Fitness Disadvantage of Transitional Intermediates Contributes to Dynamic Change in the Infecting-Virus Population during Coreceptor Switch in R5 Simian/Human Immunodeficiency Virus-Infected Macaques

Cell culture.Rhesus peripheral blood mononuclear cells (RhPBMC) were obtained by Ficoll-Hypaque gradient purification followed by stimulation with 3 μg/ml staphylococcus enterotoxin B (SEB) (Sigma-Aldrich, St. Louis, MO) in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 20 U/ml of interleukin-2 (IL-2) (Novartis, Emeryville, CA). 293T cells and TZM-bl cells expressing CD4, CCR5, and CXCR4 and containing integrated reporter genes for firefly luciferase and β-galactosidase under the control of the HIV-1 long terminal repeat (LTR) (58) were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS, penicillin, streptomycin, and l-glutamine. U87 cells stably expressing CD4 and one of the chemokine receptors (17) were maintained in DMEM supplemented with 10% FBS, antibiotics, 1 μg/ml puromycin (Sigma-Aldrich), and 300 μg/ml G418 (Geneticin; Invitrogen, Carlsbad, CA).

DNA, RNA extraction, sequencing, and analysis.Proviral DNA was extracted from 3 × 10⁶ infected macaque PBMC or tissue cells with a DNA extraction kit, and viral RNA was prepared from 500 μl plasma using a commercially available RNA extraction kit (Qiagen, Valencia, CA) followed by reverse transcription with Superscript III reverse transcriptase (RT) (Invitrogen, Carlsbad, CA) and random hexamer primers (Amersham Pharmacia, Piscataway, NJ). The V1 to V5 region of gp120 was amplified from the viral DNA or RT products by Taq DNA polymerase (Qiagen) with primers ED5 and ED12 or ES7 and ES8 as previously described (16). PCR products were cloned with the TOPO TA cloning kit (Invitrogen) per the manufacturer's instructions, followed by direct automated sequencing of cloned gp120 amplicons (Genewiz, South Plainfield, NJ). Nucleotide sequences were aligned with ClustalX (36) and edited manually using BioEdit V7.0.9. A phylogenetic tree was constructed using the maximum likelihood method, and bootstrap values were generated with 1,000 repetitions.

Sequence-specific PCR.For detection of the Δ22-25 V3 sequence, plasma cDNA products were subjected to PCR using Hot Star Taq DNA polymerase with primers V3-del (5′-AATTAAAACTGTGCATTACAA-3′) and WR8 (5′-CGGGGAGAGCATTTTACATA-3′) using the following cycling conditions: 95°C for 10 min followed by 35 cycles of 95°C for 30 s, 57.5°C for 20 s, and 72°C for 20 s and final extension at 72°C for 10 min. The sensitivity of the detection assay for Δ22-25 V3 sequence was 1 variant copy among 10⁴ R5 targets. For detection of RRW/RRW.A V3 sequences, primers SH85 (5′-AAAAGTATACATATAAGAAGGT-3′) and V3-OAS (5′-CAGTAGAAAAATTCCCCTCCACA-3′) were used with the following cycling profile: 95°C for 10 min followed by 35 cycles of 95°C for 30 s, 47.7°C for 20 s, and 72°C for 20 s, with the final extension at 72°C for 10 min. The sensitivity of detection for RRW/RRW.A was 1 variant copy among 10⁶ R5 targets. Amplified products were visualized by electrophoresis in ethidium bromide (EtBr)-stained 2% agarose gels.

Plasmid constructs and pseudotype virus production.The generation of DG08 Env expression plasmids and luciferase-reporter viruses has been described previously (43). For expression of CA28 envelope glycoproteins, full-length gp160 coding sequence was amplified from RT products with primers SH43 (5′-AAGACAGAATTCATGAGAGTGAAGGGGATCAGGAAG-3′) and SH44 (5′-AGAGAGGGATCCTTATAGCAAAGCCCTTTCAAAGCCCT-3′) and subcloned into the pCAGGS vector. To generate luciferase reporter viruses capable of only a single round of replication, an envelope trans-complementation assay was used as previously described (12). Briefly, Env expression plasmid and the NL4.3LucE-R- vector were cotransfected by polyethyleneimine (PEI) (Polyscience, Warrington, PA) into 2.5 × 10⁶ 293T cells plated in a 100-mm plate. Cell culture supernatants were harvested 72 h later, filtered through 0.45-μm-pore-size filters, and stored at −70°C in 1-ml aliquots. Pseudoviruses were quantified for p24gag content (Beckman Coulter, Fullerton, CA).

