Effect of Rev1-Vpu expression on HIV-1 replication in PBMCs and tonsillar explant cultures.

<p>(A) PHA-stimulated PBMCs were infected with adjusted amounts of the indicated viruses. Virus replication was monitored by analyzing RT-activity in the supernatant. The means of three independent experiments +/- SEM are shown. (B, C) Surface expression levels of (B) tetherin and (C) CD4 were determined by flow cytometry at day 3 post infection. Infected cells were identified by intracellular p24 staining after surface staining of CD4 or tetherin. Dot plots indicating the gating strategy are shown in the right panels. Bar diagrams summarizing four to five independent experiments +/- SD are shown on the left. (D) Sequence analyses of viral mixtures. Wt and fs mutant virus stocks were normalized for infectivity, mixed at the indicated ratios, and the <i>rev1-vpu</i> region was sequenced after bulk amplification of cDNA. The lower panels show respective standard curves. The peak fluorescence of the T residue at position 1 (ZM246 wt) and the A residue at position 3 (ZM247 wt) is expressed as a fraction of the total fluorescence (relative peak height). (E) Sequence chromatograms of 1:1 input mixtures and viral cultures 10 days post infection (dpi). Percentages of wt and fs sequences displayed in the right panels were calculated from the standard curves shown in (D). (F) Human tonsil explant cultures were infected and analyzed as described in (A). One representative experiment for one of three independent donors is shown (***p<0.001; **p<0.01; *p<0.05; n.s. not significant).</p

Splice sites generating Rev1-Vpu encoding mRNA.

<p>The HIV-1 genome (black) and nine ORFs encoding structural, regulatory and accessory proteins are depicted on top. Splice donor (D1-6) and acceptor (A1-8) sites are indicated by dotted lines. mRNAs encoding Rev1-Vpu, Vpu and Env are shown in grey. Depending on the cell type and time point post infection, 75–90% of Vpu and Env encoding mRNA species fail to express Rev1-Vpu since the usage of splice acceptor site A5 removes an intron containing the initiation codon of <i>rev1</i>. Only 10–25% of the Vpu encoding mRNAs have the potential to be translated into Rev1-Vpu since splice acceptor sites A4a, A4b and A4c retain the complete first exon of <i>rev</i>.</p

Frequency of the <i>rev1-vpu</i> polymorphism in HIV-1 quasispecies of infected individuals over time.

<p>^a single genome sequences (SGS) per sample visit</p><p>Frequency of the <i>rev1-vpu</i> polymorphism in HIV-1 quasispecies of infected individuals over time.</p

Expression of the Rev1-Vpu fusion protein.

<p>(A) Western blot analysis of HEK293T cells co-transfected with the proviral clones described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142118#pone.0142118.g001" target="_blank">Fig 1A</a> or a <i>vpu</i>-deficient mutant thereof. Expression vectors containing <i>rev1</i>-<i>vpu</i> or <i>vpu</i> cassettes served as size controls. Vpu and Rev1-Vpu were detected with an antiserum raised against ZM247 Vpu. (B, C) Expression of Vpu and Rev1-Vpu in ZM247-infected PBMCs or SupD1 cells. Bands representing the Rev1-Vpu fusion protein are highlighted by red arrows. Detection of p55, p24 and actin served as internal controls.</p

Rev function in isogenic viruses differing only in their ability to express Rev1-Vpu.

<p>(A) Gene arrangement of a reporter construct expressing GFP in a Rev-dependent manner. The <i>GFP</i> ORF is flanked by splice donor site 4 (D4) and splice acceptor site 7 (A7). Rev mediates the export of intron-containing GFP expressing mRNA via binding to the RRE. (B) Rev-dependent gene expression was determined by co-transfection of HEK293T cells with increasing amounts of the indicated molecular clones of HIV-1, the GFP reporter construct and a BFP expressing control plasmid. 40 h post transfection, GFP expression levels of BFP positive cells were analyzed by flow cytometry. Examples of primary FACS data are shown in the lower panel. (C) Western blot, showing Rev-dependent expression of p24-capsid and Rev-independent expression of Nef in HEK293T cells transfected with the indicated proviral constructs.</p

Functional activity of Rev1-Vpu expressed from pCG expression plasmids.

