IDENTIFICATION OF HIV-1 DETERMINANTS FOR REPLICATION IN-VIVO

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, bu t probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparis on must be made between attenuated genomes and those that remain patho genic in vivo. In this study, we used the heterochimeric SCID-hu Thy/L iv mouse as an in vivo model to define human immunodeficiency virus ty pe 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 re plicate in the SCID-hu Thy/Liv model. When a lab worker was accidental ly infected by Lai/IIIB, however, HIV-1 was isolated only from infecti on of primary PBMC, and not from infection of T cell lines. We hypothe sized that the lab worker was exposed to a heterogeneous viral stock w hich had been attenuated by passage in immortalized T cell lines. Eith er a rare family member from this stock was selected for in vivo repli cation or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypo thesis, we have used the SCID-hu Thy/Liv model to study the replicatio n of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolate d from the infected lab worker. HXB2 showed no or very low levels of r eplication in the Thy/Liv organ. Replacement of its subgenomic fragmen t encoding the envelope gene with a corresponding fragment from the la b worker isolate generated a recombinant virus (HXB2/LW) which replica ted actively in SCID-hu mice. The NEF mutation in the HXB2 genome is s till present in HXB2/LW. Thus, the LW sequences encode HIV-1 determina nts which enhance HIV replication in vivo in a NEF-independent mechani sm. 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. (C) 1997 Academic Press