Cellular inhibitors with Fv1-like activity restrict human and simian immunodeficiency virus tropism

Abstract

Many nonhuman primate cells are unable to support the replication of HIV-1, whereas others are nonpermissive for infection by simian immunodeficiency virus from macaques (SIVmac). Here, we show that restricted HIV-1 and SIVmac infection of primate cell lines shares some salient features with Fv1 and Ref1-mediated restriction of murine retrovirus infection. In particular, the nonpermissive phenotype is most evident at low multiplicities of infection, results in reduced accumulation of reverse transcription products, and is dominant in heterokaryons generated by fusion of permissive and nonpermissive target cells. Moreover, in nonpermissive primate cells, HIV-1 and SIVmac infection is cooperative, and enveloped HIV-1 virus-like particles, minimally containing Gag and protease, abrogate restriction. In African green monkey cells, HIV-1 virus-like particles ablate restrictions to HIV-1 and SIVmac, suggesting that both are restricted by the same factor. Finally, a virus that contains an HIV-1 capsid-p2 domain in an SIVmac background exhibits a tropism for primate cells that is HIV-1-like rather than SIVmac-like. These data indicate the existence of one or more saturable inhibitors that are polymorphic in primates and prevent HIV and SIV infection by targeting the capsid of the incoming lentivirus particle.

Figure 1

Titration of HIV-1 and SIVmac reporter viruses on primate cells. The indicated cells lines were inoculated with increasing amounts of VSV-G-pseudotyped GFP reporter viruses. Infected cells were enumerated by FACS.

Figure 2

Reduced accumulation of HIV-1 cDNA in nonpermissive primate cells at low MOI. (A) HeLa, Rh.F, and OMK cells were inoculated with varying amounts of VSV-G-pseudotyped HIV/GFP and reverse transcripts measured by real-time PCR 48 h later. Alternatively, (B) cells were inoculated with 25 ng of HIV/GFP, and reverse transcripts were measured at the indicated times after infection.

Figure 3

Cooperative infection of nonpermissive cells at high MOI. (A) OMK and HeLa cells were inoculated with 1 ng of HIV/GFP(VSV) in the presence of increasing amounts of pseudotyped or bald HIV-1 or SIVmac virions. (B) Pindak or HeLa cells were inoculated with 1 ng of SIV/GFP(VSV) in the presence of the indicated amounts of VSV-G pseudotyped SIVmac virions. For both A and B, reporter virus infection was measured by FACS.

Figure 4

Resistance to HIV-1 infection is dominant in heterokaryons. OMK or HeLa cells expressing CAT and GFP were fused with 293T cells expressing an HTLV-I envelope protein and inoculated with HIV(MuLV-E), and infected cells were revealed by immunofluorescence. (A) Representative photomicrographs showing GFP expression (Left) and HIV immunofluorescence (Right) in the same fields of cells. (B) Titers of HIV(MuLV-E) measured on CAT and GFP expressing OMK or HeLa cells cocultivated with 293T cells that did or did not express HTLV-I envelope. Infected heterokaryons were counted separately from infected unfused cells.

Figure 5

HIV-1 VLPs enhance HIV and SIVmac infection in a target-cell-dependent manner. (A) OMK cells were inoculated with 1 ng HIV/GFP(VSV) in the presence of the indicated amounts of VSV-G enveloped or bald Gag-Pol VLPs (Left). Alternatively, enveloped Gag-Pr or Gag VLPs were used (Right). (B) The indicated cell lines were inoculated with 0.2 ng of HIV/GFP(VSV) (Left) or 1 ng SIV/GFP(VSV) (Right) reporter viruses in the presence of increasing levels of enveloped or bald HIV-1 Gag-Pol VLPs. Infected cells were enumerated by FACS.

Figure 6

The CA-p2 domain of Gag is a major target for restriction. The indicated cell lines were inoculated with varying amounts (as indicated) of VSV-G-pseudotyped HIV/GFP, SIVmac/GFP, or a chimeric virus, SIV(HIV CA-p2)/GFP. Infections were done in the absence (black symbols) or presence (white symbols) of 20 ng per well HIV-1 Gag-Pol VLPs. Infected cells were enumerated by FACS.