Determination of entry efficiency and coreceptor usage.For assessment of entry efficiency and coreceptor usage of reporter viruses, 7 × 10³ TZM-bl, U87.CD4.CCR5, or U87.CD4.CXCR4 cells were seeded in 96-well plates 24 h before use and infected, in triplicate, with 3 ng p24gag equivalent of the indicated pseudovirions followed by incubation for 72 h at 37°C. At the end of the incubation period, the cells were harvested, lysed, and processed for activity according to the manufacturer's instructions (Luciferase Assay System; Promega, Madison, WI). Entry, as quantified by luciferase activity, was measured with an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, VA). For the entry blocking assays, U87.CD4 indicator cells were pretreated with 4-fold serial dilutions of TAK-779, PSC-RANTES, or AMD3100 for 30 min at 37°C before infection. The cells were lysed after 72 h of incubation at 37°C and processed for β-galactosidase activity (Galacto-Star system; Applied Biosystems, Bedford, MA). The percentage of entry blocking was calculated by the amount of entry in the presence of the inhibitor relative to that in the absence of the inhibitor, and 50% inhibitory concentration (IC₅₀) was determined using the Prism 4 software (GraphPad, San Diego, CA).

Statistical analysis.Analysis of IC₅₀ comparing two groups was performed by the Mann-Whitney U test and considered statistically significant at P < 0.05.

V3 loop deletion variants predominate at the time of first X4 appearance in macaque CA28.To further extend our understanding of the spectrum of variants present at the time of coreceptor switch in R5 SHIV_SF162P3N-infected macaques, we investigated the transitional intermediates in CA28. The envelope (Env) V1-V5 sequence of circulating viruses in this animal at 11 wpi, a time corresponding to the earliest detection of X4 variants bearing the signature V3 RRW.A mutations by sequence-specific PCR, was determined. Surprisingly, clonal sequence analysis revealed that 34 of 38 Env clones amplified from plasma of CA28 at this time had four amino acid deletions in the C terminus of the V3 loop (Δ22-25) that increased the net charge of this domain to +6 (Fig. 1A). Of the remaining clones, three had wild-type (WT) sequence, while one harbored mutations in the V3 loop that changed the GPG crown loop sequence to RRW but with the downstream alanine residue intact. This RRW V3 mutant was also a minor Env variant in the PBMC (3 of 70 clones sequenced), with 21 of 70 clones in this blood compartment harboring the V3 deletion sequence and with the majority (>60%) containing WT sequence. At the time of necropsy 4 weeks later, however, the Δ22-25 V3 deletion mutant now represented a minor population in the plasma of CA28 (4 of 31 clones sequenced) and was undetectable in the PBMC. The RRW-bearing V3 variants were also undetectable among over 30 clones sequenced from the plasma and PBMC of CA28 at necropsy. Instead, 1 of 31 plasma envelope clones had the signature X4 RRW.A V3 loop motif. Sequence-specific PCR analysis on plasma of CA28 showed that the Δ22-25 V3 loop variant could be detected as early as 9 wpi, 2 weeks prior to the detection of RRW- and RRW.A-bearing variants in this animal (Fig. 1B).

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FIG. 1.

Infecting virus population in macaque CA28 during coreceptor switch. (A) V3 loop sequence comparison of representative SHIV_SF162P3N clones (P3N-1, P3N-2) and week (w)11 and w15 plasma and PBMC viruses in macaque CA28. Dots indicate gaps, dashes stand for identity in sequences, and the net positive charge of this region is shown on the right. Positions 11 and 25 within the V3 loop are indicated by arrows, and the 4-amino-acid deletion is underlined in the reference sequence. The numbers in parentheses represent the numbers of clones matching the indicated sequence per total number of clones sequenced. Amino acid residues predicted to confer CXCR4 usage are boxed. (B) Tracking the emergence of RRW/RRW.A- and Δ22-25-bearing variants in plasma using sequence-specific PCR. wpi, week postinfection; NC, negative control using plasma from an uninfected macaque; M, marker. (C) Distribution of V3 variants in tissue compartments of macaque CA28 at time of necropsy as determined by clonal sequence analysis. Numbers in parentheses indicate numbers of gp120 clones sequenced from each of the tissue sites. Ing, inguinal LN; Mes, mesenteric LN; Axi, axillary LN; Ili, iliac LN; Col, colonic LN; IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte from the jejunum.

