Session 36Poster Presentations Accessory Genes Session Day and Time: Tuesday 1:30 - 3:30 pm Room: Hall D
212 Identification of the Domain within the Subtype C Vpu that is Responsible for Efficient Transport to the Cell Surface D K Singh, E Pacyniak, D Hout, E B Stephens* Univ of Kansas Med Ctr, Kansas City
Background: Vpu is a small non-structural transmembrane protein encoded by human immunodeficiency virus type 1 (HIV-1) that is synthesized off the same mRNA as the Env protein. Using a recently developed VpuEGFP reporter system, we have compared the biological properties of Vpu proteins from subtype B and C isolates of HIV-1. Using this reporter system, we determined the Vpu from the well studied subtype B Vpu protein was localized predominantly within the RER/Golgi complex whereas fusion proteins constructed with the Vpu proteins from several subtype C isolates of HIV-1 were efficiently expressed on the cell surface. In this study, we determined what domain(s) of the subtype C Vpu was responsible for efficient transport to the cell surface.
Methods: A series of 6 chimeric Vpu proteins were created in which the N-terminal (NTD), transmembrane (TM), and cytoplasmic (CD) domains were exchanged between the subtype B and C proteins. The chimeric proteins expressed as EGFP fusion proteins and assessed for intracellular location by fluorescence confocal microscopy, intracellular stability, and CD4 down-regulation.
Results: All chimeric Vpu proteins that retained the cytoplasmic domain of the subtype B Vpu were found to be localized predominantly within the RER/Golgi complex while those chimeric proteins retaining the cytoplasmic domain of the subtype C Vpu protein were found to be efficiently transported to the cell surface. All chimeric proteins were effective in preventing CD4 from being expressed on the cell surface.
Conclusions: These results indicate that the cytoplasmic domain is responsible for the differences in the intracellular localization of these 2 Vpu proteins. These results provide additional evidence that the Vpu proteins from subtype C isolates of HIV-1, which account for approximately 50% of the HIV-1 infections world-wide, differ fundamentally from the subtype B Vpu in intracellular transport properties.
