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Journal o f General Virology (1992), 73, 633-638. Printed in Great Britain 633 Nucleotide sequences of normal and rearranged RNA segments 10 of human rotaviruses Anna Ballard, 1 M. A. McCrae 2 and U. Desselbergerl*f 1Regional Virus Laboratory, East Birmingham Hospital, Birmingham B9 5 S T and 2Department o f Biological Sciences, Warwick University, Coventry CV4 7AL, U.K. Normal and rearranged R N A segments 10 of group A rotaviruses isolated from a chronically infected immunodeficient child were amplified by the polymerase chain reaction as full-length cDNA copies, and were subsequently cloned and sequenced. Compared with the nucleotide sequence of the normal R N A segment 10, the rearranged form contains a partial non-coding duplication at its 3' end and several point mutations. The normal R N A segment 10 was similar to that of bovine rotavirus. Introduction partial duplications have been found which had been generated by reiteration of genomic sequences after the termination codon of the normal open reading frame (ORF). We have described numerous subpopulations of rotaviruses isolated from a chronically infected immunodeficient child which contain various forms of rearrangements of RNA segment 8-, 10- and 11-specific sequences (Hundley et al., 1987). Here we report the sequences of the normal (standard size) R N A 10 and of its rearranged form in one of these viruses, and compare the sequences both with each other and with those of other RNA segments 10 (Both et al., 1983; Baybutt & McCrae, 1984; Okada et al., 1984; Ward et al., 1985), as well as with the structures of other rearranged genome segments. Group A rotaviruses, the main cause of viral gastroenteritis in infants and in the young of a variety of mammals and birds (Flewett & Woode, 1978; Estes et al., 1984; Kapikian & Chanock, 1990), usually possess a genome of 11 segments of dsRNA of well conserved size (Estes, 1990). However, within Group A, rotaviruses with atypical R N A profiles have been observed in which segments of standard size are replaced by rearranged forms of larger size. Such genomes were first found in rotaviruses isolated from chronically infected immunodeficient children (Pedley et al., 1984; Eiden et al., 1985; Hundley et al., 1987), but have also been isolated from both human (Besselaar et al., 1986) and mammalian (Thouless et al., 1986; Pocock, 1987; Bellinzoni et al., 1987; Tanaka et al., 1988) immunocompetent hosts. It has also been possible to generate genome rearrangements in tissue culture-adapted bovine rotaviruses by serial passage at high m.o.i. (Hundley et al., 1985). Variants of bluetongue virus, a member of the orbivirus genus of the Reoviridae family, also exhibit rearranged genomes (Ramig et al., 1985; Eaton & Gould, 1987). Thus, genome rearrangements may be an important mechanism of variation and evolution of dsRNA viruses (Desselberger, 1989). Several of these rearranged genes have been sequenced (Gonzalez et al., 1989; Gorziglia et al., 1989; Scott et al., 1989; Matsui et al., 1990). In most cases t Present address: Public Health Laboratory, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, U.K. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession numbers D01145 and D01146. 0001-0630 © 1992 SGM Methods Viruses and cells. Rotaviruses isolated from a child with severe combined immunodeficiency carrying various forms of rearrangements of RNA segments 8, 10 and 11 (Hundley et al., 1987) were investigated. Initially, two viruses, A28 and A64, which carry normal and rearranged RNA segments 10, respectively, were studied. Both of these viruses also carry rearranged segments 8 and 11 (genotypes 5 and 7 as shown in Fig. 3 of Hundley et al., 1987). Bovine rotavirus (UK Compton strain) was propagated as a control. Viruses were grown on confluent monolayers of MA104 cells infected at low multiplicity (m.o.i., 0-1) to prepare stock suspensions (Hundley et al., 1987). Electrophoretic fraetionation of rotavirus dsRNA and of amplified D NA. Viral genomic dsRNA was extracted from virions and fractionated by electrophoresis in 10~ polyacrylamide gels as described previously (Rodger & Holmes, 1979). The dsRNA was visualized by silver staining (Follett et al., 1984). Amplified DNA bands were fractionated on 1 ~ agarose gels using TAE buffer (Sambrook et al., 1989) and visualized by staining with ethidium bromide. