SUMMARYThe complete nucleotide sequence of the RNA of an aphid non-transmissible plum pox virus (PPV-NAT) isolate has been determined from five overlapping cDNA clones, cDNA prepared by primer extension was used to determine the 5' terminus. The assembled RNA is 9741 nucleotides in length, excluding a 3' terminal poly(A) sequence. One large open reading frame starts at nucleotide positions 36 to 38 and is terminated with an UAG codon at positions 9522 to 9524. The putative start codon is located at positions 147 to 149. The encoded polyprotein has a predicted Mr of 353"8K. Comparison of cistrons from tobacco vein mottling virus and tobacco etch virus with those predicted for PPV-NAT indicated a similar genome organization. A highly conserved sequence of 12 nucleotides was found in the 5' non-coding region of these three potyviruses. The potential polyadenylation signal from yeast (UAUGU) was found in the 3' non-coding region of PPV-NAT and several other members of the potyvirus group.
A full-length cDNA clone of an aphid non-transmissible isolate of plum pox potyvirus (PPV) was rendered biologically active when placed under the control of the cauliflower mosaic virus 35S RNA promoter and the nopaline synthase polyadenylation signal. The cDNA was constructed so that the exact 5' end of the PPV RNA was present at the transcription initiation site.Inoculation of plasmid DNA onto Nicotiana benthamiana led to systemic infection, whereas local lesions were produced in Chenopodium amaranticolor and C.quinoa, typical of an infection with PPV. Examination of infected plants revealed PPV-specific virus particles as well as viral RNA, the coat protein and the nonstructural large nuclear inclusion protein (NIb).Potyviruses belong to the largest group of plant viruses and cause serious diseases in a number of economically important crop plants. Members of the potyvirus group are flexuous rods 720 to 900 nm in length (Hollings & Brunt, 1981) and encapsidate a single positive-sense RNA molecule of about 10 kb, attached to a 5'-linked protein (VPg) and including a poly(A) tract at the 3' end. The RNA is translated into a large polyprotein which is subsequently processed by at least two virus-encoded proteases. Upon proteolytic cleavage this polyprotein yields the viral coat protein (CP) and the non-structural proteins helper component-protease, cylindrical inclusion protein and two nuclear inclusion proteins (NIa and NIb). In addition, at least two other gene products which have not yet been identified are predicted from the nucleotide sequence of all potyviruses sequenced (Allison et al., 1986;Balint et al., 1990;Domier et al., 1986;Lain et al., 1989;Maiss et al., 1989;Robaglia et al., 1989).The in vitro synthesis of biologically active RNAs from full-length cDNA clones has been reported for two potyviruses (Domier et al., 1989;Riechmann et al., 1990). Genetic manipulation of full-length cDNA together with the subsequent transcription of infectious RNA provide a powerful tool for the examination of the molecular biology of RNA viruses. Moreover, it has been shown that infectious RNA can be transcribed in vivo by unknown promoters from plasmids containing cDNA copies of RNA 3 of alfalfa mosaic virus (Dore & Pinck, 1988), tobacco necrosis satellite RNA (van Emmelo et al., 1987) and potato spindle tuber viroid (Crees et al., 1983). Recently, Mori et al. (1991) have described the infectivity of plasmids containing the 35S RNA promoter of cauliflower mosaic virus (CaMV) upstream of fulllength cDNAs of all three brome mosaic virus (BMV) RNAs. Also, Commandeur et al. (1991) have demonstrated the infectivity of clones containing the 35S RNA promoter and cDNAs of RNA 3 and RNA 4 of beet necrotic yellow vein virus when complemented with a virus isolate which contains only RNA 1 and RNA 2. In this paper, the construction of a plasmid containing a full-length cDNA of an aphid non-transmissible isolate of plum pox potyvirus (PPV-NAT;Maiss et al., 1989) linked to the 35S RNA promoter of CaMV is described. PPV host plan...
Barley mild mosaic virus is a member of the Bymoviruses, a genus of the family Potyviridae. The virus consists of two types of flexuous rod-shaped particles. Each of them contains one single-stranded polyadenylated RNA in plus orientation of approximately 7.6 kb (RNA1) and 3.6 kb (RNA2). Complementary DNAs of both RNAs have been synthesised and cloned. The nucleotide sequence of RNA2 has been determined. It is 3524 nucleotides in length, excluding the 3' poly(A) tail, and contains one large open reading frame (2679 nts), coding for a polyprotein of approximately 98 kDa. There are indications that a putative proteolytic activity in the N-terminal part can cleave the polyprotein autocatalytically into a 25 kDa protein (putative proteinase) and a 73 kDa potypeptide of unknown function.
The sequence of 1,787 nucleotides (nts) in the genomic RNA of pelargonium leaf curl virus (PLCV) was determined. It included the entire coat protein (cp) gene (nts 585 to 1,754), 558 nts of the 3' end of the putative RNA polymerase gene, 26 nts of an intercistronic region between the two genes and 33 nts downstream of the stop codon of the cp gene. The cp gene was cloned into the expression vector pET8c and expressed in E. coli. The deduced cp amino acid sequence of PLCV was compared with those of five other tombusviruses. The closer the degree of serological relatedness between two viruses, the more similarity was found in their cp amino acid sequences not only in the protruding domains, but also in their random and shell domains and in the arm regions. Nucleic acid hybridization tests, cp amino acid comparisons and serological tests all suggest the same order of sequence for the relationships in the tombusvirus group.
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