The complete nucleotide sequence (8024 nucleotides) of the circular double-stranded DNA of cauliflower mosaic virus has been established. The DNA molecule is known to possess three discrete single-stranded discontinuities, often referred to as "gaps," two in one strand and one in the other. The sequence data indicate that gap 1, the single discontinuity in the alpha strand, corresponds to the absence of no more than one or two nucleotides with respect to the complementary beta strand. The two discontinuities in the beta strand, however, are not authentic gaps since no nucleotides are missing, but are instead regions of sequence overlap: a short sequence (19 residues for gap 2, t least 2 residues for gap 3) at one terminus of each discontinuity, probably the 5' terminus, is displaced from the double helix by an identical sequence at the other boundary of the discontinuity. Analysis of the distribution of nonsense codons in the DNA sequence is consistent with other evidence that only the alpha strand is transcribed. The coding region extends around the circular molecule from 4 map units of gap 1, the map origin, to map position 91, and consists of six long open reading frames. Our findings suggest, but do not prove, that the DNA sequence of the open reading frames is colinear with viral protein sequences. The cistron for the viral coat protein, which is probably synthesized in the form of a precursor, has been situated in coding region IV on the basis of its unusual amino acid composition.

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Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non‐propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor‐mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read‐through protein, P74, which is composed of the major capsid protein fused by translational read‐through to a long C‐terminal extension called the read‐through domain. Beet western yellows virus carrying various mutations in the read‐through domain was tested for its ability to be transmitted to test plants by aphids fed on agro‐infected plants and semi‐purified or purified virus preparations. The results establish that the read‐through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read‐through domain is important for accumulation of the virus in agro‐infected plants.

Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.Most if not all plants possess a defense mechanism against virus infection known as posttranscriptional gene silencing (PTGS; see reference 44 for a recent review). PTGS is a homology-dependent RNA degradation system to recognize and degrade viral RNA (and any homologous transgene mRNA) in the cytoplasm. Mechanistically similar phenomena exist in animals and fungi (reviewed in references 4, 13, and 35). The mechanism by which a virus infection triggers PTGS in plants is not fully understood, but double-stranded RNA is a strong inducer of PTGS (43) and such a form is produced during replication of an RNA virus. Production of small RNAs of ϳ21 to 23 nucleotides, called small interfering RNAs (siRNAs), corresponding to both the plus and minus strands of the target RNA is associated with PTGS (12,13,45). In plants, gene silencing at one site can trigger PTGS in distant tissues and across a graft union (29, 39a). The mobile signal, which must consist at least in part of homologous RNA, is thought to move from cell to cell through plasmodesmata and systemically via the vasculature. Some plant viruses have been shown to encode proteins which can counteract PTGS (8,22,39,40,42). These silencing suppressor proteins may act at different steps in the PTGS pathway. Thus, (i) the potyvirus helper component-proteinase (HCPro) interferes with initiation and maintenance of silencing at a step coincident with or upstream of siRNA production (1,7,16,23,24), (ii) the 2b protein of cucumber mosaic virus (CMV) prevents initiation of PTGS in new growth by inhibiting the long-range PTGS signaling acti...

Virions of beet western yellows luteovirus contain a major capsid protein (P22.5) and a minor readthrough protein (P74), produced by translational readthrough of the major capsid protein sequence into the neighboring open reading frame, which encodes the readthrough domain (RTD). The RTD contains determinants required for efficient virus accumulation in agroinfected plants and for aphid transmission. The C-terminal halves of the RTD are not well conserved among luteoviruses but the N-terminal halves contain many conserved sequence motifs, including a proline-rich sequence separating the rest of the RTD from the sequence corresponding to the major coat protein. To map different biological functions to these regions, short in-frame deletions were introduced at different sites in the RTD and the mutant genomes were transmitted to protoplasts as transcripts and to Nicotiana clevelandii by agroinfection. Deletions in the nonconserved portion of the RTD did not block aphid transmission but had a moderate inhibitory effect on virus accumulation in plants and abolished symptoms. Deletion of the proline tract and the junction between the conserved and nonconserved regions inhibited readthrough protein accumulation in protoplasts by at least 10-fold. The mutants accumulated small amounts of virus in plants, did not induce symptoms, and were nontransmissible by aphids using agroinfected plants, extracts of infected protoplasts, or purified virus as a source of inoculum. Other deletions in the conserved portion of the RTD did not markedly diminish readthrough protein accumulation but abolished its incorporation into virions. These mutants accumulated to low levels in agroinfected plants and elicited symptoms, but could not be aphid-transmitted. A preliminary map has been produced mapping these functions to different parts of the RTD.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).

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SUMMARYThe nucleotide sequences of cDNA clones corresponding to RNA-3 and RNA-4 of beet necrotic yellow vein virus isolates F2 and G l have been determined. The cDNA of RNA-3 of isolate F2 is 1775 residues in length and contains a coding region of 219 codons. In isolate G1 this coding region has undergone an internal deletion of 354 nucleotides in such a way as to conserve a shortened reading frame. Otherwise, the RNA-3 sequences of the two isolates were closely similar. RNA-4 of isolate F2 has an extrapolated length of 1431 residues and contains an open reading frame of 282 codons. This open reading frame has undergone an internal deletion of 324 nucleotides in one cDNA clone of RNA-4(G 1) with conservation of a shortened reading flame. Sequence analysis of other RNA-4(G1) cDNA clones revealed, however, that the exact boundaries of the deletion are not always the same. RNA-3 and RNA-4 of each isolate are more than 90 % homologous for the Y-terminal 200 nucleotides. Short homologous sequences are also present in RNA-3 and RNA-4 of isolate F2 flanking the regions deleted in each of these RNAs in the G 1 isolate. These homologous sequences probably play a role in the deletion process.