The present study is the first to describe the coreceptor usage of HIV-2 variants from individuals who had undetectable levels of viral RNA in their plasma and high and stable CD4 counts for prolonged periods. Of the eight chemokine receptors tested in GHOST cells, these HIV-2 variants used only CCR5, GPR15, and CXCR6 with high efficiency. Most HIV-2 variants isolated from individuals with progressive HIV-2 infection and/or viremia were restricted to the same chemokine receptors, while some variants from half of these individuals used CXCR4 instead. Use of CCR1, CCR2b, and CCR3 was rare.
Almost a quarter of all tested HIV-2 variants could infect the parental GHOST cells, albeit inefficiently. This could be partially explained by the presence of CXCR4, which is known to be endogenously expressed by HOS cells, from which GHOST cells are derived (11
). Every GHOST cell line contained a fraction of CXCR4-expressing cells, the magnitude of which correlated with the level of virus produced by X4-restricted HIV variants. Discrepancies exist in the literature regarding infection of parental GHOST cells by X4 HIV variants (8
). This may suggest that different batches of parental cell lines have different levels of CXCR4 expression and consequently different susceptibilities to X4 HIV infection. Other variants infected parental GHOST cells independently of CXCR4, as evidenced by their insensitivity to T22, relatively low capacity to infect GHOST-CXCR4 cells, and inability to infect CCR5−/−
PBMC. One can speculate that such variants are able to infect CD4-positive cells independently of a coreceptor. However, if it exists, coreceptor-independent infection occurs only under specific conditions, since these variants could not infect CCR5−/−
PBMC. Alternatively, GHOST cells may express a second, still unidentified HIV-2 coreceptor. If so, the low susceptibility of parental GHOST cells to only a minority of HIV-2 variants indicates either that this unidentified GHOST coreceptor is not a major HIV-2 coreceptor or that expression levels are low, allowing only variants with the highest affinity for the receptor to enter the cells. A candidate might be CXCR6, which is expressed by HOS cells (3
), but as we show here, at levels too low to be detectable by FACS analysis. However, the lack of an association between virus production in GHOST-CXCR6 cells and that in parental cells argues against CXCR6 being the second GHOST coreceptor. An alternative candidate may be CXCR5/BLR1 (31
Parental U87 cells appeared highly susceptible to infection with HIV-2, although the efficiency of infection varied among virus variants. This was observed for three different batches of parental U87 cells, among which were cells freshly obtained from an AIDS reagent distribution program and cells closely related to those used in a previous study, and is in agreement with previous studies demonstrating infection of U87.CD4 cells by HIV-2 (14
). The high frequency with which parental cells are infected and the high levels of virus produced indicate the presence of a major HIV-2 coreceptor on the surfaces of U87 cells. Although U87 cells express CXCR6 mRNA (25
), we could not detect CXCR6 on the cell surface of any of the U87 derivatives other than that of the U87-CXCR6 cell line. Given the high virus production in all U87 cell lines, it is unlikely that CXCR6 represents the unknown U87 coreceptor. Alternative candidates are GPR1 and RDC1, both of which are expressed by U87 cells (25
) and both of which confer HIV-2 susceptibility on the otherwise nonpermissive human glioma cell line NP-2 when coexpressed with CD4 (48
). Further studies are needed to reveal the identity of this HIV-2 coreceptor.
In previous coreceptor studies, parental U87 cells were not susceptible to infection by all of the 33 primary HIV-2 isolates tested (29
). Four of these variants were included in our study and appeared capable of infecting three batches of parental U87 cells tested. Our results indicate that variable susceptibilities of parental cell batches used, possibly in combination with the degree of U87.CD4 tropism of HIV-2 variants tested, are responsible for different findings regarding infection of parental U87 cells. The variable susceptibility of parental U87 cells may be due to variable expression levels of the endogenous HIV-2 coreceptor and/or of transduced CD4. In support of the latter, parental cells of batch B had lower levels of CD4+
cells than cells of batch C, and parental cells we used in pilot experiments appeared to have even lower levels of CD4+
cells. In addition, retrospective analysis of an old batch of parental cells stored in our laboratory since 1998 revealed that these cells had no detectable levels of CD4, and the cells were not susceptible to HIV-2 infection (data not shown).
