Downloaded from www.microbiologyresearch.org by IP: 54.160.119.87 On: Mon, 22 May 2017 15:41:13 Journal of General Virology (1991), 72, 9-14. Printed in Great Britain 9 The genome organization of potato virus M RNA S. K. Zavriev,* K. V. Kanyuka and K. E. Levay All-Union Research Institute of Agricultural Biotechnology, Moscow 127253, U.S.S.R. The 8534 nucleotide sequence of the genome of the carlavirus, potato virus M (PVM), has been deter- mined. The sequence contains six large open reading frames (ORFs) and non-coding regions consisting of 75 nucleotides at the 5' end, 70 nucleotides followed by a poly(A) tail at the 3' end and 38 and 21 nucleotides between three large blocks of coding sequences. The ORF beginning at the first initiation codon at nucleo- tide 76 encodes a polypeptide of 223K which, accord- ing to its primary sequence analysis, seems to be a virus RNA replicase. The next coding block consists of three ORFs encoding polypeptides of 25K, 12K and 7K. The third block consists of two ORFs encoding polypep- tides of 34K (PVM coat protein) and llK. The llK polypeptide contains a pattern resembling the consen- sus for a metal-binding nucleic acid-binding 'finger'. The nucleotide sequence EMBL accession number is X53062. Introduction Potato virus M (PVM), a member of the carlavirus group, has filamentous particles composed of multiple copies of the coat protein and a monopartite plus-sense ssRNA of approximately 8 kb in length (Proll et al., 1981; Wetter & Milne, 1981; Tavantzis, 1984). In our previous papers we published the nucleotide sequence of the 3'-proximal 2630 nucleotides of PVM genomic RNA and demonstrated a similarity between PVM and the potexviruses in the gene arrangement and encoded amino acid sequences (Rupasov et al., 1989, 1990). The nucleotide sequences of the 3' terminal regions of the genomic RNAs of two other carlaviruses, potato virus S (PVS) and lily symptomless virus (LSV), were published by MacKenzie et al. (1989) and Memelink et al. (1990), respectively. In this article we report the nucleotide sequence of PVM RNA and outline its genomic organization. Methods Isolation of virus RNA. PVM (Russian wild strain) was purified from infected tomato plants as described by ProU et al. (1981). RNA was isolated from the purified virus preparation using the phenol-SDS extraction method (Proll et al., 1981) with minor modifications. cDNA cloning and sequencing. Double-stranded eDNA for the 3'- proximal 2630 nueleotides of PVM genomic RNA has been synthe- sized and sequenced previously (Rupasov et al., 1989). Double- stranded eDNA for the Y-proximal approximately 6000 nucleotides was synthesized by a similar procedure using a synthetic deoxyoligonu- cleotide primer complementary to the viral genome as deduced from the known sequence. The double-stranded cDNA was ligated to a H/ndlI-cut plasmid pGEM-3Z (Promega) and transformed into competent Escherichia coli XL-IB cells (Stratagene Gene Cloning System) as described by Maniatis et al. (1982). Ampicillin-resistant transformants were analysed for PVM-specific inserts by colony hybridization (Maniatis et al., 1982). The insert size of selected recombinant eDNA clones was determined by restriction enzyme analysis of plasmid DNA samples. Finally, six clones containing plasmids with long overlapping eDNA inserts (see Fig. 1) were isolated and used for sequencing. A nested series of exonuelease III deletions were generated from the original eDNA clones according to the Erase-a-base system (Promega Biotec). Competent E. coli XL-1B cells were used as bacterial hosts. Transformants containing plasmids with the appropriate size of deletion were grown in 1.5 ml of selective liquid medium and plasmid DNA was isolated as described by Del Sal et al. (1988). The extent of the exonuelease III deletions was determined by restriction analysis. Dideoxynueleotide sequencing of dsDNA templates was carried out using [~t-32p]dATP or [~-35S]dATP and T7 phage-derived DNA polymerase (Sequenase; U.S. Biochemi- cal). We have sequenced more than 96% of the eDNA in both orientations and more than 47% of the sequence was determined by examining two or three different clones covering each particular region. RNA sequencingl The 5' terminus of PVM RNA was sequenced using reverse transcriptase and the 32p-end-labelled synthetic primer 5' dCTCAAGTATATGCCTGCC 3', complementary to nucleotides 246 to 263 of the final sequence. 12K 25KI~ IlK 223K _ _ 1000 2000 3000 4000 5000 6000 7000 8000 I I I I I | | | , ,/~/x "~ pVM 107 3 [~]I9K poly(A) u , pVM 9 ~pVM 30 t pVM 33 pVM~47 L pVM 28 , Fig. 1. Schematic representation of six ORFs in PVM genomic RNA, the ORF in the negative strand, and long overlapping eDNA clones used for sequencing. All are shown relative to their location in the PVM genome. The arrow indicates the position of the oligonucleotide primer used for run-off sequencing of the viral RNA 5' terminus. 0000-9830 © 1991 SGM