N6-Methyladenosine reader YTHDF3 regulate Sindbis virus release by incomplete selective autophagy flux maintenance through SQSTM1

N6-methyladenosine (m6A) is vital for RNA metabolism and function and is one of the most abundant internal RNA modifications. Recently, m6A has been indicated in regulating replication of multiple viruses; however, the role of m6A in typical arbovirus-alphavirus, is mainly elusive. Alphavirus containing several emerging and re-emerging pathogens that poses great threat to human health. Here, we show that the prototype alphavirus, Sindbis virus (SINV), its replication is regulated by m6A readers. Of which, depleting YTHDF3 results in reduced SINV release but increased viral capsid within the cell. Mechanically, YTHDF3 bind and regulate the mRNA stability of SQSTM1, the major selective autophagy receptor, which targeting of capsid into autophagosomes. Furthermore, we found interaction between YTHDF3, SQSTM1 and capsid in pairs. K27 ubiquitination is the dominant modification for SINV capsid. Besides ubiquitin-associated (UBA) domain, the N-terminal domain of SQSTM1 has stronger interaction with capsid, highlighted the role of RL peptide mimic within the capsid. Further experiments revealed that the interaction between YTHDF3 and SQSTM1 protein relies on their YTH-domain and ZZ-type Zinc finger domain respectively. Together, these results highlight the role of m6A modification reader YTHDF3 in regulating selective autophagy during virus infection and provide new insights into alphavirus replication cycle.