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 14:42:05 634 A. Ballard, M. A. McCrae and U. Desselberger Preparation ofrotavirus mRNA. MA104 cells were infected at high multiplicity (1 to 5) and grown overnight in the presence of 0.5 p-g/ml of trypsin (Sigma Type IX) until c.p.e, was observed. Cells were harvested and lysed by incubation on ice in Tris-NP40 buffer (100 mM-Tris-HC1 pH 8.0, 50 mM-NaCI, 10 mM-EDTA and 0-5% NP40). Subsequently, unlysed cells and nuclei were removed by brief centrifugation (11000 g, 15 s), and the cytoplasmic supernatant was digested for 3 h at 37 °C with 250 p-g/ml of proteinase K (Sigma) in the presence of 0-2% SDS, followed by repeated extractions with phenol (saturated with 100 m i Tris-HCl pH 8.0) and ether, and ethanol precipitation. The precipitate was centrifuged (11000g, l0 min) and resuspended in a small volume of distilled water (McCrae, 1985). Starting with one 75 cm x Roux bottle of MA104 cells, 100 gl containing a mixture of mRNA and dsRNA was obtained; the RNA content was not quantified. Synthesis o f full-length cDNA. Full-length cDNA corresponding to RNA segment 10 was made by reverse transcription of mRNA from virus-infected cells followed by amplification in a polymerase chain reaction (PCR). The oligonucleotide primers used are shown in Fig. 1 (a). In the 3' halves of the primers the nucleotide sequences are complementary to the termini of viral RNA or cDNA segment 10; in their 5' halves they contain several restriction endonuclease recognition sites as indicated (Xu et al., 1991). The oligonucleotides were produced on an ABI DNA synthesizer (model PCR-mate 391). For synthesis of cDNA from normal-length RNA segment 10, 5 to 10 p-1 of mRNA was mixed with 100 ng of each primer and dimethyl sulphoxide to a final concentration of 10 % in a final volume of l 2.5 p-l. Under these conditions, amplification from dsRNA (purified from virions) was never successful; the products obtained were likely to have originated from mRNA molecules. The reaction was then heated in a Techne PHC-2 Thermocycler to 94 °C for 2 min and cooled to 42 °C. An equal volume (12.5 p.1) of reverse transcriptase/PCR amplification mixture was added [final concentrations: 10 mM-Tris-HC1 pH 8.3, 50 mM-KCI, 1.5 mi-MgCl2, 0.3 mM-DTT, 0.05% gelatin, 0.2 mMdATP, 0.2 mM-dGTP, 0.2 mM-dCTP, 0.2 mM-TTP, 6 units avian myeloblastosis virus reverse transcriptase (Pharmacia) and 0-5 units Taq DNA polymerase (Perkin-Elmer Cetus)] and incubation was continued at 42 °C for 30 min. Reverse transcription was followed by PCR amplification of the cDNA using 25 cycles of denaturation at 94 °C for 2 min, annealing at 55 °C for 1 min and synthesis at 70 °C for 4 min (Xu et at., 1991). The product of this PCR reaction was isolated from an agarose gel, purified using Isogene (Perkin-Elmer Cetus) and resuspended in 20 pl of distilled water. The product was diluted 1 : 100 and 5 p-I was used in a second PCR amplification reaction, as described above but omitting the reverse transcription step. In this way large quantities of full-length cDNA for cloning were generated. For synthesis ofcDNA from mRNA of rearranged RNA segment 10 the procedure described above was used, but only 10 ng of each primer was added and the annealing time was increased to 5 min. The decrease in primer concentration was crucial for obtaining full-length cDNA from rearranged segment 10 mRNA. When the higher primer concentration was used, cDNA of the size of normal RNA segment 10 was always obtained (results not shown), suggesting intermolecular base-pairing of short cDNA products in the duplicated