Alterations in the V1/V2 domain of HIV-2CBL24 glycoprotein 105 correlate with an extended cell tropism.

1399 H IV-2CBL24 (CBL-24) W AS ISOLA T ED from an asymptomatic individual and characterized. 1 The cellular tropism of this isolate was restricted to cord blood mononuclear cells (CBMCs) and Molt-4 cl.8 T cells. Replication was not seen in other T cell lines (H9, C8166, CEM, and U937). 1 However, herpesvirus saimiri-im mortalized T lymphocytes (HVS T cells) support CBL-24 replication. 2 A persistently productive infection was established and at 8–10 weeks postinfection (wpi), when the p26 concentration was high ( $ 1000 ng/ml), HVS T cell-passaged CBL-24 induced syncytia in C8166 and CEM-SS T cell lines. Selection of host range mutants in HIV-1/2 has been reported in several continuous cell lines, and so we investigated this observation further. The original isolate, designated passage zero (P0-CBL-24), was obtained from CBMC/Molt-4 cl.8 cocultures. Passage 1 (P1-CBL-24) was obtained after feeding cocultures with uninfected Molt-4 cl.8 cells. Passages 2 (P2-CBL-24) and 3 (P3CBL-24) were obtained by further infections of Molt-4 cl.8. Proviral DNA was extracted at each passage. The env gene sequence of P0-CBL-24 (GenBank U05353), referred to here as the “parent sequence,” has been reported. 3 P3-CBL-24 was used in our previous study.2 We now report on the cellular tropism of P2-CBL-24, P3CBL-24, and HVS T cell-passaged CBL-24 (Kesting-CBL-24 and CB23-CBL-24) in a panel of 11 cell lines and pooled peripheral blood mononuclear cells (PBMCs) that had (CD8 2 ) or had not (CD8 1 ) been depleted of CD8-expressin g cells.2 The cell tropism of HVS T cell CBL-24 isolates was determined after short-term (3 wpi) and extended (11 wpi) passage. The cell lines were inoculated with p26 at 20, 200, or 2000 ng/ml as determined by p26 enzyme-linked immunosorbent assay (ELISA) (Murex, Dartford, UK). CBL-24 phenotype/ cell tropism was assessed by microscopic examination and p26 ELISA. After an inoculum of 20 or 200 ng of p26 per milliliter, similar results were obtained. P2-CBL-24 and P3-CBL-24 replicated in 10 of 11 cell lines, and in CD8 1 and CD8 2 PBMCs (Table 1). However, syncytia were seen in five cell lines only (H9, C8166, Molt-4 cl.8, PM-1, and MT-2), and in CD81 and CD8 2 PBMCs. In contrast, short-term passaged HVS T cell isolates, Kesting-CBL-24 and CB23-CBL-24, replicated in CD8 1 and CD8 2 PBMCs but in only five and six cell lines respectively, with syncytium formation in C8166, Molt-4 cl.8, PM-1, and MT-2. Results were similar, after extended passage (11 weeks) in HVS T cells, except that at a p26 concentration of 200 ng/ml CB23-CBL-24 appeared to lose its tropism for H9. However, by increasing the virus inoculum (p26 at 2000 ng/ml), CBL-24 replicated with syncytia in 7 of 11 cell lines, including CEMSS. These results were reproducible. We could not test the cell tropism of short-term passaged HVS T cell isolates at 2000 ng/ml because the virus titer was not high enough. Clearly, CBL-24 tropism was restricted after passage in HVS T cell lines when compared with Molt-4 cl.8 isolates, but no phenotype/ tropism differences were observed between the two HVS T cell isolates when the T cell panel was inoculated with p26 at 2000 ng/ml. The env genes of these isolates were sequenced to look for substitutions that might explain the results. DNA was prepared from cells infected with the six isolates shown in Table 1. env genes were amplified by nested polymerase chain reaction (PCR), using the primers H2POLF (CARGGAGTAGTRGAAGCAATGAA) and H2LTRR (TGCTTCTAAYTGGCAGCTTTATT) and primers H2ENVF (GACTCCGCGGAAAACTAAGRCTCATYCGTCTYCTGCAYCAGA) and H2ENVR (TTGGTGTGTCCGGAACYCCYACTAGGTCATC), as described. 3 Regions corresponding to nucleotides (nt) 4499–9671 and nt 6096–8869 of HIV-2ROD were amplified. Purified env genes were sequenced directly on an ABI-377 sequencer using dye terminator cycle sequencing Ready Reaction kits (Perkin-Elmer, Foster City, CA) and a bank of primers.3 Sequences were analyzed using GCG and GDE programs (Fig. 14). The most significant changes in all six sequences, compared