Incomplete dsRNA Genomes in Purified Infectious Pancreatic Necrosis Virus
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文摘
The dsRNA containing birnavirus, infectious pancreatic necrosis virus, possesses a virion-associated RNA-dependent RNA polymerase which acts both as primer and as polymerase duringin vitroRNA synthesis (P. Dobos, 1995,Virology208, 19–25). Using [α32P]GTP, we have radiolabeled virion RNAin vitroand 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 nucleotides, the size of genome-length single stranded RNA. Hybridization experiments using strand-specific and end-specific oligonucleotides 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 oligonucleotide 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.