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Functional Analysis of HIV-2/SIV Vpx: Identification of Determinants Essential for Subcellular Localization and Virion Packaging S. MAHALINGAM* and B. H. HAHN. Univ. of Alabama, Birmingham Vpx is a HIV-2/SIVsm-specific 112 aa protein that is selectively incorporated into virus particles, localizes to the nucleus, and is required for viral replication in macrophages. To delineate functional domains of Vpx, we introduced point mutations into the vpx gene of the infectious molecular clone SIVsmPBJ1.9. Immunofluorescence staining of eukaryotically expressed Vpx showed that the wild-type protein localized to the nucleus. Mutations designed to disrupt a predicted (N-terminal) a-helix resulted in a wild-type expression pattern. Substitution of conserved Leu74 and Ile75 impaired subcellular localization as well as virion packaging of Vpx. Interestingly, alteration of conserved Tyr66, 69, and 71 retained the ability of Vpx to translocate to the nucleus, but impaired its virion incorporation. In contrast, neither the conserved His82, Gly86, Cys87, nor the C-terminal prolines (Pro103,106) were necessary for the virion packaging but did impair subcellular localization of Vpx. Proviral PBJ1.9 Vpx variants which failed to package Vpx proteins were severely impaired in their ability to replicate in macrophages. However, PBJ1.9 variants with mutations in His82, Gly86, Cys87, and Pro103,106 of Vpx also failed to replicate in macrophages despite the fact that they contained near wild-type quantities of Vpx protein. These results suggest that aa 66 to 75 are important for virion incorporation, while the domain between aa 74–112 is essential for the nuclear localization of Vpx. Studies are underway to determine whether the latter domain is important for interaction of Vpx with components of the nuclear import machinery. Key Words: Subcellular Localization, Virion Packaging, Vpx Mutagenesis |
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© 7th
Conference on Retroviruses and Opportunistic Infections, |