Non-parametric geographic subdivision tests comparing pre- ASCT viruses with those at each rebound.

<p>Non-parametric geographic subdivision tests comparing pre- ASCT viruses with those at each rebound.</p

Diversity measured as average pairwise distance at longitudinal time points.

<p>APD measured pre- ASCT and during subsequent viral rebounds. Numbers in parentheses indicate the number of single genomes acquired at each time point. Significance was determined by two-tailed.</p

HIV-1 Vpu–mediated loss of tetherin is partially abrogated by MG132.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the presence or absence of HIV-1 Vpu-HA co-expression and proteasome inhibitor MG132 (0.8 µM for 12 hours) as shown. Errors are standard error of the mean of 2 experiments. (B) Tetherin was detected by western blot of Xpress tag in cleared RIPA extract supernatants and pellets as shown. Sizes of molecular weight markers are shown in kilodaltons. (C) Vpu-HA was detected in sonicated RIPA extract as shown (D) Blots in B have been stripped and re-probed for β actin as a loading control. (E) Measurement of HIV-1 p24 in the supernatant of transfected cells by western blot. Data are representative of 2 independent experiments.</p

Inferred neighbour-joining phylogenetic tree of single genome derived HIV-1 env sequences.

<p>Two APOBEC hypermutated sequences found in the third rebound have been removed. Pre melphalan HIV DNA (-27 to -38, orange open diamonds), first rebound HIV RNA (+6, blue closed diamonds), first rebound HIV DNA (+6, blue open diamonds), second rebound HIV RNA (+515, magenta closed diamonds), third rebound HIV DNA (+643 to +958, green open diamonds). Rooted on MJ4 (black square), branches with bootstrap support > 75% are indicated by black asterisk. Overall population APD is 1.5%. The scale represents 0.005 nucleotide substitutions per site, equivalent to 4.5nt. The tips in the boxed grey area representative sequences predicted to confer CXCR4 tropism by both Geno2Pheno and Phenoseq genotypic algorithms.</p

Mutation of a single amino acid (T45I) leads to insensitivity to Vpu and persistence of tetherin protein.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the absence of HIV-1 Vpu (black bar), or presence (white bar). Mutation T45I in the trans-membrane region of human tetherin results in insensitivity to Vpu whilst maintaining antiviral activity. The titre of the unrestricted HIV-1 was 10⁷ infectious units/ml. Errors are standard error of the mean of 2 experiments. (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in cleared RIPA extracts from transfected 293T cells. Tetherin was detected by western blot of N-terminal Xpress tag in the cleared RIPA extract supernatants (D) and pellets (F) as shown. Sizes of molecular weight markers are shown in kilodaltons. Blots in (E) and (G) have been stripped and re-probed for β actin as a loading control. Data are representative of 2 independent experiments.</p

Selection analyses reveal positively selected tetherin residues in the primate lineage.

<p>(A) Nucleotide alignment of nine primate tetherin sequences. HoSa (Homo Sapiens), Popy (Pongo Pygmaeus (Orangutan)) Patr (Pan Trolodytes (Chimpanzee)), Tan (Tantalus monkey), Ver (Vervet monkey), Mane (Macaque Nemestrina (pigtailed macaque)), Mafa (Macaque Fasicularis (cynomolgus monkey)), Mamu (Macaque Mulatta (Rhesus macaque)) Caja (Callithrix jacchus (white tufted ear marmoset). Codon positions under positive selection are indicated by shaded boxes. Secondary structure (SS) was predicted by PSIPRED <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000443#ppat.1000443-McGuffin1" target="_blank">[53]</a> and is symbolised as (+) cytoplasmic domain; (I) trans-membrane domain inner cap; (X) trans-membrane domain alpha helix; (O) trans-membrane domain outer cap; (−) extra-cellular domain. (B) Predicted structure of the trans-membrane helix performed using helical wheel projection (<a href="http://rzlab.ucr.edu/scripts/wheel/wheel.cgi" target="_blank">http://rzlab.ucr.edu/scripts/wheel/wheel.cgi</a>) suggests that residues 26, 30, and 36 are on the same side of the protein. Closed circles indicate positively selected amino acids, dashed circles indicate residues that are different between human and Tantalus monkey, but not positively selected. Human amino acids are shown in black and Tantalus monkey in grey italic.</p

Genotypically defined co-receptor usage following HIV rebounds.

<p>Tropism testing of the HIV V3 loop was performed in both Geno2Pheno and Phenoseq online genotypic algorithms. Percentage of CXCR4 sequences is plotted on the Y-axis. Only sequences found to be X4 in both programs were designated as X4. Significance was determined using a two-tailed Fishers Exact test.</p

The positively selected tetherin trans-membrane region residues impact on sensitivity to HIV-1 Vpu.

<p>(A) Co-expression of HIV-1 plasmids alone (C) with wild-type human tetherin (WT) and measurement of HIV-1 release in the absence of HIV-1 Vpu (white bar) or presence (black bar). Mutation of positively selected residues I26V, V30G, I36L, T45I (Quad) in the trans-membrane region of human tetherin results in reduced sensitivity to Vpu whilst maintaining similar antiviral activity. The effect of single mutations are also shown. Errors are standard error of the mean of 2 experiments. Equal amounts of tetherin plasmids were used (100 ng) (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in extracts from transfected 293T cells. (D) Cell extract blots in C were stripped and re-probed for β actin as a loading control.</p

Vpu expression leads to a loss of wild-type but not a quadruple mutant tetherin protein steady state levels.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the absence of HIV-1 Vpu (black bar), or presence (white bar). Mutation of positively selected residues I26V, V30G, I36L, T45I (ΔTHN) in the trans-membrane region of human tetherin results in reduced sensitivity to Vpu whilst maintaining antiviral activity. The effect of co-transfection of HIV-1 plasmids and untagged human tetherin is shown for comparison (Lane C). The titre of the unrestricted HIV-1 was 10⁷ infectious units/ml. Errors are standard error of the mean of 2 experiments. (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in extracts from transfected 293T cells. Cell extract lysates (D) or pellets (F) were blotted for the Xpress tag to detect tetherin. Sizes of molecular weight markers are shown in kilodaltons. Blots in D (E) or F (G) were stripped and re-probed for β actin as a loading control. Data are representative of 3 independent experiments and similar results were seen with an N terminal HA tag.</p

Number of WGS treated with each drug, and correlations between drugs within samples.

<p>Number of WGS treated with each drug, and correlations between drugs within samples.</p