In contrast, SIVmus did not use GPR15 from either species, even though the GPR15 molecules were functional for entry by the promiscuous SIVsmm strain (Fig 2B and 2C, right panels). CD4 and coreceptor are indicated below the graph (C, chimpanzee; M, mustached monkey;C, vacant vector). 48 hours post transfection, cells were infected with luciferase reporter pseudotypes transporting the SIVcpz EK505 Env (A) or the SIVmus1085 4C12 Env (B). Access was quantified 72 hours later by lysing cells and measuring luciferase content by relative light models (RLU). hJumpy Infections were carried out in triplicate and error bars represent one standard deviation.(TIF) ppat.1007003.s002.tif (127K) GUID:?CB120737-6C71-480B-8104-B5E981448D72 S3 Fig: Antibody 20D8 detects CXCR6 but does not cross-react with other 7TMRs of sooty mangabey origin, and selects for PBMC enriched in CXCR6 RNA. A) 293T cells were transfected with expression plasmid made up of CXCR6, CCR5, CXCR4, APJ or GPR15 of sooty mangabey origin or with vacant vector. 48 hours later, cells were stained with anti-CXCR6 antibody 20D8 followed by a goat anti-mouse secondary. CXCR6-expressing cells were also stained with the secondary antibody alone. B) Human PBMCs from two donors were sorted into 20D8 positive and negative populations, and subjected to qPCR for expression of CXCR6 RNA relative to GAPDH RNA (left panel). Expression of CXCR6 RNA in 20D8-positive cells is usually shown relative to sort-negative cells. In parallel, CXCR6 expression was decided in GHOST-CXCR6 cells, which are HOS cells stably transfected to express high level CXCR6, relative to GHOST-CD4 cells (right panel).(TIF) ppat.1007003.s003.tif (390K) GUID:?47AEEC29-CFF9-4D73-9694-769570C1BA27 S4 Fig: CXCR6 expression on rhesus macaque CD4+ T cells. A) Expression on RM resting CD4+ T cells of CXCR6 (x-axis) and CCR5 (y-axis). Figures in the quadrant are the percent of CD4+ T cells expressing the respective combination of coreceptors. B) Resting PBMC from 6 RM were stained using antibodies to define CXCR6 expression of CD4+ memory subsets: naive (Tn: CD45RA+/ CCR7+/ CD28+/ CD95-), central memory (Tcm: CD45RA-/ CCR7+) and effector memory (Tem: CD45RA-/ CCR7-). Data show individual percentages, along with imply and standard deviation. Each sign represents cells from a different individual RM.(TIF) ppat.1007003.s004.tif (233K) GUID:?D466A26D-0B5B-4DCB-9C43-BC6068D52F80 S5 Fig: Regulation of CXCR6 and CCR5 on SM CD4+ T cells upon stimulation. SM PBMC from six animals were stimulated with concanavalin A and IL-2. Staining for expression of CD4, CXCR6 and CCR5 was carried out prior to activation, and at days 5, 7 and 9 post-stimulation. Data show individual percentages, along with imply and standard deviation. Each sign represents cells from a different individual SM at each time point.(TIF) ppat.1007003.s005.tif (133K) GUID:?6E826C84-6433-403D-AAB0-2C221C314C22 Data Availability StatementThe only relevant data not within the paper and its Supporting Information files are novel DNA Sequences. All novel DNA sequences have been submitted to GenBank and have the following accession figures: MG267399-MG267416, MG450752-MG450761. Abstract Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and Basmisanil HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is usually unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is usually a major coreceptor for SIV Basmisanil in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for access and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined Basmisanil by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is usually absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of.