Functional analysis of HIV-1 Vpr: identification of determinants essential for subcellular localization.
Vpr is a conserved HIV-1 auxiliary protein that localizes to the nuclear region of cells. Vpr is also present in virions, and it is directed into the assembling virus when coexpressed with Gag. Each of these two localization activities may be important for Vpr function, and we recently identified regions of Vpr that are critical for virion incorporation. In this study we analyzed the Vpr domains involved in subcellular localization. Immunofluorescence staining of transfected cells showed that wild-type Vpr localized exclusively to the nuclear region. Mutations in the N-terminal domain that were designed to disrupt a predicted alpha-helical structure resulted in aberrant localization, while conservative substitutions showed a wild-type pattern. A region in the central portion of the protein also has the potential for helical structure, and mutagenesis of two conserved amino acids in this domain (A59, H71) impaired localization, while substitution of a third (Q65) did not. In contrast, neither the conserved Gly and Cys at positions 75-76 nor the C-terminal basic residues (R87, K95) were necessary for nuclear localization. In addition, two-residue insertions within and between the two putative helices disrupted localization but insertion in the C-terminal region did not. Thus, Vpr's subcellular localization function depends on the two putative helical domains but is independent of the conserved Gly-Cys motif and of specific C-terminal basic residues.
Mahalingam
S
S
Department of Microbiology and Immunology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Collman
R G
RG
Patel
M
M
Monken
C E
CE
Srinivasan
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry*; D017124:Conserved Sequence; D016336:Gene Products, vpr / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D054325:vpr Gene Products, Human Immunodeficiency Virus
D000596:Amino Acids; D016336:Gene Products, vpr; D054325:vpr Gene Products, Human Immunodeficiency Virus