Nidovirus Replication Complexes: How enzymes shape viral genomes

Positive-stranded RNA (+RNA) viruses constitute some of the most globally significant human pathogens, causing substantial health and economic burden. The recent emergence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has evidenced the power of these viruses to rapidly evolve and adapt, even when faced with external pressures such as vaccines or antivirals.

The success of +RNA viruses can principally been attributed to a ‘defect’ of sorts; the high error-rates, or low fidelity, of their replicative enzymes. Replication and transmission of the viral +RNA genome is mediated by a suite of viral proteins, known as the replication transcription complex (RTC), at the center of which is an RNA-dependent RNA polymerase (RdRp). The RdRp and associated RTC is responsible for copying and amplifying the genomic information for passage to progeny virions.

However, in contrast to the RTCs utilized by DNA-based life-forms, these complexes lack co- and post-replicative proofreading and repair capacities. Consequently, during replication ‘mismatched’ nucleotides (mutations) are introduced throughout the genome resulting in genetically diverse populations referred to as ‘mutant swarms’ or ‘quasispecies’. This provides viruses with an incredible evolutionary capacity, ensuring their survival and success. However, high-mutation rates is a double-edged sword, and this infidelity can be exploited for antiviral therapies using drugs that mimic native nucleotide substrates. Incorporation of these chemically modified substitutes into the viral genome can block further synthesis, or corrupt the viral genetic information.

As viral mutations occur at random, many may actually be detrimental to the virus. This limits genome-size, with most +RNA viruses restricted to 10-15 kb. Viruses larger than this presumably accumulate too many mutations to remain viable. However, a notable exception are +RNA viruses belonging to the order Nidovirales, which have managed to break the ‘genome-size barrier’. The order is comprised of 14 diverse families, with genomes ranging from ∼11 to 41 kb. They remain mostly neglected, with the exception of the large-genome Coronaviridae (CoV) family (32 kb), which includes the notorious human pathogen SARS-CoV-2.

At the other end of the size-spectrum is the small-genome Arteriviridae (ArV) family (11 kb), an understudied group of prevalent, animal-infecting pathogens. The difference in genome size is believed to be due to the gradual acquisition of novel proteins, many of which are required for the stability and maintenance of such long genomes. For example, CoVs have acquired a proofreading exonuclease, capable of repairing mistakes in the viral genome to restore synthesis.

NidoRep aims to bridge the prevalent knowledge-gap between small and large genome nidoviruses through a structural-functional comparison of their viral RTCs; at the center of which is the RdRp, and its N-terminal, nidovirus-unique nucleotidylation domain (the NiRAN). The project will probe how RdRp speed and fidelity have been fine-tuned to accommodate the distinct and conflicting replication requirements of diverse nidoviruses. This will be achieved through the development of an innovative in vitro sequencing assay, also providing a new and robust tool for screening drugs that corrupt RNA synthesis. The project will additionally explore the role of the NiRAN domain in the viral lifecycle, and how this diverse domain has structurally and functionally evolved to support remarkable genome diversification within a single viral order.