We previously found that natural single-nucleotide variations located within a proximal region of splicing acceptor 1 (SA1prox) in the HIV-1 genome could alter the viral replication potential and mRNA expression pattern, especially the vif mRNA level. Here, we studied the virological and molecular basis of nucleotide sequence variations in SA1prox for alterations of viral replication ability. Consistent with our previous findings, variant clones indeed expressed Vif at different levels and grew distinctively in cells with various APOBEC3G expression levels. Similar effects were observed for natural variations found in HIV-2 SA1prox, suggesting the importance of the SA1prox sequence. To define nucleotides critical for the regulation of HIV-1 Vif expression, effects of natural SA1prox variations newly found in the HIV Sequence Compendium database on vif mRNA/Vif protein levels were examined. Seven out of nine variations were found to produce Vif at lower, higher, or more excessive levels than wild-type NL4-3. Combination experiments of variations giving distinct Vif levels suggested that the variations mutually affected vif transcript production. While low and high producers of Vif grew in an APOBEC3G-dependent manner, excessive expressers always showed an impeded growth phenotype due to defects in single-cycle infectivity and/or virion production levels. The phenotype of excessive expressers was not due primarily to inadequate expression of Tat or Rev, although SA1prox variations altered the overall HIV-1 mRNA expression pattern. Collectively, our results demonstrate that HIV SA1prox regulates Vif expression levels and suggest a relationship between SA1prox and viral adaptation/evolution given that variations occurred naturally. IMPORTANCEWhile human cells possess restriction factors to inhibit HIV-1 replication, HIV-1 encodes antagonists to overcome these barriers. Conflicts between host restriction factors and viral counterparts are critical driving forces behind mutual evolution. The interplay of cellular APOBEC3G and viral Vif proteins is a typical example. Here, we demonstrate that naturally occurring single-nucleotide variations in the proximal region of splicing acceptor 1 (SA1prox) of the HIV-1 genome frequently alter Vif expression levels, thereby modulating viral replication potential in cells with various ABOBEC3G levels. The results of the present study reveal a previously unidentified and important way for HIV-1 to compete with APOBEC3G restriction by regulating its Vif expression levels. We propose that SA1prox plays a regulatory role in Vif counteraction against APOBEC3G in order to contribute to HIV-1 replication and evolution, and this may be applicable to other primate lentiviruses.

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.

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Human immunodeficiency virus type 1 (HIV-1) is tropic and pathogenic only for humans, and does not replicate in macaque monkeys routinely used for experimental infections. This specially narrow host range (species tropism) has impeded much the progress of HIV-1/acquired immunodeficiency syndrome (AIDS) basic research. Extensive studies on the underlying mechanism have revealed that Vif, one of viral accessory proteins, is critical for the HIV-1 species tropism in addition to Gag-capsid protein. Another auxiliary protein Vpu also has been demonstrated to affect this HIV-1 property. In this review, we focus on functional interactions of these HIV-1 proteins and species specific-restriction factors. In addition, we describe an evolutional viewpoint that is relevant to the species tropism of HIV-1 controlled by the accessory proteins.

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