Functional characterization of human immunodeficiency virus type 1 (HIV-1) subtype C transmitted/founder (T/F) viruses long terminal repeat (LTR) variants and association with disease outcome.
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Background: The persistence of latent viral reservoirs is a major roadblock to human immunodeficiency virus type 1 (HIV-1) cure development. Latent reservoirs harbour transcriptionally silent yet replication competent proviruses. However, the molecular mechanisms that govern HIV-1 latency at the transcriptional level is unknown. Therefore, we hypothesize that HIV-1 subtype C (HIV-1C) transmitted/founder (T/F) 5’ long terminal repeat (LTR) genetic variation may affect disease outcome. Methods: To address this, viral RNA was extracted from plasma samples obtained from 25 HIV-1 infected patients from the HPP and FRESH acute infection cohorts (QIAamp® Viral RNA Mini Kit, Qiagen, Hilden, Germany). Viral RNA was reverse transcribed to DNA using SuperScript™ III One Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, Massachusetts, United States). Nested PCR was performed (Platinum® Taq DNA Polymerase High Fidelity PCR Kit (Invitrogen, Massachusetts, United States) and PCR products cloned into the pGL3 Basic plasmid. LTR/pGL3 recombinant plasmids were sequenced using BigDye Terminator v3.1 Sequencing Kit (Invitrogen, Massachusetts, United States) to confirm correct sequences. The LTR-pGl3 recombinant plasmids were transfected into Jurkat cells alone or co-transfected with either consensus (wild type) subtype C Tat (conTat) or autologous tat (autoTat) to determine the effect of LTR genetic variation on expression of a luciferase reporter gene. Results: Interestingly, our data demonstrate that basal transcription activity significantly differs between LTR variants. Specifically, patients harbouring the Sp1 III: G2A mutation demonstrated significantly lower transcription compared to the wild type LTR. Although conTat co-transfection increased the LTR activity for most of the LTR variants, the T/F virus LTR containing the TATA box mutation (TATAA TAAAA) in combination with other LTR mutations was not induced. Interestingly, the transactivation activity of the autologous Tat was variable among patients. Specifically, the TATA box variant was marginally induced. Lastly, we observed that the majority of LTR variants were more responsive to stimulation by PMA as compared to TNF-α, SAHA and prostratin. Interestingly, our data demonstrate that autologous tat induced transcription positively correlated with viral load at transmission (p=0.0134, r=0.66) and at one-year post infection but was not significant (p=0.3905, r=0.26). Conclusion: These data suggest that the TATAA TAAAA mutation in combination with other LTR mutations may reduce transcription activity. Taken together our data suggest that HIV-1 subtype C T/F viruses LTR genetic variation may modulate viral gene transcription and impact disease outcome.