Amplicons from published studies of HIV-1 diversity, evolution, and clonal expansion.

Amplicons from published studies of HIV-1 diversity, evolution, and clonal expansion.</p

CPS of previously published amplicons.

(a) CPS for each of the primer sets listed in Table 2, averaged over all subjects within each sample type (Table 1). (b and c) Evaluation of previously published phylogenetic trees in the context of the CPS of the primer sets used to generate those trees. Part b shows proviral DNA samples and part c shows plasma RNA samples. Black diagonal lines show the relationship between the number of sequences collected and the number expected to be unique for each primer set listed in Table 2, as an estimate of the background signal level (assuming no clonality); the slopes of these lines are equal to the CPS values in part a divided by 100. The dotted red lines were calculated as the black lines plus or minus one standard deviation in CPS. Each plotted point indicates the actual number of total sequences and unique sequences present in a previously published phylogenetic tree. Phylogenetic trees containing both proviral DNA and plasma RNA sequences were counted separately for the two sequence types and plotted separately in parts b and c. Points plotted far below the black line (green-shaded region) indicate trees with more clonality than would be expected by chance from a sample of unique HIV-1 genomes. *References in which hypermutated sequences were not explicitly included in phylogenetic trees.</p

Clonal prediction scores of 1 kb amplicons spanning the HIV-1 genome.

<p>(<b>a</b>) Schematic of algorithm to calculate clonal prediction score (CPS), which quantifies the proportion of unique sequences in an alignment that are correctly identified as unique using the amplicons produced by a specific primer set. (<b>b</b>) CPS of 1 kb-wide amplicons spanning the HIV-1 genome for six Acute treated–DNA subjects. (<b>c</b>) CPS of 1 kb-wide amplicons spanning the HIV-1 genome, averaged over all subjects in five different sample categories (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005689#ppat.1005689.t001" target="_blank">Table 1</a>). (<b>d</b>) Overlay of the five plots in part <b>c</b>. Schematics of the HIV-1 genome are aligned to the charts in parts <b>b</b> through <b>d</b> to highlight viral gene locations. The amplicons in parts <b>b</b> through <b>d</b> are defined with reference to the HXB2 genome.</p

Sources and characteristics of sequence alignments analyzed to generate clonal prediction scores.

<p>Sources and characteristics of sequence alignments analyzed to generate clonal prediction scores.</p

Relationship between amplicon length and CPS.

<p>Summary statistics describing all amplicons of a given length, spanning the HXB2 reference genome at 10 bp intervals. Amplicons with undefined CPS were not included in these summary statistic calculations. (<b>a</b>) Average, median, and minimum of the CPS values for every amplicon of a specified length spanning the viral genome. Summary statistics are shown for each subject and grouped by sample type. (<b>b</b>) Proportion of all of the amplicons of a specified length that have CPS values above 80.</p

Relationship between amplicon length and PCR coverage.

<p>Percentage of sequences in each alignment that would be detectable by PCR, averaged over all amplicons of a specified length spanning HXB2 positions 2000 through 8000 at 10 bp intervals. Results are shown for 31 subjects and grouped by sample type (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005689#ppat.1005689.t001" target="_blank">Table 1</a>).</p

The standard and the MOLT-4/CCR5 viral outgrowth assays yield comparable frequencies of latent infection.

<p>(<b>A</b>) The frequency of latently infected resting CD4⁺ T cells was measured in 3 viremic patients and 14 HAART-suppressed patients using both the standard and the MOLT-4/CCR5 viral outgrowth assays, with HIV-1 p24 antigen ELISA used as the endpoint assay of viral outgrowth at day 14. (<b>B</b>) Statistical comparison of the IUPM values measured using the standard viral outgrowth assay and the MOLT-4/CCR5 viral outgrowth assay by Wilcoxon rank sum test. (<b>C</b>) The correlation of IUPM values measured using the standard viral outgrowth assay and the MOLT-4/CCR5 viral outgrowth assay (Pearson's correlation coefficient, r).</p

Increase in HIV infectivity is not co-receptor mediated.

<p>Surface expression of co-receptor CXCR4 on unstimulated CD4+ cells, CD4+ cells stimulated with phyohemaglutinin (50 mg/ml), <i>M. bovis</i> BCG (Copenhagen), <i>M. tuberculosis</i> (CDC1551) and <i>M. smegmatis</i> (MC²155), respectively, at the time of infection with pseudovirus (A). Surface expression of co-receptor CCR5 on CD4+ cells from five subjects after stimulation (B).</p

Two-step bead depletion procedure yields highly purified resting CD4⁺ T cells from HIV-1 infected patients.

<p>A two-step negative selection strategy to purify resting CD4⁺ T cells from patient PBMC. (<b>A</b>) Representative FSC/SSC plot indicating live cell population after the two-step bead depletion procedure. (<b>B</b>) Representative dot plot indicating purity of resting CD4⁺ T cells. Purified cells were stained with antibodies to CD4 and HLA-DR.</p

PCR Primers.

(DOCX)</p