Our interpretation that the restrictive use of recombinant CCR3 previously reported by many groups is due to inadequate expression of this coreceptor is supported by a recent study describing experimental variables that influence CCR3 expression from a nuclear promoter. chemokines RANTES, MIP-1, and MIP-1 suppress infection by prototypic macrophage-tropic (M-tropic) HIV-1 isolates (13), led several groups to independently identify a CC chemokine receptor, CCR5, as the major coreceptor for such strains (4, 12, 18, 20, 21). Coreceptor activity has also been observed with other CC chemokine receptors, including CCR3 and CCR2b (12, 20) and CCR8 (4a, 44), as well as with three chemokine receptor-like orphan proteins, one designated STRL33 (33) or BONZO (17) a second designated BOB (17) or GPR15 (23, 30), and a third designated V28 (44). The specificities of different Envs for this array of coreceptors, coupled with the patterns of endogenous coreceptor expression on various CD4+ target cell types, are major determinants mediating the cytotropisms of different HIV-1 strains. In the present study we examined molecularly cloned Envs from a panel of primary HIV-1 isolates representing diverse phenotypes and genetic subtypes (clades) (26). These isolates were collected at epicenters of the global AIDS pandemic by the World Health Organization and National Institute of Allergy and Infectious Diseases Networks for HIV Isolation and Characterization, with PHA-793887 the goal of generating a panel of naturally occurring primary strains to facilitate structure and function studies and vaccine development. Our findings reveal some unexpected activity patterns with recombinant and endogenous coreceptors and highlight the importance of defining the variables that contribute to the contrasting results obtained with different experimental systems. Identification of functional Envs. The primary genes (26) were obtained PHA-793887 from the National Institutes of Health AIDS Research and Reference Reagent Program (Rockville, Md.), except for clone 92UG037, which was kindly provided by B. Hahn (University of Alabama, Birmingham). Each was cloned into plasmid pCRII, which contains the bacteriophage T7 promoter. For Envs from prototypic strains, we used the following plasmids derived from pSC59, which contains a synthetic early-late vaccinia virus promoter (11a): pCB-41 (8), TCL-adapted LAV Env; pCB-43 (8), M-tropic Ba-L Env; pGA13-89.6 (2a), dual-tropic primary 89.6 Env; and pCB-16 (8), nonfusogenic uncleavable (Unc) Env (8). To prepare effector cells, HeLa cell monolayers were transfected with Env-containing plasmids with DOTAP (Boehringer Mannheim, Indianapolis, Ind.) and then trypsinized and infected in suspension with vP11T7gene1, which contains the bacteriophage T7 RNA polymerase gene linked to a natural late vaccinia virus promoter (1). To assess fusogenic activities, we used a quantitative cell fusion assay as previously described (39), with target cells infected with vCB-21R containing the gene linked to the T7 promoter (3). Peripheral blood mononuclear cells (PBMCs) were chosen as targets to identify functional Envs. Of the 31 primary Envs examined, 19 displayed detectable fusion with PBMCs (as well as with the PM1 cell line [data not shown]). Table ?Table11 PHA-793887 shows the active Envs, all of which routinely gave values at least threefold over the activity observed with the nonfusogenic Unc Env; their reported non-syncytium-inducing (NSI) versus syncytium-inducing (SI) phenotypes (26) are also indicated. To facilitate comparisons of activities against PBMCs in different experiments, the fusion activity observed with the prototypic LAV Env in each experiment was assigned a value of 100% and the activities of the other Envs were expressed as relative values. The 19 functional primary Envs showed wide variations in relative fusogenic strengths with PBMC targets. Our results are in general agreement with and expand upon a previous study in which the activities of many of these Envs were scored by infectivity assays with pseudotyped Rabbit Polyclonal to PEX14 virions containing the chloramphenicol acetyltransferase reporter gene (26); we identified five additional fusogenic Envs not analyzed in the previous report (92US711.14, 92US716.6, 93ZR001.3, 92TH022.4, and 93BR019.10). TABLE 1 Fusogenic activities of each Env with cells expressing endogenous coreceptors axes for each Env shown in Fig..