<p>(A) CMV-IE promoter-based pCG expression vectors containing <i>vpu</i> (left panel) or the <i>rev1-vpu</i> fusion gene (right panel). An enhanced version of the green fluorescent protein (eGFP) is co-expressed via an IRES. (B) Expression of Rev1-Vpu and Vpu in HEK293T cells transfected with the indicated pCG vectors. A Vpu-specific antiserum was used for detection. eGFP was detected to check transfection efficiencies. (C-F) FACS analysis of (C) CD4, (D) tetherin, (E) CD1d or (F) NTB-A receptor modulation by ZM247 Vpu and Rev1-Vpu. HEK293T cells were transfected with expression vectors for the respective surface receptor and Vpu or Rev1-Vpu. 40 h post transfection, surface receptor levels were monitored by two-color flow cytometry. Dot plots indicating the gating strategy are shown in the right panels. Bar diagrams summarizing three to five independent experiments +/- SD are shown on the left (***p<0.001; **p<0.01; *p<0.05; n.s. not significant).</p

No fitness costs of the early CD8⁺ T cell escape mutation R355K in Env.

<p>(A) The frequency of the R355K mutation in the Env_352–360T cell epitope different time points (days post Fiebig stage I/II). The viral sequences obtained by SGA were compared to the T/F virus. Amino acid substitutions at the R355K mutation site were highlighted in red. Relative fitness of the R355K mutant was determined by comparing to the cognate T/F virus in the single-passage assay (B) and the multiple-passage assay (C) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102734#pone-0102734-g003" target="_blank">Figure 3</a>. The percentage of each virus in the inoculum stock and the culture supernatant was determined by PASS. Mean ± standard deviations are shown.</p

Determination of T cell responses by ELISpot.

<p>(A) Frequencies of the mutations in the TW10 epitope at different time points (days post Fiebig stage I/II). The viral sequences obtained by SGA were compared to the T/F virus and the subtype B consensus sequence (ConB). Amino acid substitutions at positions 242, 247 and 248 are highlighted in red. (B) T cell response to the wild type and mutant TW10 peptides at day 102 in subject CH77 were determined using an <i>ex vivo</i> IFN-γ ELISpot assay. Peptides containing T242N, V247I or G248A mutation alone as well as in various combinations were analyzed. ELISpot data are expressed as the mean spot forming units (SFU) per million PBMC (SFU/10⁶PBMC)±SEM. Positive T cell responses were defined as: ≥30 SFU/million and >4 times above background (indicated by the dotted line). All assays were performed in triplicate.</p

No fitness costs of the early reversion mutation I64T in Tat/Rev overlapping region.

<p>(A) Frequencies of the I64T mutation in the Tat protein at different time points (days post Fiebig stage I/II). The viral sequences obtained by SGA were compared to the T/F virus and the subtype B consensus sequence (ConB). Amino acid substitutions at the I64T mutation site were highlighted in red. Relative fitness of the I64T mutant was determined by comparing to the cognate T/F virus in the single-passage assay (B) and the multiple-passage assay (C). The percentage of each virus in the inoculum stock and the culture supernatant was determined by PASS. Mean ± standard deviations are shown.</p

Structural modeling of mutations in the TW10 epitope in p24 Gag.

<p>(A) Homology models of the p24 monomer for the sequences of the T/F virus (cyan), the T242N mutant (magenta) and the NIA mutant (green) show similar structures of the helix 6 region with modest structural differences in the neighboring N-terminal hairpin and CypA binding loop. Side chains at mutation positions are shown as stick representation. (B) Mutations at position 242 (red) and positions 247/248 (green) in helix 6 were mapped to the hexameric p24 crystal structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102734#pone.0102734-Pornillos1" target="_blank">[49]</a>. The mutation positions did not occur at p24 subunit interfaces. Helix 6 is located between the N-terminal hairpin (magenta) and cyclophilin-binding loop (orange) on the surface of the hexamer. (C) Mutation at position 242 (red) and positions 247/248 (green) in helix 6 (yellow) were mapped to a hexamer of hexamers in the capsid assembly <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102734#pone.0102734-Zhao1" target="_blank">[40]</a>. The T242N (red) and V247I/G248A (magenta) mutations face outward in the assembly and do not occur at hexamer-hexamer interfaces.</p