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. 2023 Jun 12;19(6):e1011194.
doi: 10.1371/journal.ppat.1011194. eCollection 2023 Jun.

Genetic variation of the HIV-1 subtype C transmitted/founder viruses long terminal repeat elements and the impact on transcription activation potential and clinical disease outcomes

Paradise Madlala  1   2 , Zakithi Mkhize  1 , Shamara Naicker  1 , Samukelisiwe P Khathi  1 , Shreyal Maikoo  1 , Kasmira Gopee  1 , Krista L Dong  3   4   5 , Thumbi Ndung'u  1   2   3   6   7
Affiliations

Genetic variation of the HIV-1 subtype C transmitted/founder viruses long terminal repeat elements and the impact on transcription activation potential and clinical disease outcomes

Paradise Madlala et al. PLoS Pathog. .

Abstract

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5' long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3' LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3'LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3' LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2-12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Design of South African acute HIV-1 infection cohorts used in this study and the number of participants from each cohort used to generate HIV-1 subtype C LTR sequences.
Initiated treatment immediately refers to the study participants that initiated treatment immediately after detection of HIV RNA. Initiated treatment late refers to the study participants who initiated treatment when their CD4 T cell count was below 500 cells/μL as was the South African national treatment guidelines at the time.
Fig 2
Fig 2. Phylogenetic analysis of patient derived LTR elements obtained at the earliest timepoint (ETP) and one-year post infection later timepoint (LTP) from the FRESH and HPP cohort and transcriptional analysis of the transmitted/founder (T/F) LTR sequences.
A: The phylogenetic tree was constructed using the online tool Phyml (http://www.hivlanl.gov) and rerooted on the Indian subtype C reference sequence (EF178613.1) using Figtree Tree Figure Drawing Tool v1.4.3. The viral sequences obtained at or near transmission are denoted as (ETP) while the viral sequences obtained at one-year post infection are denoted as (LTP). The LTR sequences highlighted in blue did not have the matching sample at the LTP timepoint. Each patients LTR element clusters independently together thus demonstrating that the LTR element is variable between patients. Phylogenetic branching demonstrates that the LTR may evolve within one-year of infection within a patient.
Fig 3
Fig 3. Multiple sequence alignment of patient derived LTR sequences and percentage of similarity between patient derived and consensus LTR sequences.
A: The patient derived LTR sequences were aligned against the Indian subtype C reference sequence, EF178613.1 since it contains 4 NF-kB binding sites. The dots represent nucleotide bases that are identical to the reference sequence while the dashes indicate deletions. The doted blocks highlight the transcription factor binding sites (TFBS) within the core-enhancer and core-promoter regions and TAR element. From left to right: the USF RBE III site, the 4th NF-kB binding site (designated F-kB and highlighted in green), standard NF-kB sites designated NF-kB I and II (H- kB, II and I) and subtype C specific NF-kB site (designated C-kB), Sp1 III (highlighted in blue), II and I binding sites, 5’ E-box, Tata box and 3’ E-box and the TAR loop region. Overall, the T/F virus canonical sequences were relatively conserved within the core enhancer, nonetheless variation was observed within the RBE III site, Sp1 III binding site and TATA Box. B: Shows the percentage of similarity of each individual patients derived LTR at earlyt time point (ETP, shown in blue) and late time point (LTP, denoted in red) to the consensus sequence.
Fig 4
Fig 4. T/F LTR transcriptional activity correlate with viral load.
A: Effect of T/F LTR sequence genetic variation on basal (shown in Cyan), consensus (shown in orange) and autologous (shown in green) Tat-mediated transcriptional activity. Recombinant consensus- or T/F LTR-pGL3 Basic vectors were transfected into Jurkat cells either alone or co-transfected with consensus or autologous Tat-pTarget recombinant plasmid and the luciferase activity was measured after a 24-hour culture period. The transfection assay for each sample was performed in triplicates therefore the transfection data presented here are illustrative of the average relative light units (RLU). B: Basal T/F LTR transcriptional activity exhibit significant positive correlation with viral loads at or near transmission (shown in Cyan). C: Consensus Tat-mediated T/F LTR transcriptional activity exhibit significant positive correlation with viral loads at or near transmission (shown in orange). D: Autologous Tat-mediated T/F LTR transcriptional activity exhibit significant positive correlation with viral loads at or near transmission (shown in green) E: Consensus Tat-mediated T/F LTR transcriptional activity exhibit significant positive correlation with viral loads at one-year post infection timepoint (shown in orange). F: Autologous Tat-mediated T/F LTR transcriptional activity exhibit significant positive correlation with viral loads at one-year post infection timepoint (shown in green). G: Consensus Tat-mediated T/F LTR transcriptional activity exhibit significant positive correlation with viral load set point (shown in orange). H: Autologous Tat-mediated T/F LTR transcriptional activity exhibit significant positive correlation with viral load set point (shown in green).* One of the ten patients without the matching plasma sample at approximately one-year post infection, had longitudinal plasma viral load measurements thus making a total of 32 patients whose viral load set point could be calculated.
Fig 5
Fig 5. Interpatient LTR genetic variability translates to differential transcription activity in the presence of cell stimulants.
Patient derived LTR-pGL3 basic vector recombinants were transfected into Jurkat cells alone or in combination with Tat followed by stimulation with PMA, TNF-α, Prostratin and SAHA after 12 hours of incubation. (A) Patient derived T/F LTR-pGL3 basic vector recombinants were transfected into Jurkat cells alone to measure basal levels of gene transcription (shown in cyan), and in the presence of stimulation with PMA (shown in pink), TNF-α (shown in green), Prostratin (shown in maroon) and SAHA (shown in blue). (B) The patient derived LTR-pGL3 basic vector recombinants were co-transfected with the consensus tat into Jurkat cells to measure Tat induced levels of gene transcription (shown in orange) in the absence or presense of stimulation with PMA (shown in cerise), TNF-α (shown in green), Prostratin (shown in plum) and SAHA (shown in violet). (C) Significant positive correlation between PMA and Prostratin. (D) Positive correlation between PMA and SAHA. (E) Significant positive correlation between Prostratin and SAHA. (F) No correlation between PMA and TNF-α. (G) No correlation TNF-α and Prostratin. (H) No correlation between TNF-α and SAHA.
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