Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins

•The RNA2-ORF2-encoded polyproteins of ToMarV-M and ToChSV have a Q at the -1 position of the cleavage site.•Multiple aa sequence comparison confirmed that this Q is conserved among other torradoviruses.•Site-directed mutagenesis of the ToMarV-M RdRp or protease prevented accumulation of viral coat proteins and RNAs.

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7 kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5 kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the −1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs.