Natural Single-Nucleotide Variations in the HIV-1 Genomic SA1prox Region Can Alter Viral Replication Ability by Regulating Vif Expression Levels

Abstract

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.

Analysis of Competing HIV-1 Splice Donor Sites Uncovers a Tight Cluster of Splicing Regulatory Elements within Exon 2/2b

“…Nomaguchi et al identified natural single-nucleotide variations within different HIV-1 isolates proximal to HIV-1 A1 (SA1prox) that could be shown to regulate vif mRNA and Vif protein expression and were linked to the fact that effective viral replication critically depends on an optimal Vif/A3G ratio (17,46). Here, we found nucleotide variations predominantly within splicing regulatory elements in exon 2/2b.…”

“…Vif is essential for counteracting the host cell restriction factor A3G, and an imbalance in the Vif/A3G ratio strongly affects viral replication. It was shown that if restriction pressure is low, lower Vif levels are sufficient to counteract A3G, whereas excessive Vif impedes viral replication ability (19,46). However, conversely, HIV-1 replicates only in cells with high restriction pressure if sufficient Vif is present (17,46).…”

“…Here, we found nucleotide variations predominantly within splicing regulatory elements in exon 2/2b. Therefore, it is tempting to speculate that the vast number of SREs within exon 2/2b ensures viral replication in cells with different A3G or splicing regulatory protein concentrations, e.g., by a mechanism like mutual evolution (46).…”

Virological characterization of HIV‐1 CA‐NTD mutants constructed in a virus‐lineage reflected manner

“…Lymphocyte cell lines H9 (human) and HSC-F (cynomolgus macaque) were maintained in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (19,21).…”

In silico Analyses of Subtype Specific HIV-1 Tat-TAR RNA Interaction Reveals the Structural Determinants for Viral Activity

“…It is known that a single mutation in the viral proteins could modulate the viral replication (Nomaguchi et al, 2014 , 2016 ). Tat-TAR interaction is essential for LTR transactivation that account for the viral pathogenesis (Feng and Holland, 1988 ; Garcia et al, 1989 ; Puglisi et al, 1992 ), while any modification in the structural residues of Tat-TAR complex interaction could affect viral gene expression (Cordingley et al, 1990 ).…”