Achuthan, V. | bioRxiv Science

bioRxiv2024

Disruption of LEDGF/p75-directed integration derepresses antisensetranscription of the HIV-1 genome

AbstractDisruption of HIV-1 Integrase (IN) interactions with the host-factor Lens Epithelium-Derived Growth Factor (LEDGF)/p75 leads to decreased, random integration, increased latent infection, and described here, accumulation of HIV-1 antisense RNA (asRNA). asRNA increase was observed following interruptions of IN-LEDGF/p75 interactions either through pharmacologic perturbations of IN-LEDGF/p75 by treatment with allosteric HIV-1 integrase inhibitors (ALLINIs) or in cell lines with LEDGF genetic knockout. Additionally, by impairing Tat-dependent HIV transcription, asRNA abundance markedly increases. Illumina sequencing characterization of asRNA transcripts in primary T cells infected in the presence of ALLINIs showed that most initiate from within the HIV-1. Overall, loss of IN-LEDGF/p75 interactions increase asRNA abundance. Understanding the relationship between ALLINIs, integration sites, asRNA, and latency could aid in future therapeutic strategies.

microbiology↗

bioRxiv2021

Physiological Magnesium Concentrations Increase Fidelity of Diverse Reverse Transcriptases from HIV-1, HIV-2, and Foamy Virus, but not MuLV or AMV

Reverse transcriptases (RTs) are typically assayed in vitro using optimized Mg2+ concentrations ([~]5-10 mM) several-fold higher than physiological cellular free Mg2+ ([~]0.5 mM). Analysis of fidelity using lacZ-based -complementation assays showed that tested HIV RTs, including HIV-1 from subtype B (HXB2-derived), HIV-2, subtype A/E, and several drug-resistant HXB2 derivatives all showed significantly higher fidelity using physiological Mg2+. This also occurred with prototype foamy virus (PFV) RT. In contrast, Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV) RTs demonstrated equivalent fidelity in both low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated {approx} equal fidelity, except for PFV RT which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to accurately determine mutation rates and profiles was used to examine the types of mutations made by HIV-1 (subtype B, wild type) in low (0.5 mM) and high (6 mM) Mg2+ with DNA or RNA that coded for lacZ. Unlike the -complementation assay, which is dependent on LacZ activity, the NGS assay scores mutations at all positions and of every type. Consistent with -complementation assays, a [~]4-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g., MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RTs activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV.

biochemistry↗