A wide range of different seven-transmembrane G-protein-coupled chemokine receptors function as coreceptors for HIV infection of indicator cell lines in vitro. Many HIV-2 and SIV strains are particularly promiscuous for different coreceptors in such assays; however, HIV-1 strains can also use a variety of alternative coreceptors (16
), e.g., CCR3, CCR8, and CXCR6. Although some of these coreceptors are expressed on CD4+
primary cells (1
), there is little current evidence to suggest they are used by HIV-1 in vivo. Thus, the ability of particular HIV-1 strains to exploit alternative coreceptors for infection of cell lines in vitro does not provide a true indication of virus coreceptor use in vivo. We therefore analyzed several untransformed cell cultures for natural expression of functional alternative coreceptors that support HIV and SIV infection.
Astrocytes established from adult temporal lobectomy samples and untransformed BMVEC cultures do not express either CCR5 or CXCR4 and thus represent suitable target cells to test for the presence of alternative coreceptors. BMVECs and adult astrocytes expressing CD4 (via Ad-CD4) were resistant to infection by HIV and SIV strains that predominantly utilize CCR5 or CXCR4 alone. Both BMVECs and adult astrocytes were susceptible to infection by a subset of R5X4 HIV-1 strains, including two primary isolates (HAN-2 and P1019) as well as GUN-1v. GUN-1v is a variant virus isolated in vitro by its capacity to infect a wider range of host cells, including BT cells (human CD4+
meningioma-derived cells) (65
). GUN-1v infection of BMVECs was sensitive to inhibition by vMIP-I (an HHV8-encoded chemokine), suggesting that a receptor for this ligand is important for infection of these cells. In contrast, astrocyte infection by GUN-1v was reduced only approximately 50% by vMIP-I. This lack of complete inhibition by vMIP-I suggests either that the vMIP-I receptor (implicated for infection of BMVECs) is not solely responsible for astrocyte infection or that vMIP-I only weakly inhibits this receptor as expressed on astrocytes.
Infection of astrocytes was also reduced approximately 50% by the chemokines RANTES and eotaxin. These ligands each bind several chemokine receptors (CCR1, CCR3, CCR5, and CCR9 for RANTES and CCR3 and D6 for eotaxin), indicating a role for this subclass of receptor in the infection of astrocytes. Although CCR5 expression was not detected on these astrocytes, several groups have reported expression on astrocytes in situ in the brain, which is lost rapidly in culture (8
). The lack of inhibition by the CCR5 small-molecule inhibitor TAK-779, as well as by the CCR5-specific MAb 2D7, confirms that the weak inhibition by RANTES was not due to the presence of low levels of CCR5.
There is evidence that astrocytes become persistently infected by HIV-1 in vivo, particularly in pediatric AIDS (57
). Since R5 viruses are predominant in the brain (1
), it is possible that these astrocytes are infected in vivo via CCR5. Nevertheless, the alternative coreceptor demonstrated on these CCR5−
cultured astrocytes may potentially confer infection of astrocyte subsets critical for brain homeostasis and thus have an impact on neuropathology. In this study we used MAbs that detect p24 as a marker for HIV-1 infection. Since p24 is a late gene product, our results show that the astrocyte infection observed here in vitro is not restricted to the early phase of viral replication and expression of early gene products, thus confirming the observations of Canki et al. (10
Since BMVECs and astrocytes are not major targets for HIV-1 in vivo, we sought evidence for use of the alternative coreceptor on primary PBMCs. Other studies have demonstrated the ability of some SIV strains to infect PBMCs lacking CCR5 via an alternative coreceptor, in a donor-dependent manner (11
). In some instances CXCR6 was implicated as the coreceptor involved (79
). In confirmation, several HIV-1 and HIV-2 isolates identified here that exploit an alternative coreceptor(s) for BMVEC and astrocyte infection were able to replicate in PBMCs that lack CCR5, with CXCR4 blocked by AMD3100. The X4 isolate 2044 was consistently sensitive to inhibition by AMD3100, although low-level residual replication was sometimes detected even in the presence of AMD3100. However, AMD3100-resistant replication in PBMCs by the isolates HAN-2, P1019, TER, and Man4 was not due to residual use of CXCR4, since such replication was inhibited by vMIP-I, a chemokine that does not interact with CXCR4 (20
). Our observations therefore provide evidence that an alternative coreceptor is active for infection of PBMCs by the HIV and SIV strains described.
