Session 36Poster Presentations Accessory Genes Session Day and Time: Tuesday 1:30 - 3:30 pm Room: Hall D
209 Evidence of a Novel Nef Activity Involved in nef-Mediated Thymocyte Depletion in the Thymus R. D'Agostin*, L. Su Univ of North Carolina, Chapel Hill
Background: We have previously shown that in the human thymus the HIV-1 Nef protein functions as a pathogenic determinant in the HXB/LW virus without affecting viral replication. Nef is known to have many functions including CD4 downregulation, MHC I downregulation, and alteration of several T-lymphocyte cell signaling pathways. We hypothesize that one or more of Nef’s functions are involved in mediating thymocyte depletion. HXB/LW replication and pathogenesis in the fetal human thymus organ culture (HFTOC) model is an excellent system for examining Nef’s function in thymocyte depletion separately from Nef’s effects on viral replication.
Methods: Using site-directed mutagenesis, a series of HXB/LW mutants were created that encode nef-mutants defective in modulating signaling, MHC I or CD4 downregulation. Two (2) of these mutants failed to interact with Pak1/2 and ASK (RR106) or V1H and Raf1 (DD175). The HXB/LW nef-mutant viruses were amplified in PBMCs and used to infect HFTOC. Thymocytes were harvested at 8 dpi and 14 dpi and virus replication levels measured by cell-associated p24. FACS analysis was used to examine thymocytes for CD4 and CD8 expression as well as overall cell viability.
Results: All of the HXB/LW nef-mutant viruses replicated to similar levels as the HXB/LW nef-wild-type virus. In HFTOC, the nef-mutant virus exhibited thymocyte depletion comparable to that seen with nef-wild type virus. When examined by FACS, the CD4+ (single-positive and double-positive) thymocyte population was similarly depleted by HXB/LW nef-mutant and HXB/LW nef-wild type infections.
Conclusions: These data suggest Nef motifs are involved in CD4 or MHC Class I downregulation, and Nef interactions with Pak1/2, ASK, Raf1, and V1H are not required for nef-mediated thymocyte depletion in vivo. A novel Nef activity resulting in thymocyte depletion in the thymus is implicated.
