Regulation of HIV-2 Vpx/Vpr expression by the poly-proline motif: Enhancement of protein dimerization and translation

Human immunodeficiency virus type 2 (HIV-2) encodes the Vpx and Vpr proteins. Although these proteins are homologous, Vpx is expressed at substantially higher levels than those of Vpr. This difference can be explained by the presence of a zinc-binding site and a poly-proline motif (PPM) consisting of seven consecutive prolines, both of which are present in Vpx but absent in Vpr. This study aims to elucidate the mechanism by which the PPM regulates Vpx/Vpr expression. Experiments using point mutants at positions P¹⁰⁸ and P¹⁰⁹ (the last proline of the PPM) in Vpx showed that P¹⁰⁹ is critical for replication in macrophages and is important for achieving sufficient expression levels and dimerization capacity of the Vpx protein, compared with P¹⁰⁸. In contrast, introduction of the PPM into the Vpr protein increased its expression level in an in vitro transcription/translation system while decreasing mRNA expression. In addition, no significant increase in protein stability was detected. These results, together with additional mutational analyses, demonstrate that introduction of the PPM enhances translation of the Vpr protein. In contrast, introduction of the PPM into Human immunodeficiency virus type 1 (HIV-1) Vif did not increase expression levels. Furthermore, a synthetic peptide containing seven consecutive prolines did not bind to immunoprecipitated Vpx protein. Overall, Vpx dimerization is not considered to be caused by dimerization of the PPM region. Collectively, these findings indicate that the PPM of Vpx enhances protein dimerization, leading to increased stability, and that introduction of the PPM into Vpr enhances its translation.