The identity of the alternative coreceptor remains to be conclusively elucidated. A hallmark of the BMVEC and PBMC infection via an alternative coreceptor demonstrated here was the sensitivity to inhibition by vMIP-I. To date, vMIP-I has been reported to inhibit HIV infection mediated through CCR8 (20
) as well as GPR1 and CXCR6 (66
). GUN-1v and the other HIV and SIV strains, identified here by their capacity to use alternative coreceptors, were able to use CCR8 and GPR1 (as well as other coreceptors) in addition to CCR5 and CXCR4 as coreceptors on cell lines. Furthermore, GUN-1v has previously been reported to use alternative coreceptors for infection of astroglial U87 cells, primary mesengial kidney cells, and BT cells. CCR8 was implicated as a coreceptor for BT infection (40
) and GPR1 was implicated as a coreceptor for mesengial cell infection (73
); however, inhibition by ligands to these coreceptors was not demonstrated. Our observations that the CCR8 ligand I309 had no effect on the replication of any HIV or SIV isolate in BMVECs or Δ32/Δ32 PBMCs, alone or in combination with AMD3100, despite being able to inhibit infection of a CCR8-expressing indicator cell line (data not shown) implies that vMIP-I is able to inhibit HIV infection of PBMCs via another chemokine receptor.
We also attempted to correlate coreceptor mRNA detection in PBMCs, BMVECs, and astrocytes with infection by this subset of HIV and SIV strains. None of the presently known vMIP-I receptors (CCR8, GPR1, and CXCR6) were detected by RT-PCR in BMVECs. mRNA for CCR8 was detected in the Δ32/Δ32 PBMCs; however, the CCR8-specific chemokine I309 had no effect on viral replication in either PBMCs or BMVECs. Δ32/Δ32 CCR5 PBMCs were also positive for expression of CXCR6, a coreceptor previously implicated as being responsible for replication of a maternal HIV-1 isolate (61
). However, the CXCR6 ligand CXCL16 failed to block replication of vMIP-I-sensitive virus strains on Δ32/Δ32 CCR5 PBMCs even though infection of CXCR6-expressing cell lines was efficiently inhibited (data not shown). Finally, mRNA for GPR1 was not detected in either BMVECs or PBMCs. We therefore believe that an as-yet-unidentified vMIP-I receptor is responsible for the HIV and SIV infection of these cells. An alternative, albeit less likely, explanation is that vMIP-I induces down-modulation of a different receptor or induces intracellular signaling responses that negatively affect viral replication. However, previous studies have demonstrated that vMIP-I has no effect on chemokine-induced intracellular calcium signaling via the chemokine receptors CCR2, CCR4, CCR5, CCR7, CXCR2, CXCR3, and CXCR4 on PBMCs (20
). Although vMIP-I did desensitize CCR8-mediated signaling, its ineffectiveness against such a large range of chemokine receptors suggests that receptor desensitization is not the mechanism of inhibition observed here.
All viruses that we found to use an alternative coreceptor(s) are R5X4, with the exception of HIV-2 TER, an isolate that readily acquires CXCR4 use in culture. We believe that an alternative coreceptor(s) may be exploited late in disease in addition to, or instead of, CXCR4 (like TER). The role of the expansion in coreceptor use may therefore contribute to the broadening of cell tropism late in disease, while a role in neuropathogenesis should also be considered. Moreover, the alternative coreceptor should be considered as a potential escape route from new therapeutic CCR5 and CXCR4 inhibitors currently undergoing clinical trials (34