HIV-1 infects renal epithelium and infection is associated with the development of HIV-associated nephropathy (HIVAN) in African Americans . Recent studies have demonstrated that HIV-1 RNA persists in renal epithelial cells even when plasma RNA levels are undetectable [2–6]. Whereas the majority of primary HIV-1 isolates utilize the chemokine co-receptors CXCR4 (X4 isolates) or CCR5 (R5 isolates) , other members of the seven transmembrane receptor family can serve as co-receptors of HIV and SIV in vitro including CCR1, CCR2b, CCR3, CCR4, CCR8, GPR15 (BOB), STRL33 (BONZO/CXCR6), GPR1, V28/CX3CR1 and APJ [8–14]. Athough CD4 and the main co-receptors CXCR4 and CCR5 are not detectable by immunohistochemistry on renal epithelium in renal biopsies , CD4 and CXCR4 have been detected on human primary proximal tubular epithelial cells in vitro . In order to obtain a better understanding of the entry of HIV-1 into renal epithelium, we evaluated the tropism and co-receptor utilization of HIV-1 envelopes simultaneously derived from renal epithelium and peripheral blood mononuclear cells (PBMC) of HIV-infected patients.
Four kidney and two peripheral blood-derived gp120 envelopes from two patients (RB5 and RB23) were selected for biological characterization. The gp120 sequences cloned from DNA extracted from renal epithelial cells isolated by laser capture microdissection  were cloned in pGEM-T easy vector and sub-cloned in frame with MfeI of gp41 derived from pNL4-3 to create a full-length gp160. Subsequently, parental gp160 of pNL4-3 was excised with EcoR-I/Blp-I and replaced by recombinant gp160 using compatible end ligation to generate the chimeric plasmid. These chimeras were used to produce replication-competent viruses by the transfection of 293 T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA). The laboratory X4 strain (III-B) and R5 strain (BAL) were used as positive controls. All envelopes derived from kidney and PBMC DNA were cloned into pNL4-3 backbone and sequenced. The expression was confirmed by Western blot analysis.
Chimeric NL4-3 viruses containing RB5-1K, RB5-2K, RB5-3K, RB23-1BL, RB23-2K or RB23-3BL envelopes were used at a concentration of 0.5 pg/cell of p24 antigen to infect primary CD4T cells. The cell lines were infected for 2 h at 37°C followed by washing and replacement of the media. To study tropism, cell lines expressing the single co-receptors CXCR4 or CCR5 were treated with 10 μg/ml anti-CCR5 or anti-CXCR4 monoclonal antibodies (2D7 or 12G5, respectively; BD, Pharmingen, California, USA), 200 nmol of the R5 inhibitor TAK-779 or 2 μmol of the X4 inhibitor AMD3100 (generous gift from Dr John P. Moore, Cornell University, New York, USA), for 1 h before infection. Subsequently, the cells were challenged with virus at a concentration of 0.5 pg/cell for 2 h. Virus production was measured at different intervals after infection using the p24 enzyme-linked immunosorbent assay (Coulter, Immunotech, Hialeah, Florida, USA and NCI, Frederick, Maryland, USA).
All chimeric viruses derived from renal epithelium or PBMC envelopes replicated in primary phytohemagglutinin-activated CD4 T cells, albeit at different levels (Fig. 1a).
Replication kinetics of the chimeric viruses derived from two different compartments differed in cell lines expressing the single co-receptors CCR5 or CXCR4.
In the U87 cell line expressing CD4 and CCR5, viruses with envelopes derived from both the kidney (Fig. 1b) and blood replicated (Fig. 1c). The CCR5-specific antibody 2D7 and the small molecule antagonist TAK-779 inhibited replication of the kidney-derived viruses in U87-CD4-CCR5, confirming the use of CCR5 by these envelopes (Fig. 1b). Infection of the same cells with blood-derived viruses was completely inhibited by TAK-779 but not by 2D7 antibody at a dose of 20 μg/ml (Fig. 1c). Using a control R5 virus (BAL), anti-CCR5 antibody (2D7) completely abolished infection (data not shown).
In contrast, only the kidney-derived viruses replicated in the U87-CD4-CXCR4 cell line (Fig. 1d). The anti-CXCR4 antibody 12G5 and small molecule antagonist AMD3100 inhibited replication of kidney-derived viruses by 85–99% (Fig. 1d) as well as the control X4 virus III-B (data not shown).
Three kidney and one blood-derived viruses also productively infected the U87-CD4-CCR3 cell line (data not shown) whereas two kidney-derived viruses utilized BONZO/STRL33 and BOB/GPR15 in GHOST cell lines expressing these co-receptors (data not shown).
All chimeric viruses replicated in the HPT-1 cell line (data not shown). The latter had detectable CD4 and CXCR4 messenger RNA as well as mRNA for some of the minor co-receptors (CCR9, BONZO/STRL33, APJ, GPR1, and ChemR23) by reverse transcriptase–polymerase chain reaction (data not shown) but no detectable CCR5 mRNA. In view of the fact that the blood isolates showed only an R5 phenotype, these viruses most likely utilized one of the alternative co-receptors for entry to the HPT-1 cell line.
In this study, we analysed the phenotype of HIV-1 envelopes simultaneously derived from renal epithelium and PBMC. HIV-1 envelopes from isolates derived from renal epithelium were dual tropic (R5X4) and were able to utilize a number of alternative co-receptors. In contrast, envelopes simultaneously derived from PBMC were R5 tropic with a heterogeneous pattern of co-receptor utilization. There was a clear distinction in co-receptor utilization between the kidney and the blood-derived envelopes.
The role of alternative co-receptors in HIV-1 entry in the kidney is unclear. We found that three kidney-derived envelopes could use CCR3 and two of them could use GPR15 and BONZO/STRL33. Interestingly, we did find BONZO/STRL33 mRNA in HPT-1 cells, consistent with earlier reports of its presence in the kidney . In addition, we detected mRNA expression of CXCR4, GPR1 and APJ in HPT-1 cells.
In conclusion, this study demonstrates that HIV-1 quasispecies in the kidney are phenotypically distinct from those in the blood, as indicated by the differential use of co-receptors, consistent with renal compartmentalization. Furthermore, the use of CXCR4 by these isolates and the presence of CXCR4 on the renal epithelial cell line suggests that entry might be mediated via this co-receptor.
Further studies on HIV-1 variants from the kidney may shed more light on HIV-1 pathogenesis and compartmentalization.
The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: U87MG cells from Dr Bruce Chesebro, U87-CD4 cells from Drs Hongkui and D. Litman, and GHOST cells from Dr Vineet, N. KewalRamani and Dr D. Litman.
Sponsorship: This work was supported by NIH grant P01DK025460.
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