Session 36Poster Presentations Accessory Genes Session Day and Time: Tuesday 1:30 - 3:30 pm Room: Hall D
207 Vascular Endothelial Cells Induce Dramatically Higher Levels of HIV-1 Replication in a Nef-dependent Manner J. Choi, J. Walker, J.S. Pober, L. Alexander* Yale Univ, New Haven, CT
Background: HIV-1 infected T-cells in the peripheral blood are in frequent contact with vascular endothelial cells (VEC) that form the lining of blood vessels. We investigated if the interaction of these cells could result in an increased level of HIV-1 replication and, if so, could Nef mediate this increase.
Methods: Human umbilical-vein derived VEC monolayers were either left untreated or were treated with interferon gamma to induce HLA-DR, a stimulus for CD4 T-cell allogeneic proliferation. Unstimulated human CD4+ T-cells, purified from PBMCs, were incubated with these cultures or were incubated in wells that did not contain VECs. These cultures were infected with HIV-1 strain NL4-3 containing either wild-type Nef sequences (Nef+) or a derivative strain containing a large deletion in Nef (Nef-). The level of virus produced in these cultures was assessed by p24Gag ELISA.
Results: Low but detectable levels of HIV-1 (Nef+ or Nef-) replication were observed in cultures without VECs. Conversely, in the presence of VECs, Nef+ HIV-1 replicated to approximately 50-fold higher levels than did Nef-HIV-1. Pretreatment of the VECs with interferon gamma resulted in peak virus production 3 days earlier than with untreated VECs, but the level of peak virus production was approximately equivalent in these conditions.
Conclusions: These studies suggest that Nef responds to signal(s) provided by VECs to HIV-1 infected T-cells and results in vastly higher levels of virus replication. To our knowledge, no previously described tissue culture assay has revealed such a significant contribution of Nef to virus replication, and thus, provides a system to for structure/function studies of Nef. Furthermore, this system reflects conditions that are encountered by HIV-1 in the peripheral blood, especially during the progressive stage of infection. Thus, the observed effect of Nef likely contributes to efficient viral replication in vivo and to progression to AIDS.
