A general strategy for cloning double-stranded RNA: nucleotide sepuence of the Simian-11 rotavirus gene 8

Abstract

Using Simian-11 rotavirus RNA, a strategy has been developed for the production of full length cloned copies of the genes of double-stranded (dsRNA) viruses. Genomic RNA segments were polyadenylated and reverse transcribed to yield a mixture of full length cDNA copies of both possible polarities. The cDNAs were annealed, filled in to complete any partial copies, tailed and inserted into the PstI site of pBR322 using dG/dC tailing. Cloned rotavirus cDNA gene copies were assigned to genomic RNA segments by Northern hybridization. The complete sequence of gene 8 which codes for NCVP3, a non-structural protein of SA11 rotavirus, was determined from a cloned gene copy. It is 1059 bases in length and has an open reading frame which could code for a protein containing 317 amino acids. The apparent 5' and 3' terminal non coding regions are 46 and 59 bases in length, respectively. The sequence ATGTGACCOH at the 3' end of the plus strand is conserved in four of the eleven genes examined. The cloning procedures used should be generally applicable to viruses with segmented dsRNA genomes.

Rotavirus RNA Segments Sized by Electron Microscopy

“…Priess et al (1980) found a linear relationship between length and number of bp of dsDNA in the range of 400 to 10000 bp and there is no reason that this should not be the case for the size range of dsRNAs investigated (Vasquez & Kleinschmidt, 1968). Our measurements agree, with a difference of ~< 6~ (Table 1, column 8), with the cDNA size estimates of size classes II (segments 5 and 6), III (segments 7 to 9) and IV (segments 10 and 11) (Both et al, 1982), whereas they are 8 to 17 ~ lower for molecules of size class I (segments 1 to 4). Segments 1, 2 and 3 of SA11 virus from which the values of Both et al (1982) are obtained and the corresponding segments of the UK bovine rotavirus migrate very similarly (Pereira et al, 1983) whereas segment 4 of the UK bovine rotavirus migrates slightly more slowly, i.e.…”

“…(1978), applying Shatkin's approach to rotavirus RNAs, obtained estimates (Table 1, column 3) which for RNA segments 7 11 were closer to their real size than the estimates obtained by Newman et al (1975), Verly & Cohen (1977), Todd etal. (1980 and Rodger & Holmes cited by Both et al (1982) (Table 1, columns 1, 2, 4 and 5 respectively). However, the size estimates of the large dsRNA segments of rotavirus by Kalica et al (1978) were considerably smaller than the corresponding values of most other estimates• Since 1982, procedures have been published which have allowed cloning of the rotavirus genome (McCrae & McCorquodale, 1982;Both et al, 1982Both et al, , 1983Imai et al, 1983;Dyall-Smith et al, 1983).…”

“…However, the size estimates of the large dsRNA segments of rotavirus by Kalica et al (1978) were considerably smaller than the corresponding values of most other estimates• Since 1982, procedures have been published which have allowed cloning of the rotavirus genome (McCrae & McCorquodale, 1982;Both et al, 1982Both et al, , 1983Imai et al, 1983;Dyall-Smith et al, 1983). DNA complementary to the genomic RNA of rotavirus was obtained in a reaction step using reverse transcriptase and this eDNA has been sized on denaturing gels using plasmid pBR322 DNA fragments of known size as markers• The size values thus obtained by Both et al (1982) differed considerably from the previous ones (Table 1, compare column 6 with columns 1 to 5), and McCrae & McCorquodale (1982) had results similar to those of Both et al (1982). The definite sizes obtained from sequence data of segments 7, 8, 9 and 11 of different rotavirus strains (Both et al, 1982(Both et al, , 1983Imai et al, 1983 ;Dyall-Smith et al, 1983) * Bovine rotavirus, 5~ polyacrylamide gel; reovirus RNAs as standard (Martin & Zweerink, 1972).…”

“…DNA complementary to the genomic RNA of rotavirus was obtained in a reaction step using reverse transcriptase and this eDNA has been sized on denaturing gels using plasmid pBR322 DNA fragments of known size as markers• The size values thus obtained by Both et al (1982) differed considerably from the previous ones (Table 1, compare column 6 with columns 1 to 5), and McCrae & McCorquodale (1982) had results similar to those of Both et al (1982). The definite sizes obtained from sequence data of segments 7, 8, 9 and 11 of different rotavirus strains (Both et al, 1982(Both et al, , 1983Imai et al, 1983 ;Dyall-Smith et al, 1983) * Bovine rotavirus, 5~ polyacrylamide gel; reovirus RNAs as standard (Martin & Zweerink, 1972). t Bovine rotavirus, 5~ polyacrylamide gel; reovirus RNAs as standard (Wood, 1973).…”

“…The sequence data of rotavirus RNAs available so far are of the smaller segments [663 bp for segment 11 of human Wa strain (Imai et al, 1983); 1059 bp for segment 8 of simian SA11 virus (Both et al, 1982); 1059 bp for segment 7 of UK bovine rotavirus (Dyall-Smith et al, 1983); 1062 bp for segment 9 of SA11 virus (Both et al, 1983)]. Our data are in good agreement with these sequences (3-7~ higher for the triplet of segments 7, 8 and 9; 3"9~o lower for segment 11 of bovine rotavirus assuming its real number of bp to be very close to that of segment 11 of the human Wa strain).…”