Session 36Poster Presentations Accessory Genes Session Day and Time: Tuesday 1:30 - 3:30 pm Room: Hall D
214 Glucocorticoid Receptor Modulates HIV-1 Replication Through Interaction with the Leucine Motifs in Helix I and III of Vpr D Kumar*1, S Mahalingam2, M Wagner1, B Gray1, SP Singh3, A Srinivasan3, P Gupta1, V Ayyavoo1 1Univ of Pittsburgh, PA; 2Ctr For DNA Fingerprinting and Diagnostics, Hyderabad, India; and 3Thomas Jefferson Univ, Philadelphia, PA
Background: HIV-1Vpr has been shown to transactivate HIV-1 LTR through the GRE domain via interaction with Glucocorticoid receptor (GR). Steroid receptors are known to bind proteins with signature motifs such as LxxLL sequences. Based on the observance of such motifs in HIV-1 Vpr, in this study we propose to identify the domains involved in Vpr-GR interaction and evaluate the subcellular localization of GR and GRE-mediated transactivation activity.
Methods: A panel of Vpr mutants (LxxLL domians in Helix I and III) was generated by site–directed mutagenesis substituting Leucine residues to Alanine. All the mutants were tested for their expression by transfection and immuno-precipitation. Immuno-fluorescence analysis was performed in HeLa cells co-transfected with pGR and pVpr mutant expression plasmids to determine the domains involved in Vpr-GR translocation. HeLa cells were co-transfected with Vpr mutant plasmids and pGRE 5x-Luciferase. Luciferase activity of the lysate was measured as RLUs by using the luciferase substrate. Single round infection assay were performed on PBMCs using pseudotype viruses to evaluate the role Vpr-GR interaction in HIV replication.
Results: Mutations (L64xxL67L68) at position 67 and 68 abolished Vpr-induced GRE-transactivation significantly, where as mutations in the first helix (L20xL22L23xxL26) did not alter GRE or HIV-1 LTR induced transcriptional activity. Interestingly, substitution mutations at position L22A has resulted in a transdominant GRE and HIV-1 LTR transactivation (10-fold higher compared to wild-type Vpr) as well as increased viral production in the single round infection assay. However, immuno-fluoresence studies indicate that subcellular pattern of Vpr did not alter the GR localization.
Conclusions: HIV-1 Vpr functions as a co-activator for GR and activates GRE mediated transactivation. Mutations in L67, 68 abolished the transactivation suggests that LxxLL domian in III helix is important for Vpr-mediated GRE function. These observations link the functions of leucine rich motifs of HIV-1 Vpr to transcriptional regulation and virus replication to the host Glucocorticoid Receptor pathway. Ability of Vpr mutants to transactivate GR (GRE-Luciferase) directly correlates with the viral replication in a single round infection assay suggesting an early role for virion-associated Vpr in HIV-1 infection through the GR pathway.
