Luisa Rubino

Expression of the Cymbidium Ringspot Virus 33-Kilodalton Protein in Saccharomyces cerevisiae and Molecular Dissection of the Peroxisomal Targeting Signal

ABSTRACT Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.

A single amino acid substitution in the ORF1 of cymbidium ringspot virus determines the accumulation of two satellite RNAs.

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.

Tepovirus, a novel genus in the family Betaflexiviridae

Tepovirus is a new monotypic genus of plant viruses typified by potato virus T (PVT), a virus with helically constructed filamentous particles that are 640xa0nm long, previously classified as unassigned species in the family Betaflexiviridae. Virions have a single-stranded positive-sense polyadenylated RNA genome that is 6.5xa0kb in size, and a single type of coat protein with a size of 24xa0kDa. The viral genome contains three slightly overlapping ORFs encoding, respectively, the replication-related proteins (ORF1), a putative movement protein of the 30xa0K 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.

Characterization of a putative novel nepovirus from Aeonium sp.

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 7549 (RNA1) and 4010 (RNA2) nucleotides. A third RNA molecule 3472 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.

PRESENCE OF TOBACCO RINGSPOT VIRUS IN AEONIUM spp.

In March 2011 plants of Aeonium spp., family Crassulaceae, showing chlorotic spots and rings on both leaf surfaces were ob- served 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 parti- cles ca. 30 nm in diameter, some of which were partially or com- pletely 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, Nico- tiana benthamiana and Lycopersicon esculentum (tomato) were systemically invaded and reacted with mottling and deformation of the leaves and yellowish concentric rings and line patterns, re- spectively. 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 ana- lyzed 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 se- quenced. The viral genome was confirmed to consist of two dis- tinct RNA species which were molecularly similar, but not identi- cal, to RNA-1 and RNA-2 of Tobacco ringspot virus (TRSV) (San- facon et al., 2012). TRSV has been intercepted in Italy in import- ed gladiolus bulbs (Bellardi and Marani, 1985) but, to our knowl- edge, has never been found in a cultivated plant. Its potential danger to economical crops like tomato is to be taken into ac- count, especially should the presence of a nematode vector be as- certained.

Small Circular Satellite RNAs

Small circular satellite RNAs (sc-satRNAs) assume compact conformations, display catalytic activity mediated by hammerhead or hairpin ribozymes, and, in most cases, do not code for proteins, thus resembling viroids in several structural and functional features. However, in contrast to viroids, and similarly to other viral satellites, sc-satRNAs rely on a helper virus for replication and transmission. sc-satRNAs may modulate helper virus accumulation and symptomatology through unknown molecular mechanisms including RNA silencing.

Biology of Satellites

Abstract 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.