Identification of the (15)FRFG domain in HIV-1 Gag p6 essential for Vpr packaging into the virion

The auxiliary regulatory protein Vpr of HIV-1 is packaged in the virion through interaction with the Gag C-terminal p6 domain. Virion packaging of Vpr is critical for Vpr to exert functions in the HIV-1 life cycle. Previous studies suggest that Vpr interacts with a (Lxx)4 domain in p6 for virion packaging. In the present study, mutational analysis of HIV-1 Gag p6 domain was performed in the context of the HIV-1 genome to examine the effect on virion packaging of Vpr. Surprisingly, Ala substitutions for Leu(44 )and Phe(45 )in the (Lxx)4 domain or deletion of the whole (Lxx)4 domain (amino acid #35–52 of the Gag p6 domain) did not affect Vpr virion packaging. Vpr virion packaging was normal when amino acid #1–23 of the Gag p6 domain was preserved. Most importantly, Ala substitutions for Phe(15), Arg(16 )and Phe(17 )in the context of amino acid #1–23 of the Gag p6 domain abolished Vpr virion packaging. Single Ala substitutions for Phe(15 )and Phe(17 )also abolished Vpr virion packaging, whereas Ala substitution for Arg(16 )had no effect. Our studies have revealed a novel signal sequence for Vpr packaging into the HIV-1 virion. The (15)FRFG domain in p6 resembles the FxFG repeat sequences commonly found in proteins of the nuclear pore complex. These results have provided novel insights into the process of virion packaging of Vpr and suggest for the first time that Vpr may recognize the FxFG domain for both virion packaging and association with nuclear pores.