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.

Keywords: HIV-1; SA1D2prox; Vif expression; adaptation; nucleotide sequence; splicing.

Figure 1

SA1D2prox sequence on the HIV-1 genome and virus adaptation procedure. (A) HIV-1 (NL4-3) genome organization. The SA1D2prox region is shown by a black box. Some of the splicing sites (SD1, SA1, SD2, and SA7) of the HIV-1 genome are indicated. Unique restriction enzyme sites (SbfI and BsaBI) used to make adapted viral clones are indicated. (B) SA1D2prox sequence. Sequences of low (NL-D229gat) and excessive (NL-R224cgc and NL-P238ccg) Vif types used for the adaptation experiments in this study are shown by blue and green letters/lines, respectively. Blue, orange, and green letters without underlines indicate single-nucleotide mutants identified by us [37,38,39] which display low, high, and excessive Vif types, respectively. Boxed, bold letters show adaptive mutations that change the Vif expression levels identified in this study. Splicing sites (SA1 and SD2) are indicated. Several known splicing enhancer/silencer motifs are represented by gray letters and lines [18,19,20,21], (for review, see [16]). nSNM, naturally-occurring single nucleotide mutation. (C) Virus adaptation. Viral clones of low (NL-D229gat) and excessive (NL-R224cgc and NL-P238ccg) Vif types were inoculated into H9 cells and cultured for the indicated periods. Fresh H9 cells were infected with the SUP collected on day 118 for NL-D229gat and day 134 for NLR224cgc/NL-P238ccg. On day 18 post-infection, cellular genomic DNA was extracted and used for the construction of adapted viral clones.

Figure 2

Characteristics of low-Vif type NL-D229gat and excessive-Vif type NL-R224cgc/NL-P238ccg for Vif expression level and A3G degrading activity. (A) Vif expression kinetics. HEK 293T cells were transfected with the indicated pro-viral clones. On day 1 and day 2 (d1 and d2, respectively) post-transfection, cell lysates were prepared and subjected to Western blotting analysis using anti-Vif and anti-β-actin antibodies. Representative data from two independent experiments are shown. (B) A3G antagonizing activity. The indicated pro-viral clones along with a FLAG-tagged A3G expression vector were co-transfected into HEK 293T cells. On day 1 post-transfection, virions and cells were collected and lysed for Western blotting analysis using anti-Vif, anti-Gag-p24, anti-FLAG, and anti-β-actin antibodies. Representative data from two independent experiments are shown.

Figure 3

Characteristics of viral clones constructed from adapted NL-D229gat. (A) Growth kinetics. Viruses prepared from HEK 293T cells transfected with the indicated pro-viral clones were inoculated into H9 cells (1 × 10⁴ RT units/10⁵ cells). Virus replication was monitored by the virion-associated RT activity in the culture supernatants. This experiment was performed once to select viral clones that have the ability to grow in cells. Viral replication kinetics of WT NL4-3 and a parental NL-D229gat are presented in both panels for easy comparison. (B) Vif expression levels. HEK 293T cells were transfected with the indicated pro-viral clones. On day 1 post-transfection, cell lysates were prepared and subjected to Western blotting analysis using anti-Vif and anti-β-actin antibodies. An empty vector pUC19 and an authentic HIV-1 NL4-3 clone were used as negative and positive controls, respectively. Numbers correspond to those for the adapted clones shown in panel (A). Representative data from two independent experiments are shown.

Figure 4

Characteristics of viral clones constructed from adapted NL-R224cgc. (A) Growth kinetics. Viruses prepared from HEK 293T cells transfected with the indicated pro-viral clones were inoculated into H9 cells (2 × 10³ RT units/10⁵ cells). Virus replication was monitored by the virion-associated RT activity in the culture supernatants. This experiment was performed once to select viral clones that have the ability to grow in cells. Viral replication kinetics of WT NL4-3 and a parental NL-R224cgc are presented in both panels for easy comparison. (B) Vif expression levels. Western blotting analysis was carried out as described in Figure 3B. Numbers correspond to those for the adapted clones shown in panel (A). Representative data from two independent experiments are shown. Short, short exposure; Long, long exposure.