The poor representation of the signature X4 RRW.A V3 loop sequences in the blood of macaque CA28 at the time of necropsy (15 wpi) prompted us to examine their presence in tissue sites. This is because previous observations in macaques BR24 and DG08 suggested that peripheral lymph nodes (LNs) are the preferred sites of X4 evolution and amplification (43, 54). The results showed ready detection of the signature X4 RRW.A sequence in the axillary (35%), iliac (19%), and inguinal (20%) LN but not in the gut (intraepithelial lymphocytes [IEL] or lamina propria lymphocytes [LPL]) or in intestinal LNs such as the colonic and mesenteric LN (Fig. 1C). The RRW-bearing V3 variants, however, could not be detected in any of the tissue sites examined by clonal analysis at end-stage disease. In contrast, the Δ22-25 deletion variant has a much wider tissue distribution, detectable in both the gut and lymph nodes, particularly in the colonic LN, where 14 of 14 clones sequenced contained this V3 motif. The finding of signature X4 V3 sequences in peripheral lymph nodes and not in the gut (IELs and LPLs) of macaque CA28 is in agreement with previous observations in macaques BR24 and DG08, lending further support for secondary LNs as the preferred sites of X4 virus emergence and expansion. Furthermore, the wider distribution and greater representation of the Δ22-25 V3 variant in tissue sites are consistent with its emergence and expansion prior to that of the RRW and RRW.A mutant viruses in CA28.

Distinct dualtropic variants coexist in macaque CA28.The Δ22-25 V3 variant in CA28 harbors the same V3 deletion sequence that has been shown to confer CXCR4 usage to a variant in macaque DG08 which retained and preferred CCR5 for entry (Table 1). To determine coreceptor usage of the Δ22-25 as well as the RRW V3 mutants in CA28, full-length gp160 envelopes (Envs) were amplified and used in the generation of pseudotyped reporter viruses for infectivity studies. To enhance our chances of obtaining these variant envelopes, especially those with RRW mutations which are present at extremely low levels, viruses recovered from peripheral blood of CA28 at 15 wpi were used as a source for amplification. We find that the proportions of these underrepresented variants increased with low-passage propagation of plasma- or PBMC-associated viruses (data not shown). Pseudoviruses expressing WT and X4 RRW.A Envs amplified from propagated viruses were also obtained and served as coexisting R5 and X4 viruses, respectively. As expected, pseudoviruses expressing WT Env infected U87.CD4.CCR5 but not U87.CD4.CXCR4 cells, whereas the RRW.A-bearing virus showed the opposite phenotype, infecting U87.CD4.CXCR4 but not U87.CD4.CCR5 cells (Fig. 2A). The Δ22-25 V3 mutant in CA28 could infect U87.CD4 cells expressing either CCR5 or CXCR4, while viruses expressing Envs with the RRW V3 mutation, similar to the RRW.A-bearing variants, infected U87.CD4.CXCR4 but not U87.CD4.CCR5 cells.

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FIG. 2.

Coreceptor usage (A) and preference (B) of V3 variants in CA28. Relative entry of pseudotyped reporter viruses in U87.CD4.CCR5 and U87.CD4.CXCR4 indicator cells was determined (A), and blocking of entry in TZM-bl cells with 1 μM CCR5-specific (TAK779) and CXCR4-specific (AMD3100) inhibitors was performed to determine coreceptor preference of the reporter viruses (B). Data are the means and standard deviations of results from triplicate wells and are representative of at least two independent experiments. RLU, relative light units.

