Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr

Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include c ell cycle arrest at G(2), nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, an d interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magneti c resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic h elix (HIII) in the middle region. Though the importance of helical domains HI and Hm has been defined with respect to Vpr functions, the role of helic al domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/ or substitution mutagenesis. To enable the detection of Vpr, the sequence c orresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/tr anslation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII show ed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay i nvolving cotransfection of NL Delta Vpr proviral DNA and a Vpr expression p lasmid was employed to analyze the virion incorporation property of Vpr. Mu tant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL3 9G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also f ailed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants Vpr Delta 35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL e xhibited both cytoplasmic and nuclear localization, unlike other mutants an d control Vpr-FL. While wild-type Vpr registered cell cycle arrest at G(2), mutant Vpr showed an intermediary effect with the exception of Vpr Delta 3 5-50 and Vpr Delta 35-50-H. These results suggest that residues in the HII domain are essential for Vpr functions.