Figure 5

Characteristics of viral clones constructed from adapted NL-P238ccg. (A) Growth kinetics. Viruses prepared from HEK 293T cells transfected with the indicated pro-viral clones were inoculated into H9 cells (1 × 10⁴ RT units/10⁵ cells). Virus replication was monitored by the virion-associated RT activity in the culture supernatants. This experiment was performed once to select viral clones that have the ability to grow in cells. Viral replication kinetics of WT NL4-3 and a parental NL-P238ccg are presented in both panels for easy comparison. (B) Vif expression levels. Western blotting analysis was carried out as described in Figure 3B. Numbers correspond to those for the adapted clones shown in panel (A). Representative data from two independent experiments are shown.

Figure 6

Effects of adaptive mutations within SA1D2prox on the Vif expression level. HEK 293T cells were transfected with the indicated pro-viral clones. On day 1 post-transfection, cell lysates were prepared for Western blotting analysis using anti-Vif and anti-β-actin antibodies. Adaptive mutations that change the Vif expression levels found within the SA1D2prox region of NL-D229gat, NL-R224cgc, and NL-P238ccg are shown in Table 2, Table 3 and Table 4, respectively. Representative data from two independent experiments are shown. gat, D229gat; aag, R269Kaag; cgc, R224cgc; tat, Y227tat; ccg, P238ccg; ggg, G237ggg.

Figure 7

Vif expression levels of a newly constructed minigenome (pcNLmini-RI) vector. (A) Organization of the minigenome. The pcNLmini-RI vector was constructed as described in the Materials and Methods. Splicing (D1, A1, D2, A2, D3) and EcoRI sites in the minigenome vector corresponding to those in the authentic HIV-1 NL4-3 clone are shown. A black box indicates the SA1D2prox region. CMV promoter, cytomegalovirus enhancer-promoter; BGH pA, bovine growth hormone polyadenylation sequence. (B) Vif expression levels of the minigenome vectors carrying various mutations. HEK 293T cells were transfected with the indicated minigenome vectors, and on day 1 post-transfection, cell lysates were prepared for Western blotting analysis using anti-Vif and anti-β-actin antibodies. Mutations introduced into the minigenome vector were the same as those shown in Figure 5. An empty vector (pcDNA3.1) was used as a negative control. Representative data from two independent experiments are shown. WT, wild type; gat, D229gat; aag, R269Kaag; cgc, R224cgc; tat, Y227tat; ccg, P238ccg; ggg, G237ggg.

Figure 8

Effects of adaptive mutations on mRNA production. (A) mRNA produced from the minigenome. Splicing sites on the minigenome are presented. The primers used for the splicing pattern analysis are indicated by arrows with their names (upper part). Colored boxes show exons produced by splicing at various sites. In the lower part, splicing products analyzed using the primer sets are presented along with their lengths. Broken and solid lines show regions with and without splicing, respectively. (B,C) Changes in the splicing pattern of parental clones (gat, cgc, and ccg) by their adaptive mutations. Total RNA was prepared from HEK 293T cells transfected with the indicated minigenome vectors and subjected to semiquantitative RT-PCR using the primer pairs shown in (A). The signal intensities of semiquantitative RT-PCR products were quantitated from three independent experiments. The intensities of the indicated mRNAs in each sample were normalized to those of all viral mRNAs (D1) and gapdh mRNA. The normalized mRNA intensities in each sample relative to those of WT are presented.

