5′-terminal nucleotide sequences of the double-stranded RNA of silkworm cytoplasmic polyhedrosis virus

Abstract

The 5'-terminal nucleotide sequence of double-stranded RNA of silkworm cytoplasmic polyhedrosis virus was analyzed. In order to label the 5′-termini of the RNA with [32P]phosphate using polynucleotide kinase, it was necessary to remove two or three nucleotides from the 3′-termini, before phosphorylation, by repeating sequential operations of oxidation of the 3′-terminal ribose, β-elimination, and phosphomonoesterase treatment. These methods did not cause any chain scission, and almost all the 5′-terminal nucleotides were labeled with [32P]phosphate. On alkaline digestion, ribonuclease T2 digestion or ribonuclease T1 digestion, the same amount of 32pGp and a dinucleotide 32pYpGp (Y denotes an alkaliresistant unknown nucleoside) were detected as terminal nucleotides. 32pYpGp was split into mononucleotides by Penicillium nuclease or by a combination of Aspergillus nuclease S1 and snake venom phosphodiesterase. The unknown nucleotide 32pY seems to be a derivative of adenylic acid (pA*) on the basis of its chromatographic behavior. By analysis of two trinucleotides obtained by pancreatic ribonuclease digestion, the two terminal sequences of cytoplasmic polyhedrosis virus RNA were identified as pGpGpCp- and pA*pGpUp-. These two sequences are complementary to the 3′-terminal sequences. Analysis of separated segments of cytoplasmic polyhedrosis virus RNA showed that all ten RNA segments carry the same two 5′-terminal sequences. Therefore, each RNA segment in the virus has the following structure: With the results obtained in the accompanying paper, it appears that the transcription of cytoplasmic polyhedrosis virus RNA starts from the side where the 5′ terminus carries an unusual nucleotide, using the complementary 3′-terminal sequence as a template.