regions. Cloning o f full-length ds cDNA. Prior to cloning, all PCR products were end-filled using the Klenow fragment of D N A polymerase I (Gibco-BRL) and standard procedures (Sambrook et al., 1989). Cloning of full-length cDNA was achieved with two different procedures. Cloning of cDNA corresponding to RNA segment 10 of standard length was carried out by G-C tailing and annealing into pAT153 (Sambrook et al., 1989). cDNA corresponding to rearranged RNA segment 10 was digested with SphI, which cuts in the region of primer AB 101 only; the other end was left uncut, i.e. blunt-ended. This product was ligated into pBluescribe which had been digested with SphI and SmaI (Sambrook et at., 1989). The plasmids were transfected into Escherichia coli strains MC1061 and JMI09, respectively, and colonies with insert-containing plasmids were detected using standard procedures (Sambrook et al., 1989). DNA sequence analysis. Sequencing was carried out by the dideoxynucleotide chain termination method (Sanger et al., 1977) using the Sequenase Version 2.0 Kit (United States Biochemical Corporation), after subcloning of suitable DNA fragments into M 13mp 18 and M13mpl9 sequencing vectors. Results The RNA profiles of rotaviruses A28 and A64 with normal and rearranged RNA segments 10, respectively, are shown in Fig. 1 (b). Full-length cDNA clones of segments 10 of both viruses were obtained by combined reverse transcription-PCR (Fig. 1c) and transfected into E. coli strains as described in Methods. Cloned rotavirus cDNA inserts were isolated, and subfragments (obtained by digestion with HinclI, PstI and SphI) cloned into M13mpl8 and M13mpl9 for sequencing. All nucleotides were confirmed by sequencing on both strands, and two independent clones of the rearranged cDNA (cl 6 and cl 2) were fully sequenced. The nucleotide sequences of RNA segment 10 of normal length (A28) and of rearranged RNA segment 10 from A64 virus (cl 6) are shown in Fig. 2 and 3, respectively. It was found that the gene rearrangement consists of a partial duplication of the normal ORF which stretches from nucleotides (nt) 41 to 569. In the rearranged gene, a reiteration of the ORF had occurred two nucleotides after the termination codon. This repeated portion of the ORF start at nucleotide 81, i.e. 40 nucleotides downstream of the initiation codon of the normal ORF. Thus, nucleotide 81 becomes rearranged nucleotide 572. The sequence is then completed to the 3' end, nucleotide 751 of the normal gene, which becomes nucleotide 1242 in the rearranged gene. An interesting feature is the occurrence of a seven base direct repeat in nucleotide positions 75 to 81 and 563 to 569 (Fig. 3). The overall relationship between normal and rearranged genes is diagrammatically represented in Fig. 4. A second clone (cl 2) of the rearranged gene from the same cloning experiment was sequenced fully and the junction point 571/572(81) confirmed. When the sequences were compared, a number of point mutations were found using the sequence of the normal gene as a reference (Table 1). Comparison of the nucleotide sequences between positions 1 and 751 of the normal and rearranged genes (cl 6) revealed five point mutations in positions 76, 184, 290, 371 and 519, of which only one (nucleotide 290) was a silent mutation (Table 1). The second cDNA clone (cl 2) of the rearranged gene had different point mutations compared with the standard sequence (Table 1). Within the ORF Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 14:42:05 Sequences of rearranged rotavirus RNAs (a) Primer AB 102 5' EcoRI bide[ 4, 4, NcoI ' ,~ : GAATTCATATGGCCAI~GGTCACACTAAGACCATTCC Ball 3' , Complementary to 3' end of m R N A Primer AB101 SphI ~, 5' Narl 4' PvulI Ij ¢ , GCATGCCGGCGCCAGCT~GCTTTTAAAAGTTCTGTTc NaeI 3' I Complementary to 3' end of negative-strand R N A (c) (b) 1 2 3 1 2 3 tlO ~10 Fig. 1. (a) Oligonucleotide primers for reverse transeription-PCR of rotavirus RNA segment 10. (b) RNA profiles of human rotaviruses with normal gene 10 (A28, lane 1) and rearranged