- Volume 79, Issue 8, 1998

Full-length cDNA clones from which infectious transcripts could be generated were constructed from the genomic RNAs of two distinct strains of peanut stunt cucumovirus (PSV), PSV-ER and PSV-W. PSV- ER, a subgroup I strain, is known to support efficient replication of satellite RNA (satRNA) in infected plants, whereas PSV-W, a subgroup II strain, does not support satRNA replication. Although artificial reassortants (pseudorecombinants) of all possible combinations of infectious transcripts representing RNA1, RNA2 and RNA3 were infectious, only those having RNA1 from PSV-ER supported the replication of satRNA. These results demonstrate conclusively that support of PSV satRNA replication maps to RNA1. Comparisons of secondary structure predictions of the C-terminal helicase-like domain of the 1a proteins of four PSV strains belonging to two subgroups did not reveal any obvious differences between strains that differ in satRNA support. The complete nucleotide sequence of RNA1 from strains PSV-ER and PSV-W were determined and found to be 79% identical. Sequence comparison analysis of RNA1 sequences of cucumoviruses confirmed the placement of the PSV strains into two distinct subgroups.

The amino acid sequence of the genome-linked viral protein (VPg) of pea enation mosaic enamovirus (PEMV) has been determined. The VPg is encoded by nt 1811–1894 within ORF1 of RNA1 downstream of the proteinase motif. Direct N terminus sequencing of intact and endoproteinase Asp-N-digested VPg combined with electrospray mass spectroscopy confirmed that the VPg is composed of 28 amino acids with a molecular mass of 3138 Da. The context of the N and C terminus residues as well as the position and size of the VPg suggest that the mature VPg may be generated via post-translational proteolytic processing of the polyprotein arrangement of membrane anchor- proteinase-VPg-polymerase encoded by ORFs 1 and 2. Computer comparisons did not reveal any significant similarity between the VPg of PEMV and any other sequences including those of the VPgs of related subgroup II luteoviruses.

Based solely on the information that beet virus Q (BVQ) contains tubular particles, the entire nucleotide sequence of its tripartite genome was determined from unpurified virus in ca. 40 ml crude sap from locally infected Chenopodium quinoa. A starting sequence for RNA 1 was generated using primers corresponding to highly conserved helicase domains in the respective RNAs of furo-, pomo-, peclu-, hordei- and tobraviruses, and was extended by a walking random-primed cDNA approach. The similarity of the 3′ ends of furoviral RNAs allowed starting sequences for BVQ RNAs 2 and 3 to be obtained once the 3′ end of RNA 1 was known. BVQ RNA 1 encodes a protein with a methyltransferaselike, a variable and a helicase-like region, and for a readthrough protein which, in addition, contains an RNA-dependent RNA polymerase region. RNA 2 carries the coat protein gene, a coat protein read- through protein gene and two additional ORFs which may have arisen by deletions from an originally larger readthrough domain. RNA 3 carries a triple gene block resembling that of several other rod-shaped viruses. The 5′ UTRs of the three RNAs have the potential to form a series of hairpins with C-Aand C-C mismatches resembling those found in tymoviral RNAs. The 3′ ends can be folded into tRNA-like structures which are preceded by a long hairpin-like structure and an upstream pseudoknot domain. BVQ belongs to the recently proposed genus Pomovirus; it shows evolutionary relationships to furoviruses in sensu stricto, peclu-, hordei-, tobra-, tymo-, tobamo-, carla- and potexviruses.

Potato virus Y group C isolates (PVYC) have been characterized according to biological, molecular and genetic criteria. Two genetic strains, PVYC and PVYC , were identified on the basis of genetic distances (among them and other PVY strains), host range (ability or inability to infect pepper), MAb response (ELISA recognition with MAb 10E3) andcoat protein processing site. Some characteristics, such as aphid transmission and ELISA using other MAbs, did not correlate with classification into these two genetic strains. All isolates tested induced a hypersensitive response on potatoes bearing the Nc resistance gene, confirming the nature of PVYC isolates as a homogeneous pathotype.

Recent studies have shown that potyvirus VPg/ proteinases and RNA-dependent RNA polymerases are capable of protein-protein interactions in yeast cells. We have extended these studies in vitro. We found that tobacco vein mottling virus (TVMV) VPg is retained on glutathione-Sepharose matrices if coincubated with a glutathione S-transferase (GST)- Nlb fusion protein, but not with GST, which is suggestive of a direct physical interaction between these two proteins. However, a mutation in the VPg (Y1860S) that eliminates virus infectivity and the interaction in yeast cells had little effect on the in vitro interaction. We also found that the TVMV VPg and Nla proteins are capable of stimulating the polymerase activity of the Nlb protein. Since this stimulatory activity is retained when the proteinase domain of the Nla is removed, we conclude that the VPg is the moiety responsible for the stimulation of polymerase activity. As with the interaction revealed by co-purification, the Y1860S mutation had little or no effect on the stimulation of polymerase activity. Moreover, the VPg was able to stimulate a mutant Nlb with an altered ‘GDDߣ motif. Our studies thus provide two lines of evidence indicative of in vitro interactions between the TVMV VPg and Nlb proteins.

