RNA Modifications as Regulators of Viral Infection
2Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New York, NY, USA, USA
3Department of Infectious Diseases, Molecular Virology, Heidelberg University, Germany
RNA modifications are increasingly recognized as important regulators of RNA, including playing a role in viral infection. RNA-virus genomes serve multiple roles: they are templates for transcription, replication and translation. They are packaged into virions, infecting new cells, while also evading the host innate-immune machinery. Regulation of viral RNA molecules is therefore critical for orchestrating successful infection, making RNA viruses great models for studying how modifications regulate RNA activity and fate.
We study 5-methylcytosine (m5C), a common modification of tRNA but infrequent on mRNA, which we found on many positive-sense RNA viruses. We have focused on two viruses representing separate viral families: Sindbis virus (SINV), an alphavirus, and coxsackievirus B3 (CVB3), an enterovirus.
For both viruses m5C proved to be pro-viral. However, the details were virus-specific. We found that SINV RNA is methylated by NSUN2, a host m5C "writer". Interestingly, only viral RNA isolated from infected cells, but not RNA from extracellular virions was methylated. Moreover, m5C was enriched in ribosome-associated viral RNA, and m5C-null virus was impaired in protein translation, suggesting that methylated viral RNA is preferentially targeted to ribosomes.
For CVB3, we identified NSUN5 as the writer, and virus infection led to NSUN5 relocalization to the cytoplasm, the site of viral replication. Importantly, m5C-null CVB3 was significantly impaired in viral replication, suggesting a role in infection. By comparing proteins that specifically bound m5C-modified, but not unmodified viral RNA, we uncovered novel m5C-reader candidates, which were unique to each of the viruses.