TY - JOUR
T1 - Structure-functional analysis of human immunodeficiency virus type 1 (HIV-1) Vpr
T2 - Role of leucine residues on Vpr-mediated transactivation and virus replication
AU - Thotala, Dineshkumar
AU - Schafer, Elizabeth A.
AU - Majumder, Biswanath
AU - Janket, Michelle L.
AU - Wagner, Marc
AU - Srinivasan, Alagarsamy
AU - Watkins, Simon
AU - Ayyavoo, Velpandi
N1 - Funding Information:
HIV-1 LTR-luciferase construct, pNL43 R + E − and pNL43 R − E − , pHXB2-Env, pHIV-1 LTR-luciferase plasmids and CEM-GFP were obtained from the AIDS Research and reference reagent Program, Division of AIDS, NIAID. We thank Dr. Pavlakis for his generous gift of RU486 for our initial experiments. We thank Dr. Frank Jenkins at the University of Pittsburgh for CV-1 cells and Dr. Michelle Calos, at Stanford University, for 293T cells. We thank Dr. Indranil Mukhopadhyay, University of Pittsburgh for his help with the statistical analysis. This work was supported by AI50463 from NIAID, NIH to VA.
PY - 2004/10/15
Y1 - 2004/10/15
N2 - HIV-1 Vpr has been shown to transactivate LTR-directed expression through its interaction with several proteins of cellular origin including the glucocorticoid receptor (GR). Upon activation, steroid receptors bind to proteins containing the signature motif LxxLL, translocate into the nucleus, bind to their response element, and activate transcription. The presence of such motifs in HIV-1 Vpr has prompted us to undertake the analysis of the role of specific leucine residue(s) involved in Vpr-GR interaction, subcellular localization and its effect on Vpr-GR-mediated transactivation. The individual leucine residues present in H I, II, and III were mutated in the Vpr molecule and evaluated for their ability to interact with GR, transactivate GRE and HIV-1 LTR promoters, and their colocalization with GR. While Vpr mutants L42 and L67 showed reduced activation, substitutions at L20, L23, L26, L39, L64, and L68 exhibited a similar and slightly higher level of activation compared to Vpr wt. Interestingly, a substitution at residue L22 resulted in a significantly higher GRE and HIV-1 LTR transactivation (8- to 11-fold higher) in comparison to wild type. Confocal microscopy indicated that Vpr L22A exhibited a distinct condensed nuclear localization pattern different from the nuclear/perinuclear pattern noted with Vprwt. Further, electrophoretic mobility shift assay (EMSA) revealed that the VprL22A-GR complex had higher DNA-binding activity when compared to the wild type Vpr-GR complex. These results suggest a contrasting role for the leucine residues on HIV-1 LTR-directed transactivation dependent upon their location in Vpr.
AB - HIV-1 Vpr has been shown to transactivate LTR-directed expression through its interaction with several proteins of cellular origin including the glucocorticoid receptor (GR). Upon activation, steroid receptors bind to proteins containing the signature motif LxxLL, translocate into the nucleus, bind to their response element, and activate transcription. The presence of such motifs in HIV-1 Vpr has prompted us to undertake the analysis of the role of specific leucine residue(s) involved in Vpr-GR interaction, subcellular localization and its effect on Vpr-GR-mediated transactivation. The individual leucine residues present in H I, II, and III were mutated in the Vpr molecule and evaluated for their ability to interact with GR, transactivate GRE and HIV-1 LTR promoters, and their colocalization with GR. While Vpr mutants L42 and L67 showed reduced activation, substitutions at L20, L23, L26, L39, L64, and L68 exhibited a similar and slightly higher level of activation compared to Vpr wt. Interestingly, a substitution at residue L22 resulted in a significantly higher GRE and HIV-1 LTR transactivation (8- to 11-fold higher) in comparison to wild type. Confocal microscopy indicated that Vpr L22A exhibited a distinct condensed nuclear localization pattern different from the nuclear/perinuclear pattern noted with Vprwt. Further, electrophoretic mobility shift assay (EMSA) revealed that the VprL22A-GR complex had higher DNA-binding activity when compared to the wild type Vpr-GR complex. These results suggest a contrasting role for the leucine residues on HIV-1 LTR-directed transactivation dependent upon their location in Vpr.
KW - Glucocorticoid receptor
KW - Replication
KW - Transactivation
UR - http://www.scopus.com/inward/record.url?scp=4544322878&partnerID=8YFLogxK
U2 - 10.1016/j.virol.2004.07.013
DO - 10.1016/j.virol.2004.07.013
M3 - Article
C2 - 15380361
AN - SCOPUS:4544322878
VL - 328
SP - 89
EP - 100
JO - Virology
JF - Virology
SN - 0042-6822
IS - 1
ER -