gene 10 (A64, lane 2), and of bovine rotavirus (UK Compton strain, lane 3). RNA segments were separated by eleetrophoresis on a 10% polyaerylamide gel and silver-stained (Sambrook et al., 1989). RNA segments are designated, the prefix R indicating rearranged forms. (c) Product of reverse transeription-PCR showing full-length eDNA copies of normal and rearranged RNA 10 of human rotaviruses A28 and A64, lanes 2 and 3 respectively. In lane 1, molecular size markers (1 kb D N A ladder Gibco-BRL) are shown. Eleetrophoretic separation was on a 1% agarose get which was stained with ethidium bromide (Sambrook et al., 1989). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 14:42:05 635 636 A. Ballard, M. A. McCrae and U. Desselberger 1 51 GGCTTTTAAA AGTTCTGTTC CGAGAGAGCG CGTGCGGAAA GATGIGAAAAG ! GGCTTTTAAA AGTTCTGTTC CGAGAGAGCG CG~GCGGAAA GATGGAAAAG CTTACCGACC TCAATTACAC ATTGAGTGTG ATCACTCTAA TGAACAGTAC 51 CTTACCGACC TCAATTACAC ATTGAATGTGVATCACTCTAA TGAACAGTAC I ATTGCATACA ATACTAGAAG ACCCAGGAAT ~GCGTATTTT CCTTATATTG 10L ATTGCATACA ATACT~GAAG ACCCAGGAAT GGCGTATTTT CCTTATATTG 151 CATCTGTTCT AACAGTTTTG TTCACTTTAC ATAGAGCGTC AATTCCAACG 151 CATCTGTTCT AACAGTTTTG TTCACTTTAC ATAAAGCGTC AATTCCAACG 201 ATGAAGATCG CACTAAAGAC ATCAAAATGC TCGTATAAGG TAGTGAAGTA 201 ATGAAGATCG CACTAAAGAC ATCAAAATGC TCGTATAAGG TAGTGAAGTA 251 CTGCATTGTG ACAATTTTTA ATACACTATT GAAACTGGCA GGTTATAAAG 251 CTGCATTGTG ACAATTTTTA ATACACTATT GAAACTGGCG GGTTATAAAG 301 AACAAATTAC TACTAAAGAT GAAATAGAAA AGCAGATGGA CAGAGTCGTC 301 AACAAATTAC TACTA~AGAT GAAATAGAAA AGCAGATGGA CAGAGTCGTC 351 AAAGAAATGA CTACACGTGA 351 AAAGAAATGA GACGTCAGTT GGAAATGATT GATAAGTTGA CTACACGTGA TTGATGGTGC 401 AATTGAGC~A GTTGAACTAC TTAAACGCAT TTATGATAAA TTGATGGTGC CGGAATAGAT ATGACGAAAG AAATAAATCA AAAAAACGTA I01 GACGTCAGTT TGAAATGATT GATAAGTTGA 401 AATTGAGCAA GTTGAACTAC TTAAACGCAT 451 GAGCAACTGA CGGAATAGAT ATGACGAAAG AAATAAATCA AAAAAACGTA 451 GAGCAACTGA 501 AAAACGCTAG AAGAATGGAA GAGTGGAAAA AATCCTTATG AACCAAAGGA 501 AAAACGCTAG AAGAATGGGA GAGTGG~AAA AATCCTTATG AACCAAAGGA 551 AGTGACTGCA GCAATGTGAG AGGTTGAGCT GCCGT~GACT GTCTTCGGAA 55$ AGTGACTGCA GCAATGTGAG 601 GCGGCGGAGT TCTTTACAGT AAGCCCCATC GGACCTGATG GCTGGCTGAG bOl AATACTAGAA GACCCAGGAA 651 AAGCCACAGT CAGCCATATC GCGTGTGGCT CAAGCCTTAA TACCGTTTAA 651 TAACAGTTTT GTTCACTTTA TTAGTGTGAC 701 GCACTAAAGA CATCAAAATG CTCGTATAAG 701 CCAATCCGGT 751 C CAGCACCGGA CGTTAATGGA TTATGATAAA AGGAATGGTC Fig. 2. N u c l e o t i d e sequence o f normal R N A segment 10 ( c D N A form, positive strand) o f h u m a n rotavirus A28. Stop and start c o d o n s are underlined. were nine point mutations (at nucleotides 58, 76, 290, 292, 349, 371, 414, 470 and 476), of which only three (nucleotides 76, 290 and 371) were identical to those ofcl 6. Two changes in cl 6 (nucleotides 184 and 519) were not found in cl 2. Comparison of nt 81 to 751 of the normal gene with nt 572 to 1242 of the rearranged genes showed one further point mutation (nt 1181) in both clones of the rearranged gene (Table 1). 1. Nucleotide and predicted amino acid changes between RNA segments 10 of human rotaviruses A28 (normal RNA 10; NIO) and A64 (rearranged RNA 10; clones 6 and 2). A~ATCACTCTA ATGAACAGTA CATTGCATAC TGGCGTATTT TCCTTATATT GCATCTGTTC CATAGAGCGT CAATTCCAAC GATGAAGATC GTAGTGAAGT ACTGCATTGT 751 GACAATTTTT AATACACTAT TGAAACTGGC GGATTATAAA GAACAAATTA 801 CTACTAAAGA TGAAATAGAA AAGCAGATGG ACAGAGTCGT CAAAGAAATG 851 AGACGTCAGT TGGA~ATGAT TGATAAGTTG ACTACACGTG AAATTGAGCA 901 AGTTGAACTA CTTAAACGCA TTTATGATAA ATTGATGGTG CGAGCAACTG 951 ACGGAATAG~ TATG~CGAAA GAAATAAATC AAAAAAACGT ~AAAACGCTA iOO1 GAAGAATGGA AGAGTGGAAA AAATCCTTAT GAACCAAAGG AAGTGACTGC 1051 AGCAATGTG~ GAGGTTGAGC TGCCGTCG~C TGTCTTCGGA ii01 TTCTTTACAG TAAGCCCCAT CGGACCTGAT GGCTGGCTGA GAAGCCACAG ~1~I TCAGCCATA, CGCGTGTGGC TCAAGCCTTA ATCCCGTTTA ~CAATCCGG 1201 TCAGCACCGG ACGTTAATGG AA~GAATG6~ CTTAGTGTGA CC AGCGGCGGAG Fig. 3. N u c l e o t i d e sequence o f rearranged R N A segment 10 (cl 6; c D N A form, positive strand) o f h u m a n rotavirus A64. Start and stop c o d o n s are underlined. T