Co-receptor Usage of BOB/GPR15 in Addition to CCR5 Has No Significant Effect on Replication of Simian Immunodeficiency Virus In Vivo

Stefan Po hlmann 2 3 Nicole Stolte 1 2 Jan Mu nch 2 3 Peter Ten Haaft 0 2 Jonathan L. Heeney 0 2 Christiane Stahl-Hennig 1 2 Frank Kirchhoff 2 3 0 Biomedical Primate Research Center, Department of Virology , Rijswijk , Netherlands 1 German Primate Center , Go ttingen , Germany 2 Received 12 April 1999; revised 23 June 1999; electronically published 8 October 1999. Presented in part: Annual spring meeting of the German Society for Virology , Bremen, Germany, March 1999 (abstract 8P31). The macaques used in this study were housed in a biosafety level 2 facility at the German Primate Center in Go ttingen. The monkeys were maintained and specimens were collected in accordance with institutional guidelines. Financial support: Deutscheforschungsgemeinschaft (grants Ki 548/1-2, SFB 466); Bundesministerium fu r Forschung und Technologie. and Molecular Virology, University of Erlangen-Nu rnberg , Schlossgarten 4, 91054 Erlangen , Germany 3 Institute for Clinical and Molecular Virology, University of Erlangen- Nu rnberg , Erlangen Human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency viruses (SIVs) frequently use the orphan receptor BOB/GPR15 in addition to the chemokine receptor CCR5 for efficient entry and replication. However, the role of BOB/ GPR15 in replication and pathogenesis of HIV-2 and SIV in vivo is unclear. This study shows that a single amino acid substitution in the V3 loop of the pathogenic SIVmac239 clone, 321PrS, impaired the ability to use BOB/GPR15 for entry and replication but had little effect on the ability to use CCR5. This envelope variant replicated with an efficiency comparable with the parental SIVmac239 isolate in rhesus macaques. Furthermore, the mutant genotype and phenotype remained stable even after the onset of immunodeficiency. These results suggest that this cofactor plays only a minor role for the pathogenicity of the HIV-2/SIVmac/SIVsm group of primate lentiviruses. - Human and simian immunodeficiency viruses (HIV and SIV) infect CD4 target cells by fusion at the plasma membrane. Entry involves binding of the viral envelope (Env) protein to the CD4 molecule and subsequent interactions with seven transmembrane-spanning G proteincoupled receptors (7TM GPCRs) of the chemokine receptor family that mediate fusion between the viral and host cell membrane [1, 2]. The CC chemokine receptor, CCR5, and the CXC chemokine receptor, CXCR4, are considered to be the major HIV-1 co-receptors, because all HIV1 strains described to date use one or both of these molecules as second receptors. CCR5 mediates efficient entry of primary macrophage-tropic (R5-tropic) HIV type 1 (HIV-1) isolates [38], and CXCR4 serves as fusion cofactor for entry of T celltropic (X4-tropic) HIV-1 isolates [9]. In addition to CXCR4 and CCR5, several other chemokine and orphan receptors of the 7TM domain receptor family, namely, CCR1, CCR2B, CCR3, CCR4, CCR8, CCR9, GPR15/BOB, STRL33/Bonzo, GPR1, V28, US28, ChemR23, and Apj can be used by some HIV and SIV isolates [5, 6, 1017]. Use of co-receptors other than CCR5 and CXCR4 is infrequent and often inefficient [1820]. HIV-2 isolates, however, are less restricted to CCR5 and CXCR4 and often use additional cofactors for entry into target cells. Several studies suggest that the orphan receptor BOB/GPR15 may be more commonly and efficiently used by HIV-2 isolates than the CXCR4 receptor is [14, 21]. An expanded co-receptor usage of HIV-2 may correlate with increased pathogenesis [14]. Infection of rhesus macaques with SIV provides one of the best animal models for HIV pathogenesis in humans [22]. SIVmac and HIV-2 belong to the same phylogenetic group of lentiviruses and probably both originate from SIVsm in naturally infected sooty mangabeys [23, 24]. SIVmac isolates can use CCR5, but not CXCR4 [2528]. Similar to many strains of HIV-2, most SIVmac strains can use BOB/GPR15 nearly as efficiently as CCR5 [14, 21]. This orphan receptor is expressed in lymphoid and colon tissues and may contribute to viral spread in vivo [16]. Analysis of SIV variants with differential co-receptor tropism in nonhuman primate models allows the evaluation of the relevance of specific co-receptors for viral replication and pathogenesis. We previously demonstrated that specific changes in the V3 loop of the molecular SIVmac316 clone had differential effects depending on the cell type [28, 29]. Further analysis revealed that a single amino acid substitution in the Env protein, 321PrS, impaired the ability to use BOB/GPR15 but had little effect on use of CCR5 or GPR1 and only a moderate effect on the ability to use Bonzo/STRL33. The SIVmac239 321PrS variant replicated with kinetics undistinguishable from wild type virus in peripheral rhesus blood mononuclear cells and in infected rhesus macaques. No variants with restored ability to use BOB/GPR15 emerged in infected animals, indicating that use of BOB/GPR15, in addition to CCR5, does not provide a major replicative advantage in vivo. Methods Construction of SIVmac239 Env variants. A modified pBR322vector containing the full-length SIVmac239 proviral DNA was provided by Toshiaka Kodama (Oregon Regional Primate Research Center, Beaverton, OR). First, a single ArC nucleotide change was introduced at position 7444 of the SIVmac239 (239wt) genome. Numbering refers to the published 239wt sequence [30]. This change generates a unique XhoI restriction site. It did not alter the deduced Env amino acid sequence and had no effect on replication or co-receptor tropism (data not shown). Site-directed mutagenesis was performed by splice overlap extension polymerase chain reaction (PCR). In brief, we amplified the env region was amplified using primers pSphI (50-AACGAGCGCTCTTCATGCAT-30) and p3V3Xho (50-GCTCGAGTTCCATTAAAGCCAAA-3), respectively, p3outV3 (50-TCCTCTGCAATTTGTCCA30), and p5V3Xho (50-CTTTAATGGAACTCGAGCAGAAAATAGAACTTATAT-30) (the XhoI site is underlined). PCR products were gel purified, mixed, subjected to a second round of PCR with primers pSphI and p3outV3, and cloned into the proviral SIVmac239 genome by use of the unique SphI and MroI restriction sites. Codon 321 in the SIVmac239 env gene was changed from CCArAGT (PrS) by use of p5outV3 (50-GAGTCTTGTGACAAACA-30) and p3outV3 as outer primers and p3mV3-1 (50-GGTGACACTTAAAACTGTCTTATTTCC-30) and p5mV3-1 (50-ACAGTTTTAAGTGTCACCATTATGTCTGGATTGGTT-30) as inner primers for splice overlap extension PCR. The unique XhoI and MroI restriction sites in the SIV env gene were used for cloning into the full-length provirus. All PCR-derived inserts were sequenced to ensure that only the intended changes were present. By use of standard cloning techniques, the deleted env gene of pBR239DenvLuc [28] was replaced with the intact 239wt and 321 PrS env genes to generate replication-competent luciferase reporter viruses. Virus stocks. We generated virus stocks by the calcium phosphate method essentially, as described elsewhere [7]. In brief, 293 (...truncated)