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 nucle ar region of cells. Vpr is also present in virions, and it is directed into the assembling virus when coexpressed with Gag. Each of these tw o localization activities may be important for Vpr function, and we re cently identified regions of Vpr that are critical for Virion incorpor ation. In this study we analyzed the Vpr domains involved in subcellul ar localization. Immunofluorescence staining of transfected cells show ed that wild-type Vpr localized exclusively to the nuclear region. Mut ations in the N-terminal domain that were designed to disrupt a predic ted alpha-helical structure resulted in aberrant localization, while c onservative substitutions showed a wild-type pattern. A region in the central portion of the protein also has the potential for helical stru cture, and mutagenesis of two conserved amino acids in this domain (A5 9, H71) impaired localization, while substitution of a third (Q65) did not. In contrast, neither the conserved Gly and Cys at positions 75-7 6 nor the C-terminal basic residues (R87, K95) were necessary for nucl ear localization. In addition, two-residue insertions within and betwe en the two putative helices disrupted localization but insertion in th e C-terminal region did not. Thus, Vpr's subcellular localization func tion depends on the two putative helical domains but is independent of the conserved Gly-Cys motif and of specific C-terminal basic residues . (C) 1995 Academic Press, Inc.