Search

50 Pág., In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratifcation vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV, This work was supported in part through Laulima Govern ment Solutions, LLC prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Gov ernment Solutions, LLC under Contract No. HHSN272201800013C. This work was also funded in part by Contract No. HSHQDC 15-C-00064 awarded by DHS S&T for the management and operation of The National Biodefense Analysis and Countermeasures Center, a federally funded research and development center operated by the Battelle National Biodefense Institute (V.W.); and NIH contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (N.V., R.B.T.). S.S. acknowledges support from the Mississippi Agri cultural and Forestry Experiment Station (MAFES), USDA-ARS pro ject 58-6066-9-033 and the National Institute of Food and Agriculture, US Department of Agriculture, Hatch Project, under Accession Num ber 1021494.

Unfortunately, the inclusion of original names (in non-Latin script) of the following authors caused problems with author name indexing in PubMed. Therefore, these original names were removed from XML data to correct the PubMed record. Mengji Cao, Yuya Chiaki, Hideki Ebihara, Jingjing Fu, George Fú Gāo, Tong Han, Jiang Hong, Ni Hong, Seiji Hongo, Masayuki Horie, Dàohóng Jiāng, Fujio Kadono, Hideki Kondō, Kenji Kubota, Shaorong Li, Longhui Li, Jiànróng Lǐ, Huazhen Liu, Tomohide Natsuaki, Sergey V. Netesov, Anna Papa, Sofia Paraskevopoulou, Liying Qi, Takahide Sasaya, Mang Shi, Xiǎohóng Shí, Zhènglì Shí, Yoshifumi Shimomoto, Jin‑Won Song, Ayato Takada, Shigeharu Takeuchi, Yasuhiro Tomitaka, Keizō Tomonaga, Shinya Tsuda, Changchun Tu, Tomio Usugi, Nikos Vasilakis, Jiro Wada, Lin‑Fa Wang, Guoping Wang, Yanxiang Wang, Yaqin Wang, Tàiyún Wèi, Shaohua Wen, Jiangxiang Wu, Lei Xu, Hironobu Yanagisawa, Caixia Yang, Zuokun Yang, Lifeng Zhai, Yong‑Zhen Zhang, Song Zhang, Jinguo Zhang, Zhe Zhang, Xueping Zhou. In addition, the publication call-out in the supplementary material was updated from issue 11 to issue 12. The original article has been corrected.

The Panel on Plant Health performed a listing of non‐EU viruses and viroids (reported hereinafter as viruses) of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L. A systematic literature review identified 197 viruses infecting one or more of the host genera under consideration. Viruses were allocated into three categories (i) 86 non‐EU viruses, known to occur only outside the EU or having only limited presence in the EU (i.e. reported in only one or few Member States (MSs), known to have restricted distribution, outbreaks), (ii) 97 viruses excluded at this stage from further categorisation efforts because they have significant presence in the EU (i.e. only reported so far from the EU or known to occur or be widespread in some MSs or frequently reported in the EU), (iii) 14 viruses with undetermined standing for which available information did not readily allow to allocate to one or the other of the two above groups. Comments provided by MSs during consultation phases were integrated in the opinion. The main knowledge gaps and uncertainties of this listing concern (i) the geographic distribution and prevalence of the viruses analysed, in particular when they were recently described; (ii) the taxonomy and biological status of a number of poorly characterised viruses; (iii) the host status of particular plant genera in relation to some viruses. The viruses considered as non‐EU and those with undetermined standing will be categorised in the next steps to answer a specific mandate from the Commission to develop pest categorisations for non‐EU viruses. This list does not imply a prejudice on future needs for a pest categorisation for other viruses which are excluded from the current categorisation efforts., This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5590/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5669/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5735/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5766/full

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Vitis L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of grapevine virus 101-14.N.23.9.1/South Africa/2009 for which very limited information exists, the pest categorisation was completed for 30 viruses or viroids having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long-distance dispersal and thus considered as the major pathway for potential entry. Depending on the virus, additional pathway(s) can also be seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Grapevine yellow speckle viroid 2, blueberry leaf mottle virus, grapevine Ajinashika virus, grapevine Anatolian ringspot virus, grapevine berry inner necrosis virus, grapevine deformation virus, grapevine fabavirus, grapevine red blotch virus, grapevine stunt virus, grapevine Tunisian ringspot virus, grapevine vein-clearing virus, temperate fruit decay-associated virus, peach rosette mosaic virus, tobacco ringspot virus, tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). With the exception of impact for the EU territory, on which the Panel was unable to conclude, blackberry virus S, grapevine geminivirus A, grapevine leafroll-associated virus 7, grapevine leafroll-associated virus 13, grapevine satellite virus, grapevine virus E, grapevine virus I, grapevine virus J, grapevine virus S, summer grape enamovirus, summer grape latent virus satisfy all the other criteria to be considered as potential Union QPs. Australian grapevine viroid, grapevine cryptic virus 1, grapevine endophyte endornavirus and wild vitis virus 1 do not meet all the criteria evaluated by EFSA to be regarded as potential Union QPs because they are not known to cause an impact on Vitis. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically non-EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as a potential Union regulated non-quarantine pests.

