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All available HIV-1 infected individual-derived Rev or Rev exon2 sequences for clade A, B, C, D, AE, G, AG and BC were obtained from the Los Alamos HIV Sequence Database (http://www.hiv.lanl.gov), and one sequence was chosen per infected individual. The number of selected sequences of each clade is shown in Table 1. BioEdit v7.0.9 was used to adjust alignment and to produce consensus sequence for each clade.
Clade Number of sequences presenting allele variance Total number Stop codon Q E S K R P H V I CRF07_BC 140 135 1 4 0 0 0 0 0 0 0 CRF08_BC 65 62 2 1 0 0 0 0 0 0 0 CRF57_BC 7 7 0 0 0 0 0 0 0 0 0 CRF60_BC 5 3 2 0 0 0 0 0 0 0 0 CRF61_BC 3 3 0 0 0 0 0 0 0 0 0 CRF62_BC 3 3 0 0 0 0 0 0 0 0 0 CRF64_BC 8 8 0 0 0 0 0 0 0 0 0 C(Asia) 182 160 13 9 0 0 0 0 0 0 0 C(Africa) 1306 1217 37 45 2 1 1 1 0 1 1 B 7023 8 6780 198 0 4 0 31 2 0 0 CRF01_AE 961 9 941 7 0 0 1 2 1 0 0 D 194 1 192 0 0 1 0 0 0 0 0 CRF02_AG 271 16 251 2 0 0 0 2 0 0 0 A 18 8 9 0 1 0 0 0 0 0 0 G 123 7 111 3 0 1 0 0 1 0 0 Table 1. Number of Rev sequences presenting allele variance at residue 101 in the different clades.
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Rev gene of CRF07_BC isolate (pXJDC13), carried the stop codon at the end of C-terminal, was cloned into pcDNA3.1-TOPO-V5-His vector (Invitrogen, Catalog: K4900-01, USA) to generate clade BC wild type Rev clone (BC WT Rev). The full length Rev gene in which the stop codon at residue 101 was replaced by Q, was chemically synthesized and named BC Rev Stop101Q. Rev mutant of Clade B (pSV-Rev Q101Stop) was generated based on the backbone of BH10 isolate Rev expression vector pSV-Rev (Smith et al. 1990).
Rev genes of pXJDC13 and BH10 and their mutations (without stop codon at the end of C-terminal) were separately cloned into pcDNA3.1-TOPO-V5-His vector to generate Rev expression plasmids with His-tag (BC WT Rev-His/BC Rev Stop101Q-His and B WT Rev-His/B Rev Q101Stop-His).
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Rev expression plasmids with His-tag (5 μg) were transfected into 5 × 105 HEK 293T cells. Fourty-eight hours after transfection, transfected cell samples were homogenized in a lysis buffer containing complete protease inhibitor cocktail (Kangweishiji, Nanjing, China) and then centrifuged at 12, 000g, 4 ℃ for 15 min for supernatant collection. Protein concentrations were determined by the use of the BCA protein assay kit (Kangweishiji, Nanjing, China). Equal amount of total protein (30 μg) were resolved on 10% SDS PAGE gel and transferred to PVDF membranes. Membranes were blocked with 5% skim milk/PBST overnight, then were incubated with anti- 6 × His mouse monoclonal antibody (1:1000 dilution) (Kangweishiji, Nanjing, China) and anti-β-actin mouse monoclonal antibody (1:1000 dilution) (Boaolong, Beijing, China), respectively. Horseradish peroxidase-linked antimouse-IgG (1:10, 000) (Boaolong, Beijing, China) were employed to detect the results with PierceTM ECL Western Blotting Kit (Pierce, USA) and membrane was imaged in a Molecular Imager CheminDocTMXRS + Systems (Biorad, USA). Image was analyzed using Image J-win32 software. The Rev signal was normalized to β-actin levels measured in the same lane of the same Western blot.
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HEK 293T cells (105/well) were seeded into 24-well tissue culture plates in 1 mL of culture medium. After 17 h, using the Sino-infection transfection reagent (Sino Biological Inc., China), each well was transfected with 1 μg Gag-Pol-RRE plasmid plus 15 ng, 30 ng or 60 ng of Rev expression plasmid (B WT Rev, B Rev Q101Stop, BC WT Rev or BC Rev Stop101Q), and 200 ng CMV-EGFP plasmid (transfection control), respectively. Supernatants were harvested at 48 h post transfection and filtered by 0.45 lm filter for p24 quantification using ELISA. The p24 antigen was normalized to transfection efficiency as determined by percentage of EGFP expressing cells measured using FACSCalibur (Becton–Dickinson, USA).
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A 1800 bp fragment spanning from V3 to Nef was amplified by high-fidelity PCR from a CRF07_BC infectious clone pXJDC13 (Wang et al. 2013), using pairs of primers carrying restriction enzyme sites (ClaI-F and BamHI-R) (Supplementary Table S1). Then the PCR products were sub-cloned into the pGEM-T vector (Promga, USA), and mutations were introduced subsequently using QuikChange Multi Site-Directed Mutagenesis Kit with two paired primers (BC-E117Stop-F and BC-E117Stop-R combined with BC-Stop101Q-F and BC-Stop101Q-R) (Supplementary Table S1). The mutated fragment was digested by Cla I and BamH I and ligated into plasmid pXJDC13 to generate CRF07_BC mutated clone pXJDC13-Stop101Q.
