In vivo analysis of Nef’s role in HIV-1 replication, systemic T cell activation and CD4+ T cell loss

Richard L Watkins, John L Foster, J Victor Garcia

Nef is a multifunctional HIV-1 protein critical for progression to AIDS. Humans infected with nef(-) HIV-1 have greatly delayed or no disease consequences. We have contrasted nef(-) and nef(+) infection of BLT humanized mice to better characterize Nef's pathogenic effects. Mice were inoculated with CCR5-tropic HIV-1JRCSF (JRCSF) or JRCSF with an irreversibly inactivated nef (JRCSFNefdd). In peripheral blood (PB), JRCSF exhibited high levels of viral RNA (peak viral loads of 4.71 × 10(6) ± 1.23 × 10(6) copies/ml) and a progressive, 75% loss of CD4(+) T cells over 17 weeks. Similar losses were observed in CD4(+) T cells from bone marrow, spleen, lymph node, lung and liver but thymocytes were not significantly decreased. JRCSFNefdd also had high peak viral loads (2.31 × 10(6) ± 1.67 × 10(6)) but induced no loss of PB CD4(+) T cells. In organs, JRCSFNefdd produced small, but significant, reductions in CD4(+) T cell levels and did not affect the level of thymocytes. Uninfected mice have low levels of HLA-DR(+)CD38(+)CD8(+) T cells in blood (1-2%). Six weeks post inoculation, JRCSF infection resulted in significantly elevated levels of activated CD8(+) T cells (6.37 ± 1.07%). T cell activation coincided with PB CD4(+) T cell loss which suggests a common Nef-dependent mechanism. At 12 weeks, in JRCSF infected animals PB T cell activation sharply increased to 19.7 ± 2.9% then subsided to 5.4 ± 1.4% at 14 weeks. HLA-DR(+)CD38(+)CD8(+) T cell levels in JRCSFNefdd infected mice did not rise above 1-2% despite sustained high levels of viremia. Interestingly, we also noted that in mice engrafted with human tissue expressing a putative protective HLA-B allele (B42:01), JRCSFNefdd exhibited a substantial (200-fold) reduced viral load compared to JRCSF. Nef expression was necessary for both systemic T cell activation and substantial CD4(+) T cell loss from blood and tissues. JRCSFNefdd infection did not activate CD8(+) T cells or reduce the level of CD4(+) T cells in blood but did result in a small Nef-independent decrease in CD4(+) T cells in organs. These observations strongly support the conclusion that viral pathogenicity is mostly driven by Nef. We also observed for the first time substantial host-specific suppression of HIV-1 replication in a small animal infection model.