Protease gene break point positions in nucleotides and their frequency.

<p>X axis indicates the number each breakpoint was detected in our study. Break points detected in single patients are represented by Y axis bars in blue whereas; break points detected in multiple patients appear in multicolored bars. The numbers inside parenthesis are the number of different patients in which this particular break point was detected.</p

Bootscan analysis of HIV-1 multiple infections based on the protease gene.

<p>We used the Neighbor-Joining method with K2P and 1000 bootstrap replicates. This analysis was performed using the software Simplot v3.5.1, with a window of 75 bp and 5 bp increments.</p

Schematic mosaic structures and pure subtypes found in multiple infections.

<p>The number of times that a particular subtype or recombinant was found in a patient is shown inside the parenthesis to the left of each structure. Break points were mapped according to Bootscan and their positions in base pairs are followed by the number they appeared on mosaic structures (inside the parenthesis).</p

Breakpoints and Recombination Individuals.

<p>Breakpoints and Recombination Individuals.</p

HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA

<div><p>In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5) and C-X-C chemokine Receptor type 4 (CXCR4) Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.</p></div

Number of sequenced reads (50 nucleotide long in average) and of nucleotides after SOLiD sequencing per experimental condition.

<p>Number of sequenced reads (50 nucleotide long in average) and of nucleotides after SOLiD sequencing per experimental condition.</p

Coding regions and mutated codons.

<p>Original codons are the most probable codons observed in control; mutated codons are those determined by nucleotide changes in X4 pseudotyped experiments, bold capital letters indicate base substitutions within affected codons, mutated amino acids are underlined; the 2 last columns indicate codons and amino acids at the same positions in the HIV-1 reference strain (NL4-3 GenBank accession number AF324493).</p><p>Coding regions and mutated codons.</p

Influence of envelope tropism, cell type, and stimulation status over proviral nucleotide probabilities after one round of reverse transcription.

<p><b>A)</b> Quantitative and qualitative changes occurring along gag-pol and env regions from X4 versus R5 pseudotyped virus infections. <b>B)</b> Quantitative and qualitative changes occurring along gag-pol and env regions from infected T CD4 + positive cells versus PBMCs. <b>C)</b> Quantitative and qualitative changes occurring along gag-pol and env regions from non-stimulated and stimulated. Asterisks indicate statistical difference at 5% of significance level between compared numbers. Probabilities are presented as percentage (%).</p

Representative range of variational distances between nucleotide probabilities observed in the control and in each experimental condition.

<p>X axis represents nucleotide positions in the sequence and Y axis indicates variational distances. Left, infection of purified non-stimulated T CD4 positive cells by R5 pseudotyped HIV-1; right, infection of non-stimulated PBMCs by X4 pseudotyped HIV-1.</p

Depth coverage of SOLiD sequencing per experimental condition covering positions 790 to 9085 of the HIV-1 pNL4-3 reference genome^*.

<p>*HIV-1 pNL4-3 reference genome [GenBank accession number AF324493]. Presented data correspond to number of reads obtained from each condition.</p><p>Depth coverage of SOLiD sequencing per experimental condition covering positions 790 to 9085 of the HIV-1 pNL4-3 reference genome^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139037#t003fn001" target="_blank">*</a>}.</p