h e junction point b e t w e e n nucleotides 571 and 572 is indicated by an arrow. T h e arrow b e t w e e n nucleotides 80 and 81 denotes the position o f the duplication starting point. A direct s e v e n base repeat is delineated by dotted lines. Table Base position N10 58 76 184 290 292 349 371 414 470 476 519 519 692 R10 C1 6 76 184 290 371 519 1181 Base change R10 Cl 2 58 76 290 292 349 371 414 470 476 ~010 1181 * NC, N o n - c o d i n g . A m i n o acid change N10 R10 Cl 6 R10 Cl 2 A G G A G T T G T G A A A A A G G G C T A G A C G A C C G C N10 R10 C1 6 R10 C1 2 D S R A G V E E D T K K NC N K G E N¢ V N D A G K ~c* NC Comparison of the normal gene 10 of rotavirus A28 with those of human rotavirus Wa (serotype 1 ; Okada et al., 1984), simian rotavirus SA11 (serotype 3; Both et al., 1983) and bovine rotavirus UK strain (serotype 6; Baybutt & McCrae, 1984; Ward et al., 1985) showed a high degree of nucleotide and amino acid similarity (Fig. 5), especially with the bovine rotavirus genes. There was an accumulation of amino acid changes in positions 131 to 141 which has already been noted by others (Estes & Cohen, 1989). Discussion Rearrangements of rotavirus R N A segments have now been described in a number of human and animal rotavirus strains (Pedley et al., 1984; Hundley et al., Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 14:42:05 Sequences o f rearranged rotavirus R N A s Normal gene 10 1 4181 I ' 569 ' 751 , I 5' 3' Met "'. Stop "" 141 I , 569 " " 572 , "",. "1(/ " -. " ".. 1242 " ,. Met 4 3, Stop Rearranged gene 10 Fig. 4. D i a g r a m o f t h e s t r u c t u r e s o f n o r m a l and rearranged genes 10of h u m a n rotavirus, m , Complete O R F ; [[], duplicated part of O R F of normal gene (untranslated); solid lines indicate 5' and 3' untranslated regions. Normal 10 Wa U K Bovine Bovine SAI 1 N o r m a l 10 Wa U K Bovine Bovine SAIl N o r m a l 10 Wa U K Bovine Bovine SAIl Normal 10 Wa U K Bovine Bovine SA11 Wa U K Bovine Bovine SAIl l MEKLIDLNYTLSV ITLMNSTLHTILEDPGMAYFPYIASVLTVLFTLHRA5 -D--A .......... S--D---S-IQ ....... L ............. K-- .................. K-- N ............................ 30 .................................... V .......... K-.................. N ...................... G--A-NK-- 51 IPTMKIALKTSKCSYKVVKYCIVTIFNTLLKLAGYKEQITTKDEIEKQMD ................. [ . . . . . . . z. . . . . . . . . . . . .................................................. i00 v . . . . . . . Q--- .................................................. .................................................. I01 RVVKEMRRQFEM I DKLTTR E I EQVELLKR -I ....... L .................... 150 IYDKLMVRATDG I DblTKE l NQ ,,-,-,,-,,-,---,--,-- ......... ......... L ........................... L .................... H .... ......... L ........................ 151 KNVKTLEEWKSGKNPYEPKEVTAAM --I---D--E ---R ..... S-GE ......... I-TV-E ......... T-QT~GE ......... 175 ........ S .... SE ............... ---R ...... N-R ............ ---R .............. R ...... Nucleic acid similarity (~) 83 91 90 89 A m i n o acid similarity (~o) 83 95 94 92 Fig. 5. Comparison of the predicted amino acid sequence of the R N A 10 product of h u m a n rotavirus serotype 10 with those of other group A rotaviruses: Wa, serotype 1 (Okada et al., 1984); U K bovine, serotype 6 (Baybutt & McCrae, 1984); bovine, serotype 6 (Ward et al., 1985); SA 11, serotype 3 (Both et al., 1983). The percentage nucleotide and a m i n o acid similarity is indicated at the bottom. 1985; Thouless et al., 1986; Besselaar et al., 1986; Bellinzoni et al., 1987; Pocock, 1987; Tanaka et al., 1988) and are not, as initially observed, confined to viruses isolated from chronically infected immunodeficient hosts (Pedley et al., 1984; Eiden et al., 1985; Hundley et al., 1987). A number of these rearrangements have been sequenced (Gonzalez et al., 1989; Gorziglia et al., 1989; Scott et al., 1989; Matsui et al., 1990) and, with a few exceptions, the normal ORF was found to be retained. 