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

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

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

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

Depth coverage of SOLiD sequencing per nucleotide position, observed nucleotide changes per position and amplification strategy.

<p><b>A)</b> X axis indicates positions along the HIV-1 genome and Y axis the number of times each position was sequenced. Shown here the sequence depth coverage obtained from stimulated purified CD4 positive cells infected by R5 pseudotyped virus. <b>B)</b> Nucleotide changes affecting the majority of sequences from the proviral population. Data obtained from non-stimulated purified CD4 positive cells infected with X4 pseudotyped HIV-1 after a single round of reverse transcription. Diagrammatical location of changes: blue lines = mutated positions; green lines = boundaries of the investigated region. <b>C)</b> Amplification strategy was performed with 4 overlapping sub-genome fragments (A,B,C,D).</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

Nucleotide changes leading to non synonymous amino acids substitutions in the <i>env</i> coding region and selected known domains in the translated protein.

<p>Numbers indicate nucleotide positions. V2: variable region 2, FP: fusion peptide, HP1 and 2: heptad repeat regions 1 and 2, LLP3 and 1: lentivirus lytic peptide region 3 and 1.</p

Expression of Activation Markers using flow cytometry assays.

<p>Histograms showing the expression of CD25, CD69, CD38 and HLA-DR from gated CD3+CD4+ cell populations from PBMCs are shown in the upper row. Histograms indicating the expression of CD25, CD69 from gated CD3+CD4+ cell populations from purified T CD4+ cells appear in the lower row. X axis indicates the fluoresce intensity of a specific marker and Y axis indicates the percentage of cells in the population expressing this specific marker. Graphics under each histogram represent the MFI (Median of Fluorescence Intensity) of each activation marker in the gated CD3+CD4+ cell populations. Results are shown as mean values ± SD and are representative from three independent experiments. Statistical significance was assessed by the Two-tailed Student’s t-test yielding * when p < 0.05; and **, when p < 0.005. Gray area: unstained control cells, green/blue line: non-stimulated cells, red line: stimulated cells.</p

Example of six nucleotide probabilities per position obtained from non-stimulated purified T CD4 positive cells infected by X4 pseudotyped HIV-1^*.

<p>*Numbers in parenthesis indicate the nucleotide probabilities as percentages at the specified positions.</p><p>Example of six nucleotide probabilities per position obtained from non-stimulated purified T CD4 positive cells infected by X4 pseudotyped HIV-1^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139037#t004fn001" target="_blank">*</a>}.</p