Rice grassy stunt virus (RGSV, IRRI isolate) has six genomic RNA segments. The nucleotide (nt) sequences of RNAs 1 –4 were determined. The cumulative length of the RGSV genome, including RNAs 5 and 6, was 25142 nt. All six RNA segments had an ambisense coding strategy and almost identical terminal sequences over 17 nt. The virus complementary (vc) sequence of the largest segment, RNA1, had an open reading frame encoding a protein of M r 339 133 (the 339 · 1K protein), while the virus sense (v) sequence encoded a protein of M r 18910 in the 5 ′proximal region. The predicted 339 ·1K protein contained the highly conserved motifs of the RNA-dependent RNA polymerase and a short but distinct Arg/Gly-rich stretch at the C terminus. The putative RNA polymerase showed strong similarity with that of rice stripe tenuivirus (RSV); they shared 37 ·9% % amino acid identity over 2140 residues. The predicted proteins of M r 23 280 on vRNA2 and 93879 on vcRNA2 were only slightly similar in sequence to the proteins encoded by vRNA2 and vcRNA2 of other tenuiviruses. The predicted proteins encoded by RNA3 and RNA4 did not show significant similarity to any database proteins. Only the putative RNA polymerase encoded on RNA1 was well-conserved between RGSV and RSV. The low sequence similarities in proteins encoded by RNAs 2, 5 and 6, together with the unique RNA segments 3 and 4, indicate that RGSV may be distinct from other tenuiviruses.

Rupestris stem pitting (RSP), a component of the rugose wood complex, is one of the most widespread graft-transmissible diseases of grapevines. Here we report on the consistent association of a high molecular mass dsRNA (ca. 8· 7 kbp) with RSP. The dsRNA was reverse-transcribed and cDNAs generated were cloned into Lambda ZAP II. Sequence analysis of the cDNA clones showed that the dsRNA was of viral origin and the putative virus was designated rupestris stem pitting associated virus- 1 (RSPaV-1). The genome of RSPaV-1 consists of 8726 nt excluding a poly(A) tail at the 3′ terminus. It has five potential open reading frames which have the capacity to code for the replicase (ORF1), the triple gene block (ORF2-4) and the coat protein (ORF5). Comparison of the genome structure and nucleotide and amino acid sequences indicated similarities of RSPaV-1 to apple stem pitting virus, and to a lesser extent, to potato virus M carlavirus. The possibility that different strains of RSPaV-1 or other viruses are associated with RSP is discussed.

Temperature-sensitive (ts) mutants of Bombyx mori nucleopolyhedrovirus (BmNPV), which exhibited no polyhedra formation at the non-permissive temperature of 33 °C, wereproduced using the base analogue 5-bromodeoxyuridine. A unique ts mutant, designated ts-S1, was characterized in terms of gene mutation and virulence in cultured cells and silkworm larvae. Mutant-infected BmN4 cells at 33 °C showed normal viral DNA synthesis but defective budded virus production and polyhedrin synthesis, suggesting the absence of late and very late gene expression. Silkworm larvae were injected with ts-S1 and reared at 33·5 °C. At 7 days postinjection, none of the larvae exhibited nucleopoly- hedrosis but some possessed viral DNA, detected by PCR using virus-specific primers. Continued rearing of the larvae at a permissive temperature of 25°C showed that, while most developed into normal adults, some developed nucleopolyhedrosis, indicating that the former larvae had aborted the virus infection during the course of rearing at 33·5°C. No viral DNA was detected in the adults. Marker rescue tests to identify the lesion involved in the ts phenotype of ts-S1, and nucleotide sequencing of the identified genome region, showed a single nucleotide mutation of a putative RNA polymerase gene, late expression factor-8 (lef-8). These results indicate that lef-8 is essential for BmNPV replication in vitro and in vivo.

As natural scrapie occurs only in sheep of specific PrP genotypes, one proposed aetiology was that scrapie is simply a genetic disease. However, Cheviot and Suffolk sheep of scrapie-susceptible genotypes are found in Australia and New Zealand, both generally accepted to be scrapie-free countries. A study of more common Australia and New Zealand sheep breeds (Merinos and Poll Dorsets) was carried out in order to obtain more generally applicable estimates of Australia and New Zealand sheep PrP genotype frequencies. We have confirmed that animals of highly susceptible PrP genotypes are found in Australia and New Zealand. Interestingly, the Poll Dorset sheep, although born in New Zealand, were brought to the UK as young adult animals and subsequently remained free of clinical scrapie despite 21 % of the sheep having scrapie-susceptible genotypes. These results have implications for the genetic control of occurrence of the equivalent human diseases.