Incomplete dsRNA genomes in purified infectious pancreatic necrosis virus.
The dsRNA containing birnavirus, infectious pancreatic necrosis virus, possesses a virion-associated RNA-dependent RNA polymerase which acts both as primer and as polymerase during in vitro RNA synthesis (P. Dobos, 1995, Virology 208, 19-25). Using [alpha 32P]GTP, we have radiolabeled virion RNA in vitro and found that after deproteinization most of the labeled product comigrated in agarose gels with the 3-kbp viral genome, while the remainder migrated faster than the dsRNA and as a heterogeneous smear. Agarose gel electrophoresis (AGE) of denatured labeled virion RNA showed a radioactive smear ranging from approximately 100 nucleotides to up to 3000 nucleotide the size of genome length single stranded RNA. Hybridization experiments using strand-specific and end-specific obligonucleotides on Northern blots revealed that the radioactivity which migrates with the dsRNA during AGE represents small, 5' end plus RNA molecules of 100-500 nucleotides. The radioactivity in the faster migrating smear denotes incomplete dsRNAs where full-length, unlabeled minus strands are base-paired with labeled plus strands that are 3' truncated to different extents. This was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using end- and strand-specific obligonucleotide primers. The results indicated that 95% of incomplete dsRNA molecules consisted of full-length minus strands and 3' truncated plus strands. The implications of these findings are discussed in light of RNA replication mechanisms of dsRNA viruses belonging to other families.
Kordyban
S
S
Department of Microbiology, College of Biological Science, University of Guelph, Ontario, Canada.
Magyar
G
G
Chung
H K
HK
Dobos
P
P
D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D016679:Genome, Viral; D018061:Infectious pancreatic necrosis virus / Q000235:genetics*; D008969:Molecular Sequence Data; D012330:RNA, Double-Stranded / Q000235:genetics*; D012367:RNA, Viral; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012330:RNA, Double-Stranded; D012367:RNA, Viral