Decrypting RNA Synthesis by Pneumoviruses – DecRisP

The pneumovirus family contains several viruses that infect the respiratory tract and induce severe or fatal pneumonia and bronchiolitis in humans and animals. Pneumoviruses include human respiratory syncytial virus (RSV), bovine RSV, human metapneumovirus, avian metapneumovirus, and the recently discovered swine orthopneumovirus (SOV). There are no effective vaccines against these viruses and antivirals are restricted to Ribavirin, a toxic and nonspecific nucleoside analogue. Developing new strategies against these viruses requires improving our knowledge of the molecular mechanisms of viral replication.

The genome of Pneumoviruses is composed of an non segmented, single-stranded RNA with a negative polarity of around 15 kb and which encodes 9-11 genes. This RNA genome is always enwrapped by the viral nucleoprotein which prevents any detection of RNA by the cellular innate immunity sensors and protects it against cellular attacks. Pneumoviruses are enveloped viruses that replicate in the cytoplasm of the cells. Once the viral and cellular membranes are fused together, the virus is released into the cytoplasm in the form of a holo-nucleocapsid including the viral polymerase L, capable on the one hand of synthesizing viral mRNAs that will be translated by the cellular machinery, and on the other hand to replicate its genome to create new viral particles that will bud at the plasma membrane thanks to the neo-synthesized glycoproteins. In this project we want to elucidate the mechanisms of viral RNAs synthesis as well as the mechanisms of encapsidation of genomes by nucleoproteins.

The viral machinery synthesizing the viral RNAs is autonomous and specific and formed by a multi-protein complex (no cellular equivalent). This complex consists of 3 central proteins: the large polymerase L (250 kDa), the phosphoprotein P, the nucleoprotein N which surrounds the genomic RNA. Pneumoviruses also have transcription (M2-1) or replication (M2-2) co-factors. To synthesize the mRNAs or replicate the genome, the polymerase must temporarily access the viral RNA by "opening" the nucleocapsid. Mirroring, after synthesis of the new genomes, they will be encapsidated by neosynthesized N proteins.

In this project, we want to elucidate the molecular mechanisms and regulation of this complex process by associating 4 teams with complementary skills and expertise:

(1) an INRA team with the molecular tools to synthesize and purify N, P, M2-1 and L proteins in recombinant form as well as genomic RNAs.
(2) an IBS team able to solve structures by electron microscopy and cryo-microscopy.
(3) an IBMM team (CNRS, Montpellier University, ENSCM) specialized in the synthesis of modified and varied RNAs at the level of their sequence (methylation at different nucleoside sites) or at the 5 'end by adding various caps.
(4) a CNRS-U Aix-Marseille team with the tools and know-how to measure enzymatic activities carried by L proteins.

Our main specific objectives will be:

To determine the mechanisms controlling the specificity (sequence, chemical nature) of encapsidation of the RNAs by the N proteins

To characterize the atomic structure of the N-RNA protein complexes

To characterize the epitranscriptomic modifications of the RNAs mediated by the L protein (capping and methylations).

Ultimately, we want to develop an autonomous system of in vitro synthesis of RNAs that will permit studying the functioning of this complex and testing antiviral compounds targeting the replication / transcription steps in vitro.