Mapping of the large RNA genome segment of infectious pancreatic necrosis virus by hybrid arrested translation

Abstract

Segment A, the larger dsRNA segment of IPNV which encodes three of the four virus-coded polypeptides (preVP2, VP3, and NS) was cloned and physically mapped. The plus and minus RNA strands of the virus genome were separated and the A+ and B+ RNA strands were identified. A nested set of cDNA subclones, coterminal with the 5' end of A+ RNA, were used in hybrid arrested translation experiments. Hybrid arrest conditions which blocked the 5' two-thirds of A+ RNA allowed the in vitro synthesis of only VP3, while hybridization of the RNA to cDNA representing the 5' half of A+ RNA allowed the synthesis of both NS and VP3 but not of preVP2. In vitro translation of A+ RNA yielded all three polypeptides. It is, therefore, concluded that the order of the three polypeptides on A+ RNA is 5'-preVP2-NS-VP3-3'. These results imply that internal initiation of translation could take place on the RNA at least in vitro at sites located hundreds of nucleotides downstream from the first in-phase AUG codon near the 5' end.