Figure 9

SA1D2prox sequences of NL4-3, its derivatives, and clinical samples. Sequence alignments were carried out using Genetyx Ver. 15. Numbers and underlines above the sequences represent Pol-IN amino acid positions (NL4-3 numbering) and codons, respectively. (A) Sequences of NL4-3, NL-pC2, and NL-pC3. NL-pC2 and NL-pC3 clones have 2 and 3 nucleotide substitutions, respectively, which are major nucleotides at each position in the SA1D2prox sequence within the HIV-1 subtype B population. (B,C) Chronological changes of the SA1D2prox sequence in samples obtained at different time points from PI-resistant patients PI1 (B) and PI4 (C). Sequence data were published previously [55] (DDBJ; PRJDB3502). Clone name was designated PI1-A, -B, and -C (B) and PI4-A, -B, -C, and -D (C) based on the sequence identity. (D,E) Chronological changes of the SA1D2prox sequence in samples obtained at different time points from RAL-resistant patients RAL4 (D) and RAL5 (E). Sequence data were published previously [55] (DDBJ; PRJDB3502). Clone name was designated RAL4-A, -B, and -C (D) and RAL5-A, -B, and -C (E) based on the sequence identity and the chronological order.

Figure 9

SA1D2prox sequences of NL4-3, its derivatives, and clinical samples. Sequence alignments were carried out using Genetyx Ver. 15. Numbers and underlines above the sequences represent Pol-IN amino acid positions (NL4-3 numbering) and codons, respectively. (A) Sequences of NL4-3, NL-pC2, and NL-pC3. NL-pC2 and NL-pC3 clones have 2 and 3 nucleotide substitutions, respectively, which are major nucleotides at each position in the SA1D2prox sequence within the HIV-1 subtype B population. (B,C) Chronological changes of the SA1D2prox sequence in samples obtained at different time points from PI-resistant patients PI1 (B) and PI4 (C). Sequence data were published previously [55] (DDBJ; PRJDB3502). Clone name was designated PI1-A, -B, and -C (B) and PI4-A, -B, -C, and -D (C) based on the sequence identity. (D,E) Chronological changes of the SA1D2prox sequence in samples obtained at different time points from RAL-resistant patients RAL4 (D) and RAL5 (E). Sequence data were published previously [55] (DDBJ; PRJDB3502). Clone name was designated RAL4-A, -B, and -C (D) and RAL5-A, -B, and -C (E) based on the sequence identity and the chronological order.

Figure 9

SA1D2prox sequences of NL4-3, its derivatives, and clinical samples. Sequence alignments were carried out using Genetyx Ver. 15. Numbers and underlines above the sequences represent Pol-IN amino acid positions (NL4-3 numbering) and codons, respectively. (A) Sequences of NL4-3, NL-pC2, and NL-pC3. NL-pC2 and NL-pC3 clones have 2 and 3 nucleotide substitutions, respectively, which are major nucleotides at each position in the SA1D2prox sequence within the HIV-1 subtype B population. (B,C) Chronological changes of the SA1D2prox sequence in samples obtained at different time points from PI-resistant patients PI1 (B) and PI4 (C). Sequence data were published previously [55] (DDBJ; PRJDB3502). Clone name was designated PI1-A, -B, and -C (B) and PI4-A, -B, -C, and -D (C) based on the sequence identity. (D,E) Chronological changes of the SA1D2prox sequence in samples obtained at different time points from RAL-resistant patients RAL4 (D) and RAL5 (E). Sequence data were published previously [55] (DDBJ; PRJDB3502). Clone name was designated RAL4-A, -B, and -C (D) and RAL5-A, -B, and -C (E) based on the sequence identity and the chronological order.

Figure 10

Vif expression levels of pro-viral clones carrying different SA1D2prox sequences in the HIV-1 NL4-3 genome. HEK 293T cells were transfected with the indicated pro-viral clones, and on day 1 post-transfection, cell lysates were prepared and subjected to Western blotting analysis using anti-Vif and anti-β-actin antibodies. Representative data from two independent experiments are shown.