Exploration of the Epitranscriptomic landscape of HIV infected cells

HIV establishes latency in a minority of cells that are infected. These cells represent one reservoir from where viral particles can be produced potentially at any time thereby initiating new rounds of infection, and thus representing a major obstacle to HV cure. Reactivating viral particle production from these latently infected cells may help purging the HIV-1 reservoir and help eradicating HIV-1, a strategy referred to as “shock and kill”.

HIV latency is characterized by the absence of infectious viral particle production, and can result from multiple mechanisms, such as repression of HIV transcription or post-transcriptional blocks. Understanding the multiple molecular processes leading to HIV latency may help design more targeted strategies and will likely require a combination of latency reversing agents (LRA) to finally purge the reservoir of latently infected cells.

In the past few years, the rise of a new field, epitranscriptomics, provided a new perspective to understand cellular biology, and thus HIV biology as well. Epitranscriptomics investigates post-transcriptional RNA modifications. To date, more than 100 post-transcriptional modifications have been described (3). Indeed, every position of pyrimidine and purine rings can be post-transcriptionally modified, mostly through methylation. Similarly to histone and DNA modifications in epigenetics, RNA modifications have been shown to impact cellular processes, including RNA splicing, export, stability and translation.

Among these modifications, N6-methyladenosine (m6A), which is also the most abundant, and 5 -methylcytosine (m5C) are of particular interest for mRNA. These two modifications are reversible marks that are co-transcriptionally added by specifics methylases (writers) and eventually removed by demethylases (erasers). These modifications are recognized by specific proteins (readers) that will dictate the fate of the modified mRNA.) .

In 2016 three studies described, for the first time, the presence of m6A modifications on HIV RNA . These studies mapped up to 14 m6A methylation sites (although the resolution did not allow the precise identification of the modified nucleotide) on the HIV genome, resulting in the enhancement of viral particle production.

To date there is still no evidence of the presence of m5C marks on HIV RNA, but its presence on other viruses may leave open the possibility that HIV will be no exception.

The discovery of HIV RNA methylation provides a new layer of regulation of the HIV replication cycle that should be further explored.

The aim of this PhD project is to characterize the m6A and m5C epitranscriptomic landscape of the viral RNA genome in two infection settings, a productive infection and a latent infection. In particular, we will explore viral epitranscriptomics in the context of (i) dynamic analysis of productively infected cells (cell line model), (ii) analysis of HIV particles, (iii) analysis of HIV latency in a primary cell model, and (iv) characterization of latency reversing agent impact on reactivation of latently infected cells.

During the first year of my PhD I optimized the techniques required for epitranscriptome analysis, including RNA immunoprecipitation, RNA bisulfite conversion and library preparation for next generation sequencing.