MUTAGENIC ANALYSIS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 VPR - ROLE OF A PREDICTED N-TERMINAL ALPHA-HELICAL STRUCTURE IN VPR NUCLEAR-LOCALIZATION AND VIRION INCORPORATION

The Vpr gene product of human immunodeficiency virus type I is a virio n associated protein that is important for efficient viral replication in nondividing cells such as macrophages. At the cellular level, Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins, Incorporation of Vpr into viral particles requi res a determinant within the p6 domain of the Gag precursor polyprotei n Pr55(gag). In the present study, we have used site-directed mutagene sis to identify a domain(s) of Vpr involved in virion incorporation an d nuclear localization, Truncations of the carboxyl (C)-terminal domai n, rich in basic residues, resulted in a less stable Vpr protein and i n the impairment of both virion incorporation and nuclear localization , However, introduction of individual substitution mutations in this r egion did not impair Vpr nuclear localization and virion incorporation , suggesting that this region is necessary for the stability and/or op timal protein conformation relevant to these Vpr functions, In contras t, the substitution mutations within the amino (N)-terminal region of Vpr that is predicted to adopt an alpha-helical structure (extending f rom amino acids 16 to 34) impaired both virion incorporation and nucle ar localization! suggesting that this structure may play a pivotal rol e in modulating both of these biological properties. These results are in agreement with a recent study showing that the introduction of pro line residues in this predicted alpha-helical region abolished Vpr vir ion incorporation, presumably by disrupting this secondary structure ( S. Mahalingam, S. A. Khan, R. Murali, M. A. Jabbar, C. E. Monken, R. G . Collman, and A, Srinivasan, Proc, Natl. Acad. Sci. USA 92:3794-3798, 1995). Interestingly, our results show that two Vpr mutants harboring single amino acid substitutions (L to F at position 23 [L23F] and A30 F) on the hydrophobic face of the predicted helix coded for relatively stable proteins that retained their ability to translocate to the nuc leus but exhibited dramatic reduction in Vpr incorporation, suggesting that this hydrophobic face might mediate protein-protein interactions required for Vpr virion incorporation but not nuclear localization, F urthermore, a single mutation (E25K) located on the hydrophilic face o f this predicted alpha-helical structure affected not only virion inco rporation but also nuclear localization of Vpr, The differential impai rment of Vpr nuclear localization and virion incorporation by mutation s in the predicted N-terminal alpha-helical region suggests that this region of Vpr plays a role in both of these biological functions of Vp r.