A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells.

A long-lived latent reservoir of HIV-1-infected CD4 T cells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 T cells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory T cells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.

Multiplexed next-generation sequencing and de novo assembly to obtain near full-length HIV-1 genome from plasma virus.

Analysing the HIV-1 near full-length genome (HIV-NFLG) facilitates new understanding into the diversity of virus population dynamics at individual or population level. In this study we developed a simple but high-throughput next generation sequencing (NGS) protocol for HIV-NFLG using clinical specimens and validated the method against an external quality control (EQC) panel. Clinical specimens (n=105) were obtained from three cohorts from two highly conserved HIV-1C epidemics (India and Ethiopia) and one diverse epidemic (Sweden). Additionally an EQC panel (n=10) was used to validate the protocol. HIV-NFLG was performed amplifying the HIV-genome (Gag-to-nef) in two fragments. NGS was performed using the Illumina HiSeq2500 after multiplexing 24 samples, followed by de novo assembly in Iterative Virus Assembler or VICUNA. Subtyping was carried out using several bioinformatics tools. Amplification of HIV-NFLG has 90% (95/105) success-rate in clinical specimens. NGS was successful in all clinical specimens (n=45) and EQA samples (n=10) attempted. The mean error for mutations for the EQC panel viruses were <1%. Subtyping identified two as A1C recombinant. Our results demonstrate the feasibility of a simple NGS-based HIV-NFLG that can potentially be used in the molecular surveillance for effective identification of subtypes and transmission clusters for operational public health intervention.