RESUMO
In this study, we performed a mutational analysis to determine whether the mechanism by which HIV-2 Vpx confers the capacity for infectivity and viral replication in macrophages is solely dependent on its ability to degrade the host antiviral factor SAMHD1. Contrary to expectations, we demonstrated that P(109) in the C-terminal poly-proline motif of HIV-2 Vpx has two unique roles: to facilitate the specific degradation of SAMHD1 in macrophages, and to facilitate multimerization of Vpx, therefore preventing SAMHD1 degradation in the presence of high levels of Vpx.
Assuntos
HIV-2/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Prolina/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Células HEK293 , HIV-2/genética , Humanos , Immunoblotting , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Prolina/genética , Proteólise , Proteína 1 com Domínio SAM e Domínio HD , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
Human immunodeficiency virus type 2 (HIV-2) carries an accessory protein Vpx that is important for viral replication in natural target cells. In its C-terminal region, there is a highly conserved poly-proline motif (PPM) consisting of seven consecutive prolines, encoded in a poly-pyrimidine tract. We have previously shown that PPM is critical for Vpx expression and viral infectivity. To elucidate the molecular basis underlying this observation, we analysed the expression of Vpx proteins with various PPM mutations by in vivo and in vitro systems. We found that the number and position of consecutive prolines in PPM are important for Vpx expression, and demonstrated that PPM is essential for efficient Vpx translation. Furthermore, mutational analysis to synonymously disrupt the poly-pyrimidine tract suggested that the context of PPM amino acid sequences is required for efficient translation of Vpx. We similarly analysed HIV-1 and HIV-2 Vpr proteins structurally related to HIV-2 Vpx. Expression level of the two Vpr proteins lacking PPM was shown to be much lower relative to that of Vpx, and not meaningfully enhanced by introduction of PPM at the C terminus. Finally, we examined the Vpx of simian immunodeficiency virus from rhesus monkeys (SIVmac), which also has seven consecutive prolines, for PPM-dependent expression. A multi-substitution mutation in the PPM markedly reduced the expression level of SIVmac Vpx. Taken together, it can be concluded that the notable PPM sequence enhances the expression of Vpx proteins from viruses of the HIV-2/SIVmac group at the translational level.