Virus taxonomy emerged as a discipline in the middle of the twentieth century. Traditionally, classification by virus taxonomists has been focussed on the grouping of relatively closely related viruses. However, during the past few years, the International Committee on Taxonomy of Viruses (ICTV) has recognized that the taxonomy it develops can be usefully extended to include the basal evolutionary relationships among distantly related viruses. Consequently, the ICTV has changed its Code to allow a 15-rank classification hierarchy that closely aligns with the Linnaean taxonomic system and may accommodate the entire spectrum of genetic divergence in the virosphere. The current taxonomies of three human pathogens, Ebola virus, severe acute respiratory syndrome coronavirus and herpes simplex virus 1 are used to illustrate the impact of the expanded rank structure. This new rank hierarchy of virus taxonomy will stimulate further research on virus origins and evolution, and vice versa, and could promote crosstalk with the taxonomies of cellular organisms., Here, the International Committee on Taxonomy of Viruses describe a new, expanded virus classification scheme with 15 ranks that closely aligns with the Linnaean taxonomic system and better encompasses viral diversity.

"A universal feature of the replication of positive-strand RNA viruses is the association with intracellular membranes. Carnation Italian ringspot virus (CIRV) replication in plants occurs in vesicles derived from the mitochondrial outer membrane. The product encoded by CIRV ORF1, p36, is required for targeting the virus replication complex to the outer mitochondrial membrane both in plant and yeast cells. Here the yeast Saccharomyces cerevisiae was used as a model host to study the effect of CIRV p36 on cell survival and death. It was shown that p36 does not promote cell death, but decreases cell growth rate. In addition, p36 changed the nature of acetic acid-induced cell death in yeast by increasing the number of cells dying by necrosis with concomitant decrease of the number of cells dying by programmed cell death, as judged by measurements of phosphatidylserine externalization. The tight association of p36 to membranes was not affected by acetic acid treatment, thus confirming the peculiar and independent interaction of CIRV p36 with mitochondria in yeast. This work proved yeast as an invaluable model organism to study both the mitochondrial determinants of the type of cell death in response to stress and the molecular pathogenesis of (+)RNA viruses."

Contains fulltext : 202661.pdf (Publisher’s version ) (Closed access) This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in October 2018. Of note, the ICTV has approved, by an absolute majority, the creation of additional taxonomical ranks above those recognized previously. A total of 15 ranks (realm, subrealm, kingdom, subkingdom, phylum, subphylum, class, subclass, order, suborder, family, subfamily, genus, subgenus, and species) are now available to encompass the entire spectrum of virus diversity. Classification at ranks above genus is not obligatory but can be used by the authors of new taxonomic proposals when scientific justification is provided.

International audience; This article lists the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2018. A total of 451 species, 69 genera, 11 subfamilies, 9 families and one new order were added to the taxonomy. The current totals at each taxonomic level now stand at 9 orders, 131 families, 46 subfamilies, 803 genera and 4853 species. A change was made to the International Code of Virus Classification and Nomenclature to allow the use of the names of people in taxon names under appropriate circumstances. An updated Master Species List incorporating the approved changes was released in March 2018 ( https://talk.ictvonline.org/taxonomy/ ).

A virus was isolated from potted plants of an unidentified species of Aeonium, a succulent ornamental very common in Southern Italy, showing chlorotic spots and rings on both leaf surfaces. It was successfully transmitted by sap inoculation to a limited range of hosts, including Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30nm in diameter, have a single type of coat protein (CP) subunits 54kDa in size, that encapsidate single-stranded positive-sense RNA species of 7,549 (RNA1) and 4,010 (RNA2) nucleotides. A third RNA molecule 3,472 nts in size entirely derived from RNA2 was also found. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to, but serologically distinct from tobacco ringspot virus (TRSV). Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed.

