Organization and Interviral Homologies of the 3'-terminal Portion of Potato Virus S RNA

Abstract

SUMMARYThe sequence of 3553 nucleotides corresponding to the 3'-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames (ORFs) encoding proteins of Mr 10 734, Mr 32 515, Mr 7222, Mr 11 802, Mr 25092 and at least Mr 41052. The sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from plasmids which were isolated by colony hybridization with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lystates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59~ identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORFs displayed significant sequence homology with the similarly sized ORFs from a number of potexviruses. The partial 41K ORF product was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus.

Transgenic Nicotiana debneyii expressing viral coat protein are resistant to potato virus S infection

“…The viral genome consists of one single-stranded, positive sense RNA molecule with an estimated Mr of 2.4 × 106 which contains a T-terminal polyadenylated region. Recent nucleotide sequence data from PVS (MacKenzie et al, 1989) and a related carlavirus, potato virus M (PVM) (Rupasov et al, 1989), have demonstrated that these two viruses have genome organizations similar to that of potato virus X (PVX) (Huisman et al, 1988) and other members of the potexvirus group. The viral genome of PVS contains six open reading frames (ORFs) which encode polypeptides with Mr of 10734, 32515, 7222, 11802, 25092 and more than 41052.…”

“…Samples of leaf tissue (100 mg) were homogenized with 100 ~tl of SDS-PAGE sample buffer (4~ SDS, 125 mMTris-HCl pH 6-8, 10~ 2-mercaptoethanol, 0.04~ bromophenol blue, 20~ glycerol) and incubated at 95 °C for 5 min. Following centrifugation at 13000 r.p.m, for 5 rain in an Eppendorf microcentrifuge, 15 ~tl aliquots were loaded onto a 12~ polyacrylamide gel and, after electrophoresis using the buffer system of Laemmli (1970), separated proteins were blotted onto Immobilon membranes (Millipore) and probed with 1G 11 MAb and l zSI-labelled goat anti-mouse Ig as previously described (MacKenzie et al, 1989). Approximately 40~ of the transgenic plants obtained expressed detectable levels of PVS coat protein as judged by immunoblotting.…”

“…The preparation and characterization of a complementary DNA (cDNA) clone (pVS57), which contains the coding sequence of the entire PVS coat protein gene as an in-frame fusion with the lacZ ~-peptide, has been previously described (MacKenzie et al, 1989). The insert contained in clone pVS57 corresponds to the T-terminal 1284 nucleotides from PVS RNA and the 5' terminus of this insert is located 16 nucleotides upstream from the initiating ATG codon for the 33K viral coat protein gene.…”

“…The ME strain of PVS which infects N. debneyff was used in these tests as the Peruvian strain used in the cloning did not readily infect N. debneyii. The ME strain was purified as described previously (MacKenzie et al, 1989) from inoculated leaves of Chenopodium amaranticolor. Groups of F2 progeny plants (12 plants each group), which had been selected by germination on medium containing 200 mg/ml kanamycin, together with equal numbers of non-transformed N. debneyii, were mechanically inoculated with a preparation of purified virus at concentrations of 0.5, 1.0, 2.0 and 5-0 lag/ml.…”

A note on four nonradioactive labeling systems for dot hybridization detection of potato viruses

“…Pairwise computer alignments between available nucleotide sé-quences of PVY (Robagliaef a/. 1989) and PVS (McKenzie et al 1989) revealed a 72% homology for a région of 93 residues located at 2500 (PVY) and 2800 (PVS) residues downstream their 3' end (data not shown). Homology even reached 81% for a stretch of 26 nucleotides.…”