Structure-functional analysis of human immunodeficiency virus type 1 (HIV-1) Vpr: role of leucine residues on Vpr-mediated transactivation and virus replication.
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 Vprwt. 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.
Thotala
Dineshkumar
D
Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Schafer
Elizabeth A
EA
Tungaturthi
Parithosh K
PK
Majumder
Biswanath
B
Janket
Michelle L
ML
Wagner
Marc
M
Srinivasan
Alagarsamy
A
Watkins
Simon
S
Ayyavoo
Velpandi
V
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D016336:Gene Products, vpr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015658:HIV Infections / Q000821:virology*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007930:Leucine / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D015728:Receptors, HIV / Q000378:metabolism; D013997:Time Factors; D015533:Transcriptional Activation; D014779:Virus Replication; D054325:vpr Gene Products, Human Immunodeficiency Virus
D004268:DNA-Binding Proteins; D016336:Gene Products, vpr; D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid; D015728:Receptors, HIV; D054325:vpr Gene Products, Human Immunodeficiency Virus; C422457:vpr peptide (13-33), Human immunodeficiency virus 1; D007930:Leucine