In the present study, use of CCR1, CCR2b, and CCR3 by HIV-2 was rarely detected using GHOST cells. This contrasts particularly with previous studies using U87 cells, which demonstrated frequent use of CCR1, CCR2b, and CCR3, albeit with different efficiencies in different studies (29
). In line with our data, with GHOST cells, use of these coreceptors was less frequently detected, and if detected, was mainly associated with use of CXCR4 (42
). It is conceivable that GHOST cells are less sensitive than U87 cells and that the differences between studies and different systems reflect weak use of these coreceptors. In our hands, virus production levels at least 100 times lower than that in CCR5-expressing GHOST cells could still have been detected for most variants, indicating that if CCR1, CCR2b, and CCR3 are used, then it is only with relatively low efficiency. Inefficient use of these coreceptors might be revealed using more sensitive cell-to-cell fusion assays, as was previously demonstrated for the HIV-2ROD
Alternatively, underestimation of nonspecific entry in coreceptor-transduced GHOST and U87 cells, concealed due to the relative lack of susceptibility of parental cells, may have resulted in overestimation of coreceptor use. In support of this, our conclusions based on U87 cells are similar to those based on GHOST cells when virus production in the parental U87 cells is considered to represent nonspecific infection in the other cell lines. Nevertheless, analysis in U87 cells confirmed efficient use of a broad spectrum of coreceptors for two out of four previously tested variants, suggesting that at least some of the discrepancies between previous studies and our own reflect true differences in virus properties.
The use of CXCR6 and/or GPR15 appeared to be a common property of HIV-2 and has also been observed by others (9
). HIV-2 shares this property with SIV (9
), which differentiates these two viruses from HIV-1 (8
). The capacity to use these two chemokine receptors, and also the use of the unidentified U87 coreceptor, may explain the broader tropism of HIV-2 than HIV-1 observed in vitro (14
). The demonstration that HIV-2 variants isolated from individuals with undetectable levels of virus in their plasma efficiently use these chemokine receptors argues against a role in enhancing HIV virulence. The paradox of broad in vitro tropism and low in vivo virus production may be explained by the absence of significant numbers of target cells expressing both CD4 and sufficient levels of the chemokine receptors in vivo. Both CXCR6 and GPR15 are expressed by activated T cells (20
), but none of the HIV-2 variants analyzed here could infect CCR5−/−
PBMC in vitro independently of CXCR4. However, others did observe low levels of infection of CCR5−/−
PBMC by HIV-2 variants that cannot use CXCR4. The inconsistencies may partially result from the different genetic background and CXCR6 and GPR15 expression levels of the donor of the CCR5−/−
In contrast to CXCR6 and GPR15, use of CXCR4 was observed only in variants isolated from individuals with viremia, three of whom had overt progressive disease. The association of HIV-2 variants capable of using CXCR4 with progressive infection is reminiscent of what is known for HIV-1 (16
). Variants from one asymptomatic individual with viremia could infect some, but not all, CXCR4-positive cell types, which may suggest that X4 HIV-2 variants with different CXCR4 binding properties and different pathogenic potentials exist. In analogy to HIV-1 infection, X4 variants were not observed in all individuals with progressive infection, indicating that although the capacity to use CXCR4 is likely a determinant of HIV-2 virulence, it is not the only one.
In conclusion, we find that HIV-2 variants from nonprogressing individuals have a broader coreceptor repertoire than HIV-1. Moreover, we find that the number of coreceptors used per HIV-2 variant does not increase with disease progression. These findings establish for the first time that promiscuous coreceptor usage per se is not a determinant of HIV pathogenicity.