Genetically divergent strains of human immunodeficiency virus type 2 use multiple coreceptors for viral entry

Abstract

Several members of the seven-transmembrane chemokine receptor family have been shown to serve, with CD4, as coreceptors for entry by human immunodeficiency virus type 1 (HIV-1). While coreceptor usage by HIV-1 primary isolates has been studied by several groups, there is only limited information available concerning coreceptor usage by primary HIV-2 isolates. In this study, we have analyzed coreceptor usage of 15 primary HIV-2 isolates, using lymphocytes from a donor with nonfunctional CCR5 (CCR5 -/-; homozygous 32-bp deletion). Based on the infections of PBMCs, seven of these primary isolates had an absolute requirement for CCR5 expression, whereas the remaining eight exhibited a broader coreceptor usage. All CCR5-requiring isolates were non-syncytium inducing, whereas isolates utilizing multiple coreceptors were syncytium inducing. Blocking experiments using known ligands for chemokine receptors provided indirect evidence for additional coreceptor utilization by primary HIV-2 isolates. Analysis of GHOST4 cell lines expressing various chemokine receptors (CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR4, BONZO, and BOB) further defined specific coreceptor usage of primary HIV-2 isolates. The receptors used included CXCR4, CCR1-5, and the recently described receptors BONZO and BOB. However, the efficiency at which the coreceptors were utilized varied greatly among the various isolates. Analysis of V3 envelope sequences revealed no specific motif that correlated with coreceptor usage. Our data demonstrate that primary HIV-2 isolates are capable of using a broad range of coreceptors for productive infection in vitro. Additionally, our data suggest that expanded coreceptor usage by HIV-2 may correlate with disease progression.

FIG. 1

Infection of CCR5 +/+ and CCR5 −/− normal donor PBMCs by primary HIV-2 isolates. (A) Isolate A2267 was not capable of replicating in CCR5 −/− PBMCs. The data shown are representative of the remaining six isolates (A1958, SLRHC, A2270, 310072, 310340, and 60415K) that require CCR5 for replication. (B) Isolate 77618 showed comparable replication abilities in CCR5 +/+ and CCR −/− donor PBMCs. The data are representative of isolates 7924A, GB122, GB87, and 310319. (C) Isolate 7312A showed delayed kinetics and decreased antigen production in the CCR5 −/− donor PBMCs. This was also observed with isolate 310248.

FIG. 2

Phylogenetic relationships of the newly derived HIV-2 isolates with representatives of HIV-2 subtypes A and B. The tree was constructed from partial env nucleotide sequences in the C2/V3 region (consensus alignment, 300 to 350 bp). Phylogenetic relationships were determined by the neighbor-joining method as described in Materials and Methods. Horizontal branch lengths are drawn to scale, while vertical branches are for clarity only. The numbers on the nodes represent the percentage bootstrap samples with which the cluster to the right is supported; only values over 80% are shown. The tree was rooted by using SIV_{MAC 251} as an outgroup. Brackets denote HIV-2 subtypes as reported previously (31). Newly derived isolates are in boxes.

FIG. 3

Chemokine effects on infection with HIV-2 primary isolates. CCR5 −/− donor PBMCs were infected with HIV-2 in the presence or absence of SDF-1 (A) or a cocktail of RANTES, eotaxin, and MCP-3 (B). The values shown are p27 antigen concentrations from day 7 of culture. The values in panel A represent means ± standard errors of the means of two independent experiments.

FIG. 4

Kinetics of p27 antigen production by HIV-2 isolates in GHOST4 cells expressing various coreceptors. The values given are p27 antigen (picograms/milliliter) present in the supernatants of cultures on days 3, 7, 10, and 15 from different GHOST4 cell lines infected with the same infectious dose of virus. Values given as 784 pg/ml actually represent antigen values which were above the linear scale of the assay. (A) Isolates which produce large quantities of antigen in only a few cell lines; (B) isolates which produce large quantities of antigen in many cell lines.