The Expression Level of HIV-1 Vif Is Optimized by Nucleotide Changes in the Genomic SA1D2prox Region during the Viral Adaptation Process

Abstract

HIV-1 Vif plays an essential role in viral replication by antagonizing anti-viral cellular restriction factors, a family of APOBEC3 proteins. We have previously shown that naturally-occurring single-nucleotide mutations in the SA1D2prox region, which surrounds the splicing acceptor 1 and splicing donor 2 sites of the HIV-1 genome, dramatically alter the Vif expression level, resulting in variants with low or excessive Vif expression. In this study, we investigated how these HIV-1 variants with poor replication ability adapt and evolve under the pressure of APOBEC3 proteins. Adapted clones obtained through adaptation experiments exhibited an altered replication ability and Vif expression level compared to each parental clone. While various mutations were present throughout the viral genome, all replication-competent adapted clones with altered Vif expression levels were found to bear them within SA1D2prox, without exception. Indeed, the mutations identified within SA1D2prox were responsible for changes in the Vif expression levels and altered the splicing pattern. Moreover, for samples collected from HIV-1-infected patients, we showed that the nucleotide sequences of SA1D2prox can be chronologically changed and concomitantly affect the Vif expression levels. Taken together, these results demonstrated the importance of the SA1D2prox nucleotide sequence for modulating the Vif expression level during HIV-1 replication and adaptation.

Altered HIV-1 mRNA Splicing Due to Drug-Resistance-Associated Mutations in Exon 2/2b

“…The 3′ end of HIV-1 pol is of particular interest since it is interspersed with a dense network of splicing regulatory elements that have been shown to mediate the use of splice acceptor SA1 (MaxEnt 6.41) and splice donors SD2 (HBond score 10.7) and SD2b (HBond score 12.4) ( Figure 1 B) [ 22 , 26 , 34 , 35 ]. HEXplorer plots show the predicted impact of the two secondary resistance-associated mutations on the ESE2b SRE.…”

“…Recently, Koma et al described naturally occurring single nucleotide mutations in the terminal pol region that are caused by adaptation in an APOBEG restricted setting that led to varied Vif expression levels. They show that excessive or insufficient expression of Vif has detrimental effects on viral replication and suggest that the adaptive mutations alter Vif expression mainly via splicing [ 34 , 35 ]. However, at exon 2, the Vif mRNA overlaps with the integrase reading frame.…”

Involvement of a Rarely Used Splicing SD2b Site in the Regulation of HIV-1 vif mRNA Production as Revealed by a Growth-Adaptive Mutation

“…Proviral clones pNL4-3 [31], pNL-IN-Y226tac (Y226tac) [28][29][30], and pcNLmini-RI [32] have been described previously. The introduction of mutations into these clones was done by PCR-based site-directed mutagenesis.…”

“…The virion-associated reverse transcriptase (RT) activity was measured as previously described [37,38] to quantify virus amounts. Virus samples were inoculated into H9 cells and the infected cells were maintained as the long-term culture in Figure 2 as previously described [32]. Potentially adapted proviral clones derived from the long-term culture were constructed by introducing the PCR-amplified fragments from SbfI to BsaBI sites (Figure 1) into the corresponding sites of pNL4-3 as described previously [32].…”

“…Virus samples were inoculated into H9 cells and the infected cells were maintained as the long-term culture in Figure 2 as previously described [32]. Potentially adapted proviral clones derived from the long-term culture were constructed by introducing the PCR-amplified fragments from Sbf I to BsaBI sites (Figure 1) into the corresponding sites of pNL4-3 as described previously [32]. The sequence analysis of adapted viral clones was done for the region from Sbf I to BsaBI sites.…”