RNA Viruses Reshape the RNA Modification Landscape in the Cell

Ribonucleic acid (RNA) has a breathtaking variety of biological functions, which far exceeds its classical role as a carrier of genetic information. The repertoire of this versatile molecule is further diversified by covalent post-transcriptional modifications (PTMs) or chemical marks that decorate all four canonical nucleotides in mRNAs and noncoding RNAs. Although, methylation of the N6-position of adenosine (m6A) was recently shown to be a key regulator of viral gene expression and modulate nuclear export, expression, stability and packaging of viral genomic RNA, the role of the more than 170 other RNA modifications during viral infection is largely unknown. To investigate these other RNA modifications we employed an unbiased and comprehensive analytical approach based on mass spectroscopy to characterize the changes in the landscape of RNA modifications in the cell during virus infection as well as identify the epitranscriptomic marks on the RNA genomes of Zika virus. Zika virus is a re-emerging mosquito-borne flavivirus that during the 2016 outbreak in the Americas was associated with developmental and neurological defects in newborns. Our data show that the RNA modification landscapes change significantly during viral infection. Moreover, during infection of mammalian cells we observed the appearance of two dimethylcytosine modifications namely 5,2’-O-dimethylcytosine (m5Cm) and N4,N4-dimethylcytosine (m44C). Our studies with other RNA viruses show that this dimethylcytosine mark also appears following infection with human immunodeficiency virus 1 (HIV-1), poliovirus, hepatitis C virus and Dengue virus. Remarkably when we analyzed the viral RNA, we identified not just m6A but more than 30 different epitranscriptomic marks on Zika virus genomes. To investigate whether epitranscriptomic profile would similarly change following Zika virus infection in mosquitoes, we fed Aedes aegypti mosquitoes a bloodmeal without and with Zika virus. Mosquitoes were collected 14 days post-infection for analysis. We found that particular dimethyl modifications were more prevalent among infected mosquitoes and may impart a role during the mosquito immune response to infection. While the localization of specific RNA modifications, the function and putative modifying enzymes remains to be identified, our research demonstrates a way in which RNA viruses interface with cellular RNA pathways that may present new targets for bioassays and antiviral treatments.

Learning Objectives:

1.    To be able to define what an RNA modification is and the function of these chemical marks.

2.    To be able to describe one experimental approach to identifying RNA modifications.