Functional analysis of the simian immunodeficiency virus Vpx protein: Identification of packaging determinants and a novel nuclear targeting domain

The vpx gene products of human immunodeficiency virus type 2 (HIV-2) and of the closely related simian immunodeficiency viruses from sooty mangabeys ( SIVsm) and macaques (SIVmac) comprise a 112 amino-acid virion-associated pr otein that is critical for efficient virus replication in nondividing cells such as macrophages. When expressed in the absence of other viral proteins , Vpx localizes to the nuclear membrane as well as to the nucleus; however, in the context of virus replication Vpx is packaged into virions via inter action with the p6 domain of the Gag precursor polyprotein (p55(gag)). To i dentify the domains essential for virion incorporation and nuclear localiza tion, site-directed mutations were introduced into the vpx gene of SIVsmPBj 1.9 and functionally analyzed. Our results show that (i) mutation of two hi ghly conserved L74 and I75 residues impaired both virion incorporation and nuclear localization of Vpx; (ii) substitution of conserved H82, G86, C87, P103, and P106 residues impaired Vpx nuclear localization but not virion in corporation; (iii) mutations of conserved Y66, Y69, and Y71 residues impair ed virion incorporation but not the translocation of Vpx to the nucleus; an d (iv) a mutation at E30 (predicted to disrupt an N-terminal alpha -helix) had no effect on either virion incorporation or nuclear localization of Vpx . Importantly, mutations in Vpx which impaired nuclear localization also re duced virus replication in macaque macrophages, suggesting an important rol e of the carboxyl terminus of Vpx in nuclear translocation of the viral pre integration complex. Analyzing this domain in greater detail, we identified a 26-amino-acid (aa 60 to 85) fragment that was sufficient to mediate the transport of a heterologous protein (green fluorescent protein [GFP]) to th e nucleus. Taken together, these results indicate that virion incorporation and nuclear localization are encoded by two partially overlapping domains in the C-terminus of Vpx (aa 60 to 112). The identification of a novel 26-a mino-acid nuclear targeting domain provides a new tool to investigate the n uclear import of the HIV-2/SIV preintegration complex.