SUMMARYThe sequence of the 4662 nucleotides [excluding poly(A)] of RNA-2 of tomato black ring virus (TBRV) has been determined. Most of the sequence (4074 nucleotides) encodes a polypeptide of mol. wt. 150000 (150K). The 5' and Y non-coding sequences are 287 and 301 nucleotides in length, differ from the coding sequence in base composition, and contain repeated sequences, some of which resemble oligonucleotides in the 3' non-coding sequence of M-RNA of cowpea mosaic virus (CPMV). From its amino acid composition, the coat protein of TBRV was tentatively located in the Cterminal third of the 150K polypeptide. The amino acid sequence of the 150K polypeptide immediately N-terminal to the putative coat protein sequence was found to resemble parts of the 30K polypeptides of tobamoviruses and, to a lesser extent, part of the 105K polypeptide translation product of CPMV M-RNA.

). ² These authors contributed equally to this work. SummaryIn plants, post-transcriptional gene silencing (PTGS) is part of a defence mechanism against virus infection. Several plant viruses have been shown to encode proteins which can counteract PTGS. In this paper it is demonstrated that P15 of peanut clump pecluvirus (PCV) has anti-PTGS activity. P15 is a small cysteine-rich protein with no sequence similarity to previously described PTGS-suppressor proteins which has several novel properties. It possesses four C-terminal proximal heptad repeats that can potentially mediate a coiled-coil interaction and is targeted to peroxisomes via a C-terminal SKL motif. The coiled-coil sequence is necessary for the anti-PTGS activity of P15, but the peroxisomal localization signal is not, although it is required for ef®cient intercellular movement of the virus.

SUMMARYThe sequence of the 7356 nucleotide [excluding the 3' poly(A)] RNA-1 of tomato black ring virus (TBRV) was determined from overlapping cDNA clones. A putative initiation codon at nucleotide 26l was considered to be the start of an open reading frame which terminates at a UAG codon at position 7053. The predicted translation product is a protein of 2264 amino acids with an Mr of 253 680 (254K). Comparison of the amino acid sequence of this 254K protein with other viral proteins revealed three regions, each having about 60~ homology with a region of the three cowpea mosaic virus (CPMV) proteins (58K, 24K, 87K) which are thought to be involved in replication of the CPMV RNAs. The same regions show similarities, although less extended, with proteins 2C, 3C and 3D of picornaviruses. From these results we suggest that the genome organization of TBRV is similar to that of comoviruses and picornaviruses.

The genome of peanut clump pecluvirus (PCV) consists of two messenger RNA components which contain, respectively, three and five open reading frames (ORFs). Inoculation of transcripts from full-length cDNA clones derived from the PCV RNAs showed that RNA-1 is able to replicate in the absence of RNA-2 in protoplasts, but both RNAs are necessary for plant infection. To investigate the role of different gene products in viral RNA replication and movement, transcripts from mutant cDNA clones were inoculated to protoplasts and to Chenopodium quinoa or Nicotiana benthamiana plants, and progeny RNA was detected by Northern blot analysis. The protein P15, encoded by the third ORF of RNA-1, is essential for efficient replication of the viral genome. The three proteins, P51, P14, and P17, of the triple gene block contained in RNA-2 are involved in localized movement of the viral genome, whereas the coat protein (P23) is also required for vascular movement. Insertion of the beta-glucuronidase reporter gene (GUS) in place of the P23 or P39 genes (the first and the second genes of RNA-2) allows visualization of the virus infection in inoculated leaves. Although the presence of the GUS gene resulted in a lower accumulation of progeny RNA and, despite instability of the construct in planta, histochemical detection of PCV multiplication was more sensitive than Northern blot detection.

The complete nucleotide sequence of RNA 1 of the tentative furovirus peanut clump virus (PCV) has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 5897 nucleotides in length and contains three long open reading frames (ORFs). The Y-terminal proximal ORF has the potential to encode a polypeptide of M r 130942 (P131) containing methyltransferase and RNA helicase homologous domains and displaying homology with large nonstrnctural proteins of alpha-like viruses, which are known or thought to be involved in virus replication. The P131 ORF is followed in-frame by a second ORF which is probably expressed by partial readthrough of the UGA termination codon of the P131 ORF to produce a polypeptide of M r 191044 (P191). The readthrough region of P191 contains the characteristic 'core' RNA polymerase motif, indicating that the PCV replicase proteins are expressed as a pair of overlapping proteins as in the tobamoviruses, tobraviruses and the furovirus soil-borne wheat mosaic virus (SBWMV). Sequence comparisons indicate that P 131 and P 191 are most closely related to the replicase proteins of SBWMV and the hordeivirus barley stripe mosaic virus (BSMV) but are only distantly related to the replicase of the furovirus beet necrotic yellow vein virus (BNYVV). The T-terminal proximal ORF can encode a putative polypeptide ofM r 14556 (P15) which displays homology to small cysteine-rich proteins of hordeivirnses and SBWMV. We have corrected four errors in the sequence of PCV RNA 2 published previously by Manohar et al. (Virology 195, 33-41, 1993). One of these changes causes two small ORFs near the 3' terminus of RNA 2 to be fused together to create an ORF for a putative polypeptide ofM r 16 833 (P17) which displays extensive homology with the third protein of the triple gene block of BSMV RNA fl.