IDENTIFICATION OF THE INITIATION SEQUENCE FOR VIRAL-STRAND DNA-SYNTHESIS OF WHEAT DWARF VIRUS

The intergenic region of the circular single-stranded DNA genome of ge miniviruses contains a sequence potentially able to fold into a stem-l oop structure. This sequence has been reported to be involved in viral replication by serving as the origin for rolling-circle replication. However, in wheat dwarf virus (WDV) a deletion of 128 bp, removing thi s sequence, surprisingly does not prevent de novo viral DNA synthesis, but instead abrogates the processing of replicative intermediates int o monomeric genomes. This deletion mutant permitted us to study the in itiation of viral-strand DNA synthesis independently from its terminat ion and also to identify the sequence within which rolling-circle DNA replication of WDV begins. We have mapped the initiation site of repli cation to a pentanucleotide, TACCC, a sequence that occurs twice in th e large intergenic region of WDV: it is found in the right half of the stem-loop sequence and again 170 bases upstream where it is part of a 15 nucleotide sequence highly homologous to the right half of the ste m-loop sequence. Here we show that viral-strand DNA synthesis efficien tly initiates at both sequences.