To investigate coreceptor preference of the Δ22-25 virus in CA28, blocking experiments with CCR5 and CXCR4 antagonists in TZM-bl cells that express both coreceptors were performed. The results confirmed the coreceptor preference of viruses pseudotyped with WT or RRW.A V3 loop-bearing envelopes (Fig. 2B). Infection with WT V3 pseudovirus was blocked with the CCR5 inhibitor TAK779 but not with the CXCR4 inhibitor AMD3100, while the converse was observed for viruses containing RRW.A mutations in the V3 loop. Infection of TZM-bl cells with pseudovirus expressing the Δ22-25 V3 sequence was blocked to a greater extent by TAK779 (56%) than by AMD3100 (25%). Thus, similar to the Δ22-25 V3 variant in DG08 (Table 1), this Env variant in CA28 could function with CXCR4 but showed a preference for CCR5 usage and could be classified as dual-R as well. Interestingly, while AMD3100 efficiently blocked infection of TZM-bl cells with the RRW pseudovirus (70%), partial inhibition was also seen with TAK779 for this virus (26%). Dualtropic HIV-1 variants that use CXCR4 more efficiently than CCR5 have also been reported (30), and in this respect, the RRW variant in CA28 could be designated dual-X. Table 2 summarizes the transitional intermediates identified in CA28, showing a spectrum of dualtropic phenotypes similar to those seen in HIV-1-infected individuals.

To examine the evolutionary relationship of the transitional intermediates in macaque CA28, neighbor-joining tree analysis of Env V1-V5 sequence was performed. The results showed that while the dual-X RRW- and X4 RRW.A-bearing sequences clustered together on the tree, they sat on branches that differed from those of the dual-R Δ22-25-bearing sequences (Fig. 3). These findings in CA28 therefore are consistent with those in DG08 (43), supporting the notion that multiple switch events leading to the emergence of diverse tropism variants can evolve in parallel in macaques infected with R5 SHIV_SF162P3N.

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FIG. 3.

Phylogenetic tree showing the relationship between Env variant sequences (V1 to V5) in macaque CA28. A neighbor-joining tree rooted on four functional sequences of the inoculating virus SHIV_SF162P3N was generated. The scale bar indicates the genetic distance along the branches in nucleotides, and the asterisk at the node represents a bootstrap value of >70%. ⧫, representative clones in the inoculum; ▴, WT variants; •, RRW/RRW.A-bearing variants; ○, Δ22-25-bearing variants.

Change in dominance of tropism variants during the course of coreceptor switching in R5 SHIV_SF162P3N-infected macaques.Clonal Env sequence analysis of variants in tissues obtained from macaque DG08 at the time of first X4 appearance (13 wpi) and at end-stage disease (20 wpi) suggested that while the dual-R Δ22-25 switch preceded the X4 HR switch event temporally, its presence diminished over time, implying a loss of competitive fitness (43). Clonal Env sequences of viruses in longitudinal plasma and PBMC samples from DG08 confirmed this change in dominance of the V3 loop variant (Fig. 4). Of 44 plasma Env clones amplified at 13 wpi, 34% had an ATGD deletion in the V3 loop, with the remaining being WT, but over 97% of the 41 clones sequenced 1 week later harbored this mutation. Dominance of the Δ22-25 V3 variant also increased in PBMC within this same time period, from 17% to 98%. Notably, the remaining clone sequenced at 14 wpi in the PBMC compartment had insertions of histidine and isoleucine (HI) upstream of the GPG crown. Variants bearing these V3 insertions had been shown to be the evolutionary, functional, and antigenic intermediates of the HR-bearing X4 virus in macaque BR24 (27, 54). At the time of necropsy 6 weeks later (20 wpi), the dual-R Δ22-25 V3 was present at much lower frequency in the PBMC (3.5%) and not at all in the plasma, while variants with HI insertions could not be detected in either compartment. However, the dual-R Δ22-25 variant was present in both PBMC (16%) and plasma (3.3%) 2 weeks prior to necropsy (18 wpi), and variants with the signature X4 HR insertions could be detected in PBMC (13%) but not in the plasma at this time point. The transitory nature of the HI variant in DG08 and its detection prior to that of the HR variant in PBMC suggest that, as with macaque BR24, it also could be the R5X4 transitional intermediate of the HR-bearing X4 virus in this macaque (Table 2).

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FIG. 4.

Distribution of V3 variants in plasma and PBMC of macaques DG08 and CA28 over time. Numbers in parentheses indicate numbers of Env clones sequenced at indicated time points.

