Parvovirus RNA processing: Elucidating the localization, expression, and role of m6A methylation proteins and m6A modifications in parvovirus infection

Abstract

Viral mRNA processing is an essential gene expression node exploited by DNA viruses for efficient genome replication, RNA metabolism, and virion production. Parvoviruses are linear single-stranded DNA viruses that infect animals and humans and represent an excellent tractable model to understand virus-host cell interactions. Carnivore bocaparvovirus 1 (minute virus of canine, MVC) is a parvovirus with a single promoter that generates multiple viral RNA transcripts through alternative splicing and alternative polyadenylation. NP1, a nonstructural protein, suppresses MVC proximal polyadenylation and facilitates the splicing of the upstream intron for capsid protein production. The mechanism of this NP1-mediated alternative polyadenylation and splicing has not been comprehensively explored. An NP1 pulldown assay identified RNA processing proteins, including Wilms Tumor 1-Associating Protein (WTAP), a critical component of the N6-methyladenosine (m6A)-methyltransferase complex as potential interacting partners. m6A RNA modifications have been shown to impact gene expression, genome replication, and virion production of many DNA viruses, but the role of these modifications has not yet been described in linear single-stranded DNA viruses. Our results indicate the recruitment and localization of WTAP, METTL3, METTL14, YTHDF3, and ALKBH5 to parvovirus autonomous replication sites (APAR bodies). There is a marked depletion of YTHDF1 (m6A reader) 48 hours after MVC infection. m6A modifications are reorganized and have increased expression in infected cells, and there is colocalization of these modifications with viral non-structural proteins. Our results suggest the m6A methylation proteins and potential parvoviral mRNA m6A modification may be essential for parvoviral RNA processing, DNA replication, and capsid protein production.