AT1 and AT2 are two pNL4-3 based clade B infectious clones in which nef was substituted by Thy1.1 (Rat CD90.1) and Thy 1.2 (Mouse CD90.2) (Dykes et al. 2006) separately. A subclone carrying Rev from clone AT1 was initially constructed using the primers, B-BmtI-F and B-HpaI-R (Supplementary Table S1). The mutations were introduced using Quicession plasmids with Hisk-change Multi Site-Directed Mutagenesis Kit (Stratagene, USA) with primers (B-BH10- Q101Stop-F and B-BH10-Q101Stop-R). Following double digestion of Bmt I and Hpa I enzymes, mutated Rev was ligated into the AT1 to generate clade B mutated clone AT1-Q101Stop.
HEK 293T cells were maintained in the DMEM containing 10% FCS, 100 mg/mL of streptomycin, 100 IU/mL of penicillin, full-length genome plasmid (5 μg) was transfected into 5 × 106 HEK 293T cells using the Sinoinfection transfection reagent (Sino Biological Inc., China) in a six-well plate to generate clone-derived viruses. The supernatant was collected 48 h post infection, filtered through a 0.2 lm filter, and stored at - 80 ℃ for p24 quantification.
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Viruses pXJDC13 and pXJDC13-Stop101Q were propogated in PBMCs for they were CCR5-tropic primary isolates (Wang et al. 2013). PBMCs were isolated from the healthy HIV-1 seronegative donors using the Ficoll-Hypaque gradient method. The p24 antigen quantified virus (10 ng) was used to infect 3 × 106 phytohemaglutinin (PHA)-stimulated PBMCs in triplicate for 2 h at 37 ℃ in 1 mL total volume, then the supernatant was removed and cells were washed twice by 10 mL phosphate buffer saline (PBS). The cells were then cultured in 3 mL of complete RPMI 1640 medium (containing 10% FCS and 10 IU/mL IL-2) in a 25 cm2 flask (Corning, USA). Supernatant (1 mL) was substituted by fresh medium every 3–4 days, and 3 × 106 PHA-stimulated fresh PBMCs were seeded into the flasks every 7 days, and the supernatant obtained was passed through a 0.2 lm filter and stored at - 80 ℃.
Viruses AT1 and AT1-Q101Stop were replicated in a human T cell type, PM1 cells. The p24 antigen quantified virus (10 ng) was inoculated into 7 × 105 PM1 cells in 1 mL total volume for 2 h at 37 ℃, after which cells were washed with 5 mL PBS twice. Then the cells were grown in 2 mL of complete RPMI 1640 medium, and 0.5 mL medium was replaced by fresh medium at days 3, 5 and 7 post-infection for p24 quantification.
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The growth competition assay was conducted as previously described (Dykes et al. 2006; Wang et al. 2017). Cells were harvested by centrifuging on days 3, 4, 5 and 6 post-infection. For each sample, cells (5 × 105) were stained with anti-Thy1.1-FITC antibody (BD Biosciences, USA) plus 7AAD (BD Biosciences, USA), anti-Thy1.2-PE antibody (BD Biosciences, USA) plus 7AAD, and antiThy1.1 antibody combined with anti-Thy1.2 antibody plus 7AAD, respectively. The anti-CD4 antibody-PE plus 7AAD, anti-CD4 antibody-FITC plus 7AAD, and 7AAD stained cells were set as controls, separately. Following the antibodies incubation in the dark for 30 min on ice, stained cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for flow cytometry performed as described (Dykes et al. 2006; Wang et al. 2017). The data was analyzed using CellQuest software (Becton–Dickinson, San Jose, CA) and FlowJo software (Treestar, USA). The percentages of cells infected with virus mutant, wild-type virus and both viruses are labeled in the lower right, upper left and upper right quadrants, respectively.
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Relative fitness of virus was calculated using the formula: Fitness: 2-Point Calculation in Competitive Virus Fitness (http://bis.urmc.rochester.edu/TFitness/FitnessTwo.aspx) (Ma et al. 2010) using the formula:
(Wang et al. 2010). Where Tm (t6) and Tw (t6) represent the infected cell number by the mutant and wild-type virus at day 6, separately; Tw (t3) and Tm (t3) represent the cell number infected by virus at day 3, separately.$1 + {\rm{s}} = {\rm{exp}}\left({\frac{1}{3} \times {\rm{ln}}\frac{{{\rm{Tm}}({\rm{t}}6){\rm{Tw}}({\rm{t}}3)}}{{{\rm{Tw}}({\rm{t}}6){\rm{Tm}}({\rm{t}}3)}}} \right)$ -
Student's t test was used to assess the statistical difference between two groups using Graphpad Prism (version 5.0, GraphPad, Inc.). Two-way ANOVA was applied to determine the statistical significance of replication curves of virus, set as P < 0.05 (two tailed value). Results are shown as the mean ± SD of independently triplicate experiments.