The dynamics in the change in dominance of the V3 tropism variants was similar in macaque CA28. Although sequence-specific PCR showed that the Δ22-25 variant first emerged in the plasma at 9 wpi (Fig. 1B), none of the 17 PBMC and 23 plasma Env clones sequenced at this time point harbored this deletion, suggesting that it is present at <5% frequency. Two weeks later, however, this variant represented ∼90% and 30% of the plasma and PBMC envelope clones sequenced, respectively. The frequency of the Δ22-25 V3 variant continued to increase in PBMC but dropped to 15% in the plasma at 13 wpi. At the time of necropsy, however, the Δ22-25 V3 variants could not be found in 36 PBMC clones sequenced but maintained the same representation in the plasma. The dual-X RRW-bearing variant was present at low frequencies (<4%) in both the plasma and PBMC at 11 and 13 wpi but was undetectable at the time of necropsy. Instead, variants bearing the signature X4 RRW.A sequence now appeared in the plasma, albeit at low frequencies (∼3%). These changes in virus population in the two macaques during and following the time of first X4 appearance prompted us to examine the underlying basis.

Differences in fitness of the dualtropic transitional intermediates contribute to the change in infecting virus population during coreceptor switching in macaques CA28 and DG08.To determine if fitness disadvantage of the dual-R Δ22-25 variant explains its diminished dominance with time, the entry efficiency and sensitivity of the V3 variants in macaques DG08 and CA28 to CCR5 as well as CXCR4 inhibitors were determined and compared, with sensitivity to the inhibitors serving as surrogate markers for coreceptor utilization efficiencies. We were not able to obtain functional HI-bearing gp160 from DG08 for these analyses, however, because of its transitory nature and extremely low frequency. The results showed differences in infection of TZM-bl cells that likely reflect differences in the Envs to interact with target cells. For DG08, entry of the Δ22-25 and HR V3 variants was less efficient than those of the coevolving WT viruses, with the Δ22-25 variant being the most inefficient (WT>HR>Δ22-25) (Fig. 5A). Moreover, the dual-R Δ22-25 virus in DG08 used CCR5 less efficiently than the coevolving WT viruses (Fig. 5A), as reflected by lower concentrations of the CCR5 inhibitors TAK779 and PSC-RANTES required to inhibit 50% of infection (IC₅₀) with this virus than with the WT virus in U87.CD4.CCR5 cells. However, the difference did not reach statistical significance. The dual-R Δ22-25 virus, however, was significantly less efficient in engaging CXCR4 than the X4 HR variant (P = 0.0495). Similar observations were made with the CA28 viruses (Fig. 5B). Compared to results for the coevolving WT virus, entry into TZM-bl cells of all the V3 loop mutants was attenuated, with the following rank order of efficiency: WT > RRW.A > RRW > Δ22-25 V3 mutants. Furthermore, the dual-R Δ22-25 virus in CA28 was significantly less efficient in using the CCR5 coreceptor than the coevolving WT virus (P = 0.0209 and P = 0.0495 for TAK779 and PSC-RANTES, respectively). It was also significantly less efficient in using CXCR4 than the dual-X RRW (P = 0.0433) and the X4 RRW.A (P = 0.0209) viruses. We noted that while the dual-R viruses in DG08 and CA28 harbored the same V3 deletions, the V3 mutant in CA28 was less efficient in using CCR5 as well as CXCR4 than the variant in DG08, suggesting that a region(s) outside the V3 loop modulates coreceptor usage efficiency. Regardless, the results show that the dual-R Δ22-25 variant in both CA28 and DG08 is at a disadvantage in entry and in engaging CCR5 and CXCR4 compared to the coevolving R5, dual-X, and X4 viruses. Furthermore, the observations that the RRW-bearing virus still retains residual CCR5 engaging ability (Fig. 2C) and is genetically linked to but uses CXCR4 less efficiently than the final X4 RRW.A virus suggest that it serves as an intermediate in this pathway of tropism switch.

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FIG. 5.

Entry and coreceptor usage efficiencies of V3 variants in macaques DG08 (A) and CA28 (B). Determination of relative entry of pseudotyped reporter viruses in TZM-bl cells and blocking of pseudovirions with increasing concentrations of the CCR5 inhibitors TAK779 and PSC-RANTES and the CXCR4 inhibitor AMD3100 were performed. 50% inhibitory concentrations (IC₅₀) of the inhibitors were determined using Prism 4 software (GraphPad, San Diego, CA). RLU, relative light units. Data shown are means ± standard errors of the mean of results of at least two to four independent experiments.