 cells expressing HIV-1 receptors. First we examined a possible role of transmembrane/cytoplasmic anchor of HIV-1 Env, in observed restriction to fusion. The deletion of transmembrane anchors significantly increases the fusion mediated by HIV-1 envelope glycoprotein [2
]. Our observations that the mutant gp120-gp41 envelope also failed to support fusion, further supported the notion that block was due to inefficient interactions of HIV-1 envelope with the target membrane.
We results on lateral diffusion of CD4, lipids, and CCR5 in B16/GM95/NIH3T3 cells ( and ), show that CD4 diffusion in B16 cells is significantly slower when compared with GM95 or NIH3T3 cells. Since we did not observe such differences in lateral diffusion of lipids or the chemokine receptor, these data confirmed that fusion was restricted at the level of CD4-envelope interactions. Slower diffusion can prolong the formation of multi-protein complexes, as is the case with HIV-1 fusion machinery comprising of HIV-1 fusion protein CD4 and co-receptor.
The ability of RA5-expressing B16-CXCR4 cells to support gp120-gp41 mediated fusion () further suggested that the restriction was at the level of wt-CD4 disposition and/or the microenvironment in its vicinity. We found that distribution of wt-CD4 and RA5 mutant into raft and non-raft regions respectively was similar in B16 and GM95 cells. These observations led us to conclude that raft-localization of CD4 is not an essential factor for HIV-1 entry, consistent with previous reports. Interestingly, we observed significant differences in lateral diffusion of wt-CD4 and RA5 in B16 cells (). In contrast, diffusion of wt-CD4 and RA5 mutant were similar when expressed in GM95 cells () or NIH3T3 cells (data not shown). These data allow us to propose () that the failure of B16 cells expressing wt-CD4 result from restriction of CD4 lateral diffusion presumably as a result of clustering [9
CD4 clustering in B16 cells and susceptibility to HIV-1 fusion
Taken together, our results suggest that over-expression of GM3 in B16 cells enhances clustering of CD4. This restricts the formation of trimolecular complex formation between CD4-corecptor (CCR5/CXCR4)-viral Envelope glycoprotein and hence block in fusion, consistent with our earlier observations [30
]. In our previous report we achieved the fusion rescue by decreasing the levels of GM3. In the current report we have bypassed the restriction by moving CD4 away from the GM3 annulus. Further, when CD4 is allowed to leave the GM3 annulus as in CD4-RA5 construct, trimolecular complex is formed and fusion proceeds as normal. Hence restricting components of multi-protein reaction as is the case for HIV envelope mediated fusion can achieve results akin of inhibitor for coreceptor -CD4 interaction.