637 The rearrangement involved partial duplication of the gene in the non-coding Y-terminal sequences and thus increased its length. This overall strategy has been confirmed by sequencing gene 10 of human rotaviruses (Hundley et al., 1987). Compared to the ORF region of the normal gene, the ORFs of the rearranged gene contained five and nine nucleotide changes in clones 6 and 2, respectively. Of the rearranged genes sequenced previously (all from R N A 11), one (Scott et al., 1989) had duplications almost identical to the original sequences, whereas the others (Gorziglia et al., 1989; Gonzalez et al., 1989; Matsui et al., 1990) had numerous point mutations and one (Gorziglia et al., 1989) had a partial deletion. The rearranged sequence reported here would fall in between. Whereas some of the 'super-short' electropherotype viruses (VMRI; Matsui et al., 1990) contain a clear partial duplication in the 3' end untranslated region, the 'short' electropherotype virus DS-1 and the 'super-short' electropherotype virus M69 contain sequences in their 3' end untranslated regions which are similar to each other, but not to any other available rotavirus gene sequence (Matsui et al., 1990). The differences in point mutations between clones 2 and 6 of cDNA of rearranged R N A segment 10 are likely to be due to errors produced by the retrovirus reverse transcriptase (Leider et al., 1987) and the double PCR procedures used before sequencing. To what extent these changes reflect true heterogeneity of m R N A remains to be elucidated. With regard to the mechanisms which produce R N A rearrangements, it has been suggested that the rotavirusspecific RNA-dependent R N A polymerase (Cohen, 1977) falls back on its template at various stages of primary transcription and retranscribes from that template (Hundley et al., 1987; Tanaka et al., 1988; Gorziglia et al., 1989; Matsui et al., 1990). It is remarkable that in all the cases sequenced (this sequence and those described in references below) the reinitiation has occurred close to the termination codon: after zero (Scott et al., 1989), two (this sequence), four (Gorziglia et al., 1989) and six nucleotides (Gonzalez et al., 1989). This could be due to selection against any other intramolecular recombination events, or to a predilection for rearrangement at this site on structural grounds. The first possibility may be true in most cases, but Hundley et al. (1985) described a rearrangement of the bovine rotavirus genome resulting in a novel R N A segment 5 product of larger than normal size, which was generated through an in-frame reiteration within the ORF (Tian Ye et al., unpublished results). With regard to the possibility of the site near the termination codon being a preferred site for recombination, it should be noted that it has been implicated in Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 14:42:05 638 A. Ballard, M. A. McCrae and U. Desselberger intramolecular base pairing (Okada et al., 1984, for R N A segment 11; Gorziglia et aL, 1989). Such base pairing may be an important signal for polymerase binding and replication, as suggested for other viruses (Hsu et al., 1987). On the other hand, it is of interest that a direct seven base repeat is found to the left of the junction and reinitiation sites (AATGTGA, positions 75 to 81 and 563 to 569, Fig. 3). It seems possible that the polymerase becomes less processive near the termination codon and preferentially jumps to a site identical to the one it has just passed. In any case, rearrangements, whether they are produced by this or a similar mechanism, do require an extended single-stranded region of transcript template, whether it is positive- or negative-strand RNA. Thus, the phenomenon of rearrangement may help to unravel the mechanism of R N A replication in rotaviruses and other dsRNA viruses. U.D. acknowledges fruitful discussions with P. Palese. This work was funded by a grant from the Medical Research Council to U.D. References BAYBUTr, H. N. & MCCRAE, M. A. 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