Satellite viruses and satellite nucleic acids depend on their helper viruses for replication and other essential functions, but are dispensable for the virus infectious cycle. Satellites may influence the host range and the transmission of the helper virus, and by adaptation to different helpers, they may induce the outbreak of new devastating diseases. The most attractive feature of satellites stands in their ability to alter helper virus-induced symptoms, either attenuating them, or exacerbating the disease caused by the helper virus alone. In addition, some satellites may produce new symptoms, different from those incited by the cognate virus. In the relationship between satellites and their helper viruses, host plants play a major role in the elicitation of diseases.

The infectivity of small circular satellite RNAs (sc-satRNAs) depends on a helper virus. However, sc-satRNAs resemble viroids in several features, including the small (220!257 nt) and single-stranded genome, the accumulation in the infected host as covalently-closed circular RNAs, the replication through rolling-circle mechanisms based on RNA intermediates only, and the inability to code for proteins, with one exception. In addition, sc-satRNAs are endowed with self-cleaving (and some with self-ligation) activity mediated by ribozymes, another feature shared with some viroids, thus reinforcing the hypothesis that these two groups of subviral infectious RNAs may have a monophyletic origin (Elena et al., 2001). However, a key biological difference exists between sc-satRNAs and viroids regarding the molecular machinery needed for replication, which is supplied by the helper virus and the host in the first case, but only by the host in the second. In addition, in contrast to viroids, sc-satRNAs are encapsidated by the helper virus coat protein. This chapter focuses on the main structural and biological features of sc-satRNAs, pointing out the role of hammerhead and hairpin ribozymes in their replication. Readers looking for additional information on sc-satRNAs may check previous reviews

Tombusviruses may support the replication of satellite (sat) RNAs. In particular, two satRNAs, sat L and Cymsat RNAs, are replicated by carnation Italian ringspot (CIRV) and tomato bushy stunt (TBSV) virus, but not by cymbidium ringspot virus (CymRSV) in vitro transcripts unless they contain a poly(A) tail at the 3' end. Conversely, the replication of both satRNAs was supported by virus particles or viral RNA of the original CymRSV inoculum even in the absence of the poly(A) tail. Sequence and mutational analyses revealed that the full-length infectious CymRSV clone contains one relevant sequence variation in the ORF 1-encoded protein (p33) compared with the original inoculum, i.e. a Ser₁₉ TCC codon instead of a Phe₁₉ TTC codon, which inhibited the replication of sat L and Cymsat RNAs. It is suggested that this amino acid is contained in a domain essential for the replication of some subviral RNAs.

The biological and molecular properties of a new satellite RNA (satRNA L) associated with Tomato bushy stunt virus (TBSV) are described. satRNA L consists of a linear single-stranded RNA 615 nucleotides in size, lacks significant open reading frames and has no sequence homology with the helper genome other than in the 5’-proximal seven nucleotides and in a central region that is also conserved in all tombusvirus genomic, defective interfering (DI) and satellite RNAs. Secondary structure analysis showed the presence of high order domains similar to those described for other tombusvirus RNAs. Shorter-than-unit-length molecules were shown not to be related to a silencing mechanism. satRNA L did not modify the symptoms induced by TBSV at all temperature conditions tested. A full-length cDNA clone was constructed and used in coinoculations with transcripts of Carnation Italian ringspot virus (CIRV) and Cymbidium ringspot virus (CymRSV). CIRV, but not CymRSV, supported the replication of satRNA L. Using CIRV/CymRSV hybrid infectious clones, two regions were identified as possible determinants of the different ability to support satRNA L replication. The first region is in the 5’-untranslated region, which folds differently in CymRSV in comparison with CIRV and TBSV; the second region is in the ORF 1-encoded protein where a more efficient satRNA L binding domain is suggested to be present in CIRV.

Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.

The p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA. Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other. DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity. Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA. DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone. By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an ‘all-or-none’ behaviour. Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined. Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.

Infection ofNicotiana benthamianacells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which thein vitrosynthesized RNA was inoculated toN. benthamianaplants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident.

