Poly-proline motif in HIV-2 Vpx is critical for its efficient translation

Abstract

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.

Molecular Determinants for Recognition of Divergent SAMHD1 Proteins by the Lentiviral Accessory Protein Vpx

“…In agreement, SIV mac Vpx lacking the poly-proline tail is still able to assemble into a ternary SAMHD1/Vpx/DCAF1 complex and to induce cullin-4-dependent SAMHD1 poly-ubiquitination (Ahn et al., 2012) and degradation (Belshan et al., 2012). Functional studies suggest involvement of VR3 in nuclear localization of Vpx and Vpr (Pancio et al., 2000; Zhou et al., 1998) with consequences for viral replication (Belshan et al., 2012), but notably, mutations in this region also influence protein expression levels significantly (Miyake et al., 2014). …”

Construction of a molecular clone of ovine enzootic nasal tumor virus

“…Vpx proteins of the HIV-2/SIVsmm/SIVmac group encode a highly conserved polyproline motif (PPM) consisting of a hepta-proline stretch in the C-terminal region [43, 44]. Recently, the PPM in HIV-2 Vpx was shown to be critical for its efficient expression in both eukaryotic and prokaryotic systems and this effect is determined by the context of PPM amino acid sequences, not the nucleotide sequences [45]. Importantly, the number and position of consecutive prolines in this PPM were important for Vpx expression.…”

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

“…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).…”

“…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 (ΔVpx).…”