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J Biol Chem, Vol. 274, Issue 13, 9083-9091, March 26, 1999

Incorporation of Vpr into Human Immunodeficiency Virus Type 1 Requires a Direct Interaction with the p6 Domain of the p55 Gag Precursor

From the Laboratoire de rétrovirologie humaine, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3C 3J7, Canada

The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55^gag), its incorporation requires an anchor to associate with the assembling viral particles. Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55^gag corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein. In order to clarify the mechanism by which the Vpr accessory protein is trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55^gag using the yeast two-hybrid system and the maltose-binding protein pull-down assay. The present study provides genetic and biochemical evidence indicating that the Pr55^gag can physically interact with the Vpr protein. Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6^gag completely abolish the interaction between Vpr and the Pr55^gag and, as a consequence, prevent Vpr virion incorporation. In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55^gag. Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal -helical structure is essential for the Vpr-Pr55^gag interaction. Vpr and the Pr55^gag demonstrate a strong interaction in vitro as salt concentrations as high as 900 mM could not disrupt the interaction. Finally, the interaction is efficiently competed using anti-Vpr sera. Together, these results strongly suggest that Vpr trans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6^gag. The development of Pr55^gag-Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions.

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