Aureusvirus is a new genus of plant viruses typified by pothos latent virus (PoLV) and comprising cucumber leaf spot virus (CLSV), previously classified as definitive species in the genus Carmovirus. Aureusviruses are soil-borne viruses readily transmitted by sap inoculation to a moderate range of hosts. Natural transmissions of CLSV is by the chytrid fungus Olpidium bornovanus, whereas PoLV infects the host without the apparent intervention of a vector. Aureusviruses have isometric particles with size (c. 30 nm) and structure similar to those of the family Tombusviridae, to which the genus belongs. The genome consists of a molecule of single-stranded, positive-sense RNA c. 4.4 kb in size comprising five ORFs. The structural organization (i.e. number and order of genes) is virtually identical to that of members of the genus Tombusvirus. However, the aureusvirus genome has a smaller size and shows distinct differences in the amino acid sequence of some of the ORFs. ORF 1 encodes a 25 kDa product and terminates with a leaky amber codon the readthrough of which results in a 84 kDa protein (ORF 2) with the conserved motifs of RNA dependent RNA polymerase. ORF 3 encodes the coat protein (40-41 kDa), ORF 4 the movement protein (27 kDa), and ORF 5 a 14-17 kDa product responsible for symptom severity. Virions accumulate in great quantity in the cytoplasm, often forming crystalline aggregates, and in bubble-like evaginations of the tonoplast protruding into the vacuole. Replication is likely to occur in the cytoplasm with a stategy based on direct expression of the 5' proximal ORF and expression of downstream ORFs through subgenomic RNAs.

Replication of positive-strand RNA viruses is commonly associated with extensive cell membrane proliferation and rearrangement and/or modification of some organelles of the infected hosts. These changes, which are induced by nonstructural proteins, provide the sites (e.g., vesicles or membrane invaginations) in which virus replication occurs. In plants infected by members of the genus Tombusvirus, this function is played by peculiar cytopathologic structures, called ‘multivesicular bodies’ (MVBs), that originate from heavily modified peroxisomes or mitochondria. The genesis, structural organization, and role of MVBs in the economy of tombusvirus infections are reviewed and commented upon.

The complete sequences of genomic and defective interfering (DI) RNAs of carnation Italian ringspot tombusvirus (CIRV) were determined. The genome (4760 nt) has an organization identical to that reported for other tombusviruses except that the pre-readthrough domain of the viral replicase encoded by the 5'-proximal open reading frame (ORF) is larger. In particular, the N-terminal region of this protein differs from the corresponding region of the other members of the genus Tombusvirus and Carmovirus. Two DI RNAs were found in infected tissues whose sequences were completely derived from genomic RNA. The smaller molecule (474 nt) is contained completely within the larger molecule (656 nt), which suggests that it is derived from the larger one.

Cymbidium ringspot tombusvirus (CyRSV) supports the replication of an RNA molecule known to be a satellite (sat-RNA). Sequence analysis of wild-type sat-RNA showed that the 3' terminus is heterogeneous and terminates with one, two, or three C residues. Transcripts from mutant clones which lacked up to eight nucleotides at the 3' end were biologically active and yielded progeny RNA that had the 3' end restored as in wild-type RNA. Deletions or substitutions at the 5' end produced nonviable molecules. Studies with mutants containing deletions in internal regions showed that replication was affected essentially by the location of deletions; viable mutants were much less encapsidated than the wild type, showing that size also may be important in the encapsidation and survival of CyRSV sat-RNA.

Tepovirus is a new monotypic genus of plant viruses typified by potato virus T (PVT), a virus with helically constructed filamentous particles that are 640 nm long, previously classified as unassigned species in the family Betaflexiviridae. Virions have a single-stranded positive-sense polyadenylated RNA genome that is 6.5 kb in size, and a single type of coat protein with a size of 24 kDa. The viral genome contains three slightly overlapping ORFs encoding, respectively, the replication-related proteins (ORF1), a putative movement protein of the 30 K type (ORF2) and the coat protein (ORF3). Its structure and organization (number and order of genes) resembles that of trichoviruses and of citrus leaf blotch virus (CLBV, genus Citrivirus) but has a smaller size. Besides potato, the primary host, PVT can experimentally infect herbaceous hosts by mechanical inoculation. No vector is known, and transmission is through propagating material (tubers), seeds and pollen. PVT has a number of biological, physical and molecular properties that differentiate it from betaflexiviruses with a 30K-type movement protein. It is phylogenetically distant from all these viruses, but least so from grapevine virus A (GVA), the type member of the genus Vitivirus, with which it groups in trees constructed using the sequences of all of the genes.

