Role of poly-proline motif in HIV-2 Vpx expression

Human and simian immunodeficiency viruses (HIV and SIVs) contain several auxiliary genes not found in other retroviruses. These genes are thought to be functionally important for optimal viral replication and persistence in infected individuals. Primate lentiviruses can be classified by the composition of these accessory genes. While viruses of the HIV type1 (HIV-1) group have vif, vpr, vpu, and nef genes, those of the HIV-2 group carry vif, vpx, vpr, and nef genes (Fujita et al., 2010). Vpx protein encoded by the vpx gene is unique to non-HIV-1 viruses, and is essential for viral replication in macrophages in contrast to its structural paralog Vpr (Fujita et al., 2010). The most outstanding sequence feature to distinguish Vpx from Vpr is the presence of poly-proline motif (PPM) at its C-terminal region. We have recently shown, by in vitro and in vivo assay systems, that the PPM in HIV-2 Vpx is critical for its efficient translation (Miyake et al., 2014). Although PPM consisting of seven consecutive prolines has been demonstrated to be required for efficient HIV-2 Vpx translation, thereby acquiring viral infectivity in macrophages, the effects of PPM mutations on the degradation of Vpx in cells was not formally analyzed as yet (Fujita et al., 2008; Miyake et al., 2014). Therefore, in this study, we asked whether the PPM plays a role in keeping away from proteasomal and/or lysosomal degradation (Figure 1). In order to assess this, we used various expression plasmids for HIV-2 Vpx (pEF-Fvpx series) described in a previous study (Miyake et al., 2014): wild-type (WT) plasmid has the vpx gene derived from HIV-2 GL-AN clone (Kawamura et al., 1994); mutants 103/4A and 106/4A have four consecutive alanine-substitutions at the site of P103-P106 and P106-P109, respectively, and have been shown to express a low/minimum level of mutant Vpx proteins in cells (Figure 1A); a negative control is a frame-shift mutant pEF-FxSt that lacks Vpx expression ( Vpx). Various expression plasmids were transfected into human 293T cells (Lebkowski et al., 1985) as described