Identification and Characterization of the Marek's Disease Virus-encoded Circular RNAs

Serving both as a carrier of genetic information and as a catalyst for chemical reactions, RNA shaped the primitive world. Later, the emergence of DNA and proteins allowed the evolution of all domains of life, in which the three major molecules of life, DNA, RNA, and proteins, are always omnipresent.

RNAs are present in various forms in the cell. Messenger RNA (mRNA) is translated into proteins, transfer RNA (tRNA) and ribosomal RNA (rRNA) are the operators of this translation,
while small RNAs serve in gene regulation, whether they are small nuclear RNAs (snRNA), microRNAs (miRNA), or small nucleolar RNAs (snoRNA). In this fine regulation, circular RNAs (circRNA) participate in several ways in cellular biology. The first way is to serve, like mRNAs, as a template for protein translation. The second way is the inhibition, by sequence interaction, of regulatory RNAs. Finally, through secondary structures, they can recruit proteins and either sequestrate them or allow them to interact with other partners to support their function.

The gallid herpesvirus 2, better known as Marek's disease virus (MDV), causes an aggressive lymphoma in chickens, leading to their death within a few weeks. This is due to the numerous virulence factors it produces. Notably, MDV produces a transcription factor from the AP-1 family that induces the transformation of cells in which it is expressed. This gene is derived from the Q fragment of the viral genome after digestion by the enzyme EcoRI, hence its name: Meq (MDV EcoRI Q). This thesis project focuses on a circRNA derived from this gene.

This study began with the identification of the numerous circRNAs encoded by MDV both in vitro and in vivo. This study demonstrated that during lymphomagenesis, circRNAs are encoded from four major loci: the origin of viral lytic replication (OriLyt), the viral telomerase RNA subunit (vTR), latency-associated transcripts (LAT), and the Meq gene. Then, through the development of the vCircTrappist program, we were able to demonstrate the expression of numerous circRNAs by viruses. Furthermore, the circularization of viral transcripts has been shown to rely on a yet unknown mechanism. Additionally, this program allowed us to investigate the viruses used in vaccination strategies against MDV. These viral MDV-related species also encode circRNAs derived from OriLyt and LATs, as well as circRNAs expressed from the viral DNA packaging gene.

Knowing the importance of Meq in MDV virulence, it was decided to focus on the circRNA produced from this locus, circMeq. The objective was to define its role in MDV-induced pathogenesis. Surprisingly, circMeq has been shown to be a factor attenuating MDV virulence. This was demonstrated by selectively inhibiting either circMeq or the translation of the associated linear transcript to the Meq protein (linMeq) through mutations introduced within the viral genome.

This thesis project has thus revealed major and previously unsuspected properties of the Meq gene, which had always been associated with MDV virulence and not its attenuation. Several studies are still necessary to conclude the story initiated in this project. For example, additional in vivo experiments could be launched to determine the potential role of circMeq in the transmission of the pathogen.