In March 2011 plants of Aeonium spp., family Crassulaceae, showing chlorotic spots and rings on both leaf surfaces were observed in a private garden in the vicinity of Salerno (southern Italy). Electron microscope observations of leaf dips from several of these plants revealed the presence of isometric virus-like particles ca. 30 nm in diameter, some of which were partially or completely penetrated by the negative stain, as if they were devoid of nucleic acid in part or totally. A number of herbaceous hosts were successfully infected after mechanical inoculation with sap expressed from symptomatic Aeonium plants. For example, Nicotiana benthamiana and Lycopersicon esculentum (tomato) were systemically invaded and reacted with mottling and deformation of the leaves and yellowish concentric rings and line patterns, respectively. A virus with isometric particles indistinguishable from those seen in leaf dips was readily purified from symptomatic N. benthamiana leaves. RNA extracted from virus particles and analyzed in ethidium bromide-permeated agarose gels migrated as two separate bands. These were recovered and used as template for synthesizing cDNAs, which were cloned and partially sequenced. The viral genome was confirmed to consist of two distinct RNA species which were molecularly similar, but not identical, to RNA-1 and RNA-2 of Tobacco ringspot virus (TRSV) (Sanfaçon et al., 2012). TRSV has been intercepted in Italy in imported gladiolus bulbs (Bellardi and Marani, 1985) but, to our knowledge, has never been found in a cultivated plant. Its potential danger to economical crops like tomato is to be taken into account, especially should the presence of a nematode vector be ascertained.

An antiserum was raised against the 33 K protein encoded by the 5' proximal gene of cymbidium ringspot tombusvirus RNA. This antiserum reacts specifically with the 33 K and 92 K proteins, which constitute the viral replicase, in CyRSV-infected Nicotiana benthamiana plants and in transgenic plants transformed with the full-length replicase gene. In inoculated leaves of infected plants, synthesis of the 33 K/92 K proteins stops ten days after inoculation, whereas in newly produced systemically infected leaves there was continuous production of these proteins. In transgenic plants, both proteins were detected showing that readthrough of the termination codon of the 33 K protein does not depend on the presence of the replicating virus. The subcellular localization of the 33 K/92 K proteins is similar in infected and transgenic plants. No correlation was found between the level of expression of integrated virus gene and level of resistance to the challenging virus.

Genomic RNA of cymbidium ringspot tombusvirus (CyRSV) contains five large open reading frames (ORFs). The two 5′ proximal ORFs encode proteins of 33 and 92 kDa and the three 3′ proximal ORFs encode proteins of 41,22, and 19 kDa. In addition, a small reading frame encoding a protein of 4 kDa was recently observed by computer analysis of the 3′ nontranslated region of CyRSV and other tombusvirus RNAs (ORF 6). The function of putative gene products was investigated with the use of several mutants. Mutants in ORFs 1 and 2 were not viable indicating that both 33- and 92-kDa proteins are required for replication. Deletion of a large portion of the coat protein gene, encoding a 41-kDa protein, did not prevent replication of viral RNA and cell-to-cell movement, but interfered severely with long-distance translocation. No virus particles or viral RNA could be detected in inoculated or upper leaves of plants inoculated with transcripts obtained from mutants not expressing the 22-kDa protein. However, replication and encapsidation occurred normally in inoculated protoplasts indicating that this gene product is a transport protein. Conversely, no role could be assigned to putative gene products of ORF 5 (19-kDa protein) and QRF 6. It was also shown that factors other than gene expression can influence the replication of RNA mutants, probably due to instability of RNA molecules.

RNA replication of Cymbidium ringspot virus (CymRSV) normally occurs in plant and yeast cells in association with membranes derived from peroxisomes. However, in yeast strains devoid of these organelles CymRSV RNA replication takes place on the perinuclear membrane and cytoplasmic endoplasmic reticulum (ER) strands. The association of the CymRSV RNA replication complex to ER is mediated by the ORF 1- encoded protein p33. To analyze which part of this protein contains the information leading to localization to ER, a deletion analysis was carried out. The behaviour of deletion mutants was studied by confocal immunofluorescence microscopy and membrane floatation gradient analysis. It was shown that the signal targeting p33 to ER membranes largely coincides with the peroxisomal targeting signal, but contains additional elements in the first 87 amino acids.

The replication ofCymbidiumringspot virus (CymRSV) defective interfering (DI) RNA in cells of the yeastSaccharomyces cerevisiaenormally takes place in association with the peroxisomal membrane, thus paralleling the replication events in infected plant cells. However, previous results with a peroxisome-deficient mutant strain of yeast had suggested that the presence of peroxisomes is not a strict requirement for CymRSV DI RNA replication. Thus, a novel approach was used to study the putative alternative sites of replication by usingS. cerevisiaestrain YPH499 which does not contain normal peroxisomes. In this strain, CymRSV p33 and p92 accumulated over portions of the nuclear membrane and on membranous overgrowths which were identified as endoplasmic reticulum (ER) strands, following immunofluorescence and immunoelectron microscope observations. The proteins were not released by high-pH treatment, but were susceptible to proteolytic digestion, thus indicating peripheral and not integrated association. ER-associated p33 and p92 proteins supportedin transthe replication of DI RNA. The capacity of plus-strand RNA viruses to replicate in association with different types of cell membranes was thus confirmed.