Identification of HIV-1 Determinants for Replicationin Vivo

Abstract

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.

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

“…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.…”

“…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).…”

“…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).…”

“…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).…”

“…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).…”