Molecular characterization of a closterovirus from carrot and its identification as a German isolate of Carrot yellow leaf virus.

A high-molecular-weight dsRNA (approximately 15 kbp) was isolated from chlorotic leaves of a carrot plant and used for determining the entire nucleotide sequence of a closterovirus. The complete genome of this carrot closterovirus (CCV) was 16.4 kb in length and contained ten open reading frames (ORFs). The genome organization of CCV resembled that of beet yellow stunt virus, but ORF2 and ORF3 were in a reversed order. Based on Hsp70h sequences, CCV is most closely related to carnation necrotic fleck virus and mint virus 1, two viruses of the genus Closterovirus (family Closteroviridae). The major coat protein gene of CCV was expressed in Escherichia coli for raising an antiserum. This permitted routine detection of CYLV by DAS-ELISA and immunoelectron microscopy and was used for demonstrating the bipolar nature of the CCV virion. Moreover, the antiserum gave a Western blot reaction with a reference sample of a Carrot yellow leaf virus (CYLV) isolate from the Netherlands, suggesting that CCV is a German isolate of CYLV.

At-4/1, an interactor of the Tomato spotted wilt virus movement protein, belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking.

The Tomato spotted wilt virus (TSWV) encoded NSm movement protein facilitates cell-to-cell spread of the viral genome through structurally modified plasmodesmata. NSm has been utilized as bait in yeast two-hybrid interaction trap screenings. As a result, a protein of unknown function, called At-4/1, was isolated from an Arabidopsis thaliana GAL4 activation domain-tagged cDNA library. Using polyclonal antibodies against bacterially expressed At-4/1, Western blot analysis of protein extracts isolated from different plant species as well as genome database screenings showed that homologues of At-4/1 seemed to be encoded by many vascular plants. For subcellular localization studies, At-4/1 was fused to green fluorescent protein, and corresponding expression vectors were used in particle bombardment and agroinfiltration assays. Confocal laser scannings revealed that At-4/1 assembled in punctate spots at the cell periphery. The protein accumulated intracellularly in a polarized fashion, appearing in only one-half of a bombarded epidermal cell, and, moreover, moved from cell to cell, forming twin-structured bodies seemingly located at both orifices of the plasmodesmatal pore. In coexpression studies, At-4/1 colocalized with a plant virus movement protein TGBp3 known to reside in endoplasmic reticulum-derived membrane structures located in close vicinity to plasmodesmata. Thus, At-4/1 belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking.

Molecular Identification, Reverse Transcription-Polymerase Chain Reaction Detection, Host Reactions, and Specific Cytopathology of Artichoke yellow ringspot virus Infecting Onion Crops.

ABSTRACT An isometric virus ca. 25 nm in diameter with angular contour was isolated from onion plants showing yellow leaf striping and necrotic tips. The virus was mechanically transmitted onto 28 species of indicator plants belonging to five families, viz. Amaranthaceae, Chenopodiaceae, Cucurbitaceae, Leguminosae, and Solanaceae where it causes ring spots, malformations, and/or tip necrosis. Cytopathological studies in infected Nicotiana benthamiana tissues revealed cytoplasmic inclusions resembling those caused by Artichoke yellow ringspot virus (AYRSV), a member of the family Comoviridae. Host range and symptomatology of the onion virus were also similar to AYRSV. A high seed transmission rate (20%) was found in onion. Reverse transcription-polymerase chain reaction using degenerate primers specific for the family Comoviridae allowed amplification of RNA-dependent RNA polymerase sequences, which upon sequence analysis and comparison with AYRSV isolates from Cynara scolymus (AYRSV-AtG) and Vicia faba (AYRSV-F) were highly similar, thus providing evidence that the nepovirus AYRSV is infecting onion in the field.

Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus.

ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His(6) was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly(588)/Gly(589) bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.

Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus.

Monoclonal antibodies (MAbs) specific to the methyltransferase (MT) and helicase (HEL) domains of the closterovirus Beet yellows virus (BYV) were used for immunogold labelling of ultrathin sections of virus-infected Tetragonia expansa plants. MAbs 4A2 and 4A5 from the MT panel, and 1C4 from the HEL panel, specifically labelled distinct closterovirus-induced membranous structures, the 'BYV-type vesicles', thus suggesting that the closterovirus MT-like and HEL-like proteins co-localize in these structures. Probing of the MT and HEL MAbs with synthetic octapeptides spanning the sequences of the recombinant MT and HEL fragments that had been used as immunogens showed that 4A5 and 4A2 recognized a single epitope, SRLLENET (aa 686-692 in the BYV 1a protein), and 1C4 reacted with the DDPF epitope (aa 2493-2496). These epitopes apparently reside on the exposed parts of the membrane-associated molecules of the closterovirus MT-like and HEL-like proteins. Two other epitopes determined for the MT MAbs that were nonreactive in the immunogold labelling, namely TMVTPGEL (aa 750-757; MAbs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806-813; MAb 2A4), are possibly buried in the MT domain fold or shielded by membranes or other proteins involved in the viral replicative complex.