Cellular and viral specificities of human immunodeficiency virus type 1 vif protein

Abstract

The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.

FIG. 1

Western immunoblot analyses of viral proteins from infected H9 cells and virion particles. At 72 h after the start of coculturing (see Materials and Methods), extracts of infected H9 cells (A) and virion particles produced from H9 cells (B and C) were prepared for Western immunoblot analyses. HIV-1 proteins electrotransferred to nitrocellulose membranes were probed with HIVIG (A and B) or sheep gp120 antiserum (C). Antibody binding was detected by incubation with chemiluminescence reagents. Molecular weights of protein standards are indicated on the left, in thousands. Wt, wild type.

FIG. 2

Infectivity of wild-type (○) and vif deletion (□) HIV-1 on HeLa-CD4 cells. The viruses produced from nonpermissive H9 cells infected with a wild-type or vif deletion strain of HIV-1, NL4-3, were used to infect HeLa-CD4 clones expressing different amounts of cell surface CD4, and infectivity was analyzed by the focal infectivity assay. The infectivity for the vif deletion NL4-3 virions ranged from 25 to 50 focus-forming units (FFU) per 0.1 ml of virus. Each point is the mean of three independent experiments, and the error bars represent standard errors. FL U, fluorescence units.

FIG. 3

Expression of HIV-1 vif does not enhance the infectivity of xenotropic MLV particles. H9 cells infected with xenotropic virus were superinfected with wild-type (wt) or vif deletion HIV-gpt virus. Expression of BV2 p30^Gag (A) or HIV-1 viral proteins (B) in infected H9 cells was monitored by Western immunoblot analyses. Molecular weights of protein standards are indicated on the left, in thousands. (C) Medium containing viral particles was collected from the infected cells and used to infect HeLa cells, and infectivity was analyzed by a focal infectivity assay using p30^Gag antiserum. The graph shows the BV2 titer in a representative experiment. FFU, focus-forming units.