Signatures of Host mRNA 5′ Terminus for Efficient Hantavirus Cap Snatching

Abstract

Hantaviruses, similarly to other negative-strand segmented RNA viruses, initiate the synthesis of translation-competent capped mRNAs by a unique cap-snatching mechanism. Hantavirus nucleocapsid protein (N) binds to host mRNA caps and requires four nucleotides adjacent to the 5′ cap for high-affinity binding. N protects the 5′ caps of cellular transcripts from degradation by the cellular decapping machinery. The rescued 5′ capped mRNA fragments are stored in cellular P bodies by N, which are later efficiently used as primers by the hantaviral RNA-dependent RNA polymerase (RdRp) for transcription initiation. We showed that N also protects the host mRNA caps in P-body-deficient cells. However, the rescued caps were not effectively used by the hantavirus RdRp during transcription initiation, suggesting that caps stored in cellular P bodies by N are preferred for cap snatching. We examined the characteristics of the 5′ terminus of a capped test mRNA to delineate the minimum requirements for a capped transcript to serve as an efficient cap donor during hantavirus cap snatching. We showed that hantavirus RdRp preferentially snatches caps from the nonsense mRNAs compared to mRNAs engaged in translation. Hantavirus RdRp preferentially cleaves the cap donor mRNA at a G residue located 14 nucleotides downstream of the 5′ cap. The sequence complementarity between the 3′ terminus of viral genomic RNA and the nucleotides located in the vicinity of the cleavage site of the cap donor mRNA favors cap snatching. Our results show that hantavirus RdRp snatches caps from viral mRNAs. However, the negligible cap-donating efficiency of wild-type mRNAs in comparison to nonsense mRNAs suggests that viral mRNAs will not be efficiently used for cap snatching during viral infection due to their continuous engagement in protein synthesis. Our results suggest that efficiency of an mRNA to donate caps for viral mRNA synthesis is primarily regulated at the translational level.