Separation of Human Immunodeficiency Virus Type 1 Replication from nef-Mediated Pathogenesis in the Human Thymus

Human immunodeficiency virus type 1 (HIV-1) diseases (AIDS) are associated with high levels of HIV-1 viremia and depletion of CD4+ T lymphocytes. In vivo, HIV-1 can infect diverse cell types, including CD4+ T cells, macrophages, dendritic cells, Langerhans cells, and hematopoietic progenitor cells (13, 24, 30, 39, 42). However, the HIV-1 isolates used in many studies have been expanded and maintained in immortalized human T-cell lines. The different selective pressures in vitro have led to the generation of variants with attenuated replication and pathogenicity in vivo (38). Many laboratory-adapted isolates of HIV-1 show defects in the functions of some genes, such as env, vpr, vpu, and nef (38). A good example of such adaptation in vitro is Lai/IIIB (human T-cell lymphotropic virus strain IIIB) (6). Initially derived from a human patient blood sample and cultured in MT2/B cells, Lai/IIIB stock was prepared by infecting the human leukemia T-cell line H9 with infected MT2/B-cell supernatant. Subsequent analyses of the genome from the Lai/IIIB isolate showed that multiple changes accumulated during expansion in vitro (38). For example, the HXB2 genome cloned from Lai/IIIB carries mutations that lead to premature termination of three of the nine open reading frames (ORFs): vpr, vpu, and nef. Many other subtle mutations also may have accumulated. These mutations do not usually affect HIV-1 replication in vitro under specific culture conditions, although some of them may enhance viral replication in certain cell lines. However, it remains unclear which mutations contribute to attenuated HIV-1 replication and pathogenesis in vivo. It has been difficult to analyze HIV-1 functions specifically involved in pathogenicity. Mutations in putative pathogenic factors such as nef or env in simian immunodeficiency virus (SIV) or HIV-1 have led to reduced viremia in vivo (1, 14, 34, 40). Thus, the reduced pathology may be due to a reduced viral load in vivo. Such pathogenic factors are therefore also replication factors. Transgenic mouse models in which nef is expressed constitutively in thymocytes and CD4+ T and macrophage cells lead to CD4+ T-cell depletion and other AIDS-like symptoms, suggesting that nef may be a principal pathogenic factor (15). However, problems arise with transgenic mouse models during attempts to correlate nef expression with virus-mediated pathogenicity. First, the level and time of transgenic nef expression are very different from those in HIV-1 infection in humans. Second, murine host cells may respond differently to HIV-1 nef proteins. Therefore, whether nef is a factor for replication and/or pathogenicity in vivo is still not clear. Comparison of HIV-1 isolates attenuated in vitro with pathogenic revertants in vivo will help to identify viral determinants important for replication and pathogenesis in vivo. One example of a factor involved in pathogenicity is found in SHIV (SIV-HIV env chimeric genome) adapted in monkeys. SHIV variants with enhanced replication and pathogenicity have been isolated from monkeys infected with SHIV recombinant viruses (19). Mutations in HIV env genes have been identified which contribute to enhanced replication in monkeys. Interestingly, env determinants have also been defined that specifically contribute to CD4+ T-cell depletion (i.e., pathogenicity) but not replication in monkeys (12). Therefore, unique env determinants have intrinsic pathogenic activity in monkeys. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T-cell lines such as H9 as well as peripheral blood mononuclear cells (PBMCs) in vitro but to be replication defective in vivo (14, 34, 40). When a laboratory worker was accidentally infected by Lai/IIIB, infectious virus was isolated from plasma by infection of primary PBMCs but not by infection of T-cell lines (22, 43). We have previously used SCID-hu Thy/Liv mice as an in vivo model (29, 31) to study the replication of HXB2 and of HXB2 recombinant viruses containing HIV-1 fragments isolated from the infected laboratory worker (40). Like Lai/IIIB, HXB2 failed to replicate in the Thy/Liv organ (4, 40). Replacement of an HXB2 subgenomic fragment containing the env ORF with the corresponding fragment from the laboratory worker isolate (LW12.3) generated a recombinant virus (HXB/LW) which replicated in SCID-hu Thy/Liv mice and in the human fetal thymus organ culture (HF-TOC) model (23, 40). The specific in vivo replication determinants were mapped to the region from V1 to V3 of the HXB/LW env gene (40). Therefore, the attenuated Lai/IIIB isolate acquired in vivo replication activity by mutational reversion of the env gene in the infected laboratory worker. We used the SCID-hu Thy/Liv model to study the pathogenic activity of HXB/LW. HXB/LW replicated to high levels in the SCID-hu Thy/Liv mouse and HF-TOC models. However, the pathogenic activity of HXB/LW was significantly reduced. Restoration of the defective nef ORF in HXB/LW resulted in enhanced viral pathogenicity, with no significant effect on viral replication, in both the SCID-hu Thy/Liv mouse and the HF-TOC models. Thus, we demonstrated in this study that it was possible to separate the replication activity of a recombinant HIV-1 clone from its pathogenicity in vivo and that an intact nef ORF was able to serve exclusively as an HIV-1 pathogenic factor in the replication-competent recombinant virus.