5′-Terminal stem-loop of carnation ringspot virus RNA1 is required for the efficient amplification of viral RNAs

Carnation ringspot virus (CRSV) is the prototype virus of the genus Dianthovirus. Full-length cDNAs of CRSV strainsPV-0097 and PV-21 were constructed and the infectivity of in vitro transcripts was analyzed. Infectivity of PV-0097 and PV-21 to several plants was markedly higher than that of 1.30, a previously reported infectious CRSV clone. Overall RNA sequences of these viruses were similar, but PV-0097 and PV-21 contained additional nucleotides at the 5′ end of RNA1. Stem-loop structures were predicted in the 5′-terminal region of PV-0097 and PV-21 RNA1 but not in 1.30 RNA1. Mutant CRSV 1.30 RNA1 that contains the terminal 4 nucleotides of PV-0097, predicted to fold a 5′-terminal stem-loop structure, recovered higher level accumulation of viral RNAs in the inoculated protoplasts and leaves of Nicotiana benthamiana. These results suggest that the 5′-terminal stem-loop structure of CRSV RNA1 plays an important role in efficient amplification of the virus