The 3' terminal region is strictly required for clover yellow vein virus genome replication

To identify the cis-element in the 3' terminal region of infectious cDNA required for replication of clover yellow vein virus (ClYVV), a series of mutants with duplications or deletions of the 3' terminal non-coding region (3'-NCR) of the genome that did not affect the ORFs in the genome was constructed. These were tested for infectivity, and the 3' terminal regions of their progeny RNAs were sequenced. Deletion mutants that lacked portions of the 3'-NCR were not infectious. Various mutants with duplicated 3' terminal sequences were infective only when the authentic 3' terminal sequence was restored, probably by recombination, and none of the constructs retained the original sequence in progeny viral RNA. When a coat protein gene sequence of bean yellow mosaic virus (BYMV) followed by a termination codon was introduced between the nuclear inclusion b and coat protein genes, infective progeny were generated. Sequence analyses of the progeny viruses showed that the coat protein gene was a chimera of the BYMV N-terminal and CIYVV C-terminal portions. These results suggest that the 3'-NCR of ClYVV contains cis-acting elements and is strictly required for genome replication.