MGC, the result of fusion between infected and uninfected microglia and macrophages, are the signature neuropathological finding in HIVD and are the main reservoir for HIV within the CNS (27
). Given the evidence of genetic sequestration within the CNS provided by postmortem studies (23
), it is likely that a subpopulation of viruses replicates in microglia and adapts to them over an indeterminate period. While studies have documented the phenotype and to some extent the evolution of viruses in the cerebrospinal fluid (6
), because of the obvious sampling problem, few studies have looked at the functional evolution of virus in the CNS parenchyma, necessitating the design of in vitro studies.
We characterized a virus, HIV-1BORI-15, that forms extensive syncytia in microglia cultures and compared it to its parental primary isolate, HIV-1BORI. The syncytium-forming phenotype could not be explained by a simple coreceptor switch or by CD4-independent entry. Rather, the HIV-1BORI-15 envelope had a quantitatively greater ability to mediate fusion of several CD4+CCR5+ cell types. In the context of pIIIB backbone, envelope sequences from HIV-1BORI-15 (VH-B15) mediated a high level of syncytium formation in microglia in comparison with an equivalent construct with the HIV-1BORI env (VH-BORI), whereas both viruses replicated equivalently in PBMCs. Furthermore, VH-B15 infected U373-MAGI-CCR5E cells with greater efficiency than VH-BORI, and pseudotypes incorporating the HIV-1BORI-15 env constructs were less sensitive to decreases in the amount of CD4 on the cell surface when CCR5 levels were held constant.
Surprisingly, VH-B15-mediated fusion was independent of viral replication, since it occurred within 24 h of exposure to the cells and was not decreased by reverse transcriptase inhibitors, indicating FFWO. However, the timing of fusion with the uncloned HIV-1BORI-15
, which occurred at 2 to 3 weeks after infection, did not suggest FFWO. FFWO, or fusion mediated by viral particles in the absence of infection, is a well-known phenomenon in other enveloped viruses with high fusion potential (2
) and has previously been described in HIV under certain circumstances (14
). We hypothesize that the high fusion activity mediated by VH-B15 is due to the intrinsically greater fusogenicity of the HIV-1BORI-15 env
. It is unlikely that the gp41 sequences from HXB-3 present in the VH-based recombinants played a major role in this fusion, since VH-BORI did not demonstrate any significant syncytium formation. We also found equivalent envelope expression among the recombinant viruses (data not shown). In any case, the syncytium-forming phenotype was clearly mapped to four amino acids in the V1/V2 region, with the bulk of the VH-B15 fusogenicity accounted for by a single-amino-acid difference, E153G. Surprisingly, the loss of two potential glycosylation sites in the same region, while perhaps associated with changes in neutralization (data not shown), had no major effect on syncytium formation when introduced independently. Moreover, the full phenotype depended on all four amino acid differences between VH-BORI and VH-B15, indicating that it is probably related to the overall conformation of the region.
Our results support the idea that envelope sequences play a major role in HIV-1 tropism for microglia, although it is probably not the exclusive determinant. Since HIV-1 isolates obtained from the CNS are predominantly R5 (i.e., use CCR5 as coreceptor), once a mechanism for its enhanced syncytium formation has been defined, the experiments with HIV-1BORI-15 will provide information regarding the interactions between HIV and these specialized cells. Specifically, there may be requirements for interaction with CD4 and CCR5 at the ratio present in microglial cells.
We believe that there are different interactions between the HIV-1BORI
and the HIV-1BORI-15
envelopes and CD4 or CCR5 and that the V1/V2 loops are critical to this difference. Although the effects of V3 on tropism, particularly in determining coreceptor use, have received the widest attention (10
), some investigators have found that sequences in V1/V2 can influence virus spread in MDM (64
). There is also an extensive literature indicating that these loops are involved in neutralization (7
) and other cellular tropisms (5
). In the current model of HIV entry, the V1/V2 loops are thought to shield the coreceptor binding site (73
). CD4 binding probably induces a conformational change involving the V1/V2 loops that exposes a conserved coreceptor binding site (53
). This conformational change is detectable through increased binding of some antibodies, like 17b (61
How could the HIV-1BORI-15
envelope influence this interaction? We can propose several potential scenarios. Firstly, the V1/V2 loops of HIV-1BORI-15
gp120 may favor the conformation triggered by CD4 binding, increasing the exposure of the chemokine receptor-binding site. On a membrane with few CD4 molecules, like those of microglia (17
), this more stable conformation may be necessary to promote a gp120-CCR5 interaction. Along the same lines, the interaction between HIV-1BORI-15
gp120 and CD4 could be stronger, resulting in a similar outcome. Indeed, HIV-1 strains may have different affinities for the CD4-coreceptor complex and demonstrate variations in infectivity of cells with different amounts of receptor and coreceptor (39
). Alternatively the HIV-1BORI-15
gp120 could interact with CCR5 more efficiently, with the more basic V1/V2 region of the HIV-1BORI-15
gp120 (in comparison with HIV-1BORI
) facilitating gp120 interaction with the acidic residues in the CCR5 amino terminus (19
). Interaction with different extracellular domains of CCR5 or different conformational states of CCR5 may also play a role (3
). Future studies with purified preparations of HIV-1BORI-15
gp120 should determine which of these possibilities is most relevant to its phenotype. If the results are generalized to other HIV strains, these findings could provide mechanistic information regarding the development of syncytia in this area of HIV pathogenesis. Indeed a recent report focused on the involvement of V1/V2 regions in HIVD (50
We were surprised that, when placed in the context of the pIIIB backbone, the HIV-1BORI-15 env did not produce a virus with high replication in microglia, in comparison with VH-BORI. This may indicate that other regions of the HIV-1BORI-15 virus are involved in its neurotropism. Alternatively, the high fusogenicity exhibited by its envelope could have affected p24gag release or viral spread. Defining the mechanism of the enhanced replication will be an area for future experimentation.
Finally, the neutralization pattern of HIV-1BORI-15
may provide additional evidence of the importance of the conformation of env
in generating an effective immune response. Preliminary studies with these viruses showed that HIV-1BORI-15
may be easier to neutralize than its parent. In light of recent findings that fusogenic intermediates of env
may generate broadly cross-reactive antibody responses (40
) and that a CD4-independent HIV-1 stably exposing its coreceptor binding site is more easily neutralized (31
), this isolate may be a candidate for a better understanding of the potential role of V1/V2 in immunity.