The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N. benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N. benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N. benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.

Molecular Characterization of Divergent Closterovirus Isolates Infecting Ribes Species.

Five isolates of a new member of the family Closteroviridae, tentatively named blackcurrant leafroll-associated virus 1 (BcLRaV-1), were identified in the currant. The 17-kb-long genome codes for 10 putative proteins. The replication-associated polyprotein has several functional domains, including papain-like proteases, methyltransferase, Zemlya, helicase, and RNA-dependent RNA polymerase. Additional open reading frames code for a small protein predicted to integrate into the host cell wall, a heat-shock protein 70 homolog, a heat-shock protein 90 homolog, two coat proteins, and three proteins of unknown functions. Phylogenetic analysis showed that BcLRaV-1 is related to members of the genus Closterovirus, whereas recombination analysis provided evidence of intraspecies recombination.

Biological, Serological, and Molecular Characterization of a Highly Divergent Strain of Grapevine leafroll-associated virus 4 Causing Grapevine Leafroll Disease.

The complete genome sequence of a highly divergent strain of Grapevine leafroll-associated virus 4 (GLRaV-4) was determined using 454 pyrosequencing technology. This virus, designated GLRaV-4 Ob, was detected in Vitis vinifera 'Otcha bala' from our grapevine virus collection at Agroscope. The GLRaV-4 Ob genome length and organization share similarities with members of subgroup II in the genus Ampelovirus (family Closteroviridae). Otcha bala was graft-inoculated onto indicator plants of cultivar Gamay to evaluate the biological properties of this new strain, and typical leafroll symptoms were induced. A monoclonal antibody for the rapid detection of GLRaV-4 Ob by enzyme-linked immunosorbent assay is available, thus facilitating large-scale diagnostics of this virus. Based on the relatively small size of the coat protein, the reduced amino acid identity and the distinct serological properties, our study clearly shows that GLRaV-4 Ob is a divergent strain of GLRaV-4. Furthermore, molecular and serological data revealed that the AA42 accession from which GLRaV-7 was originally reported is in fact co-infected with GLRaV-4 Ob and GLRaV-7. This finding challenges the idea that GLRaV-7 is a leafroll-causing agent.

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.

Complex molecular architecture of beet yellows virus particles.

Closteroviruses possess exceptionally long filamentous virus particles that mediate protection and active transport of the genomic RNA within infected plants. These virions are composed of a long "body" and short "tail" whose principal components are the major and minor capsid proteins, respectively. Here we use biochemical, genetic, and ultrastructural analyses to dissect the molecular composition and architecture of particles of beet yellows virus, a closterovirus. We demonstrate that the virion tails encapsidate the 5'-terminal, approximately 650-nt-long, part of the viral RNA. In addition to the minor capsid protein, the viral Hsp70-homolog, 64-kDa protein, and 20-kDa protein are also incorporated into the virion tail. Atomic force microscopy of virions revealed that the tail possesses a striking, segmented morphology with the tip segment probably being built of 20-kDa protein. The unexpectedly complex structure of closterovirus virions has important mechanistic and functional implications that may also apply to other virus families.

Closterovirus bipolar virion: evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5' region.

The long flexuous virions of the Closteroviridae have a unique bipolar architecture incorporating two coat proteins, with most of the helical nucleocapsid encapsidated by the major coat protein (CP) and a small portion of one end encapsidated by the minor coat protein (CPm). It is not known whether CPm encapsidates the genomic RNA and, if so, which end and what effects transition between the two coat proteins. Two other virus-encoded proteins, an HSP70 homolog (HSP70h) and an approximately 61-kDa protein, are required to augment virion assembly. In this work, we examine the in vivo encapsidation of Citrus tristeza virus by its CPm in the absence of CP. In the absence of other assembly-related proteins, CPm protected a family of 5' coterminal RNAs, apparently because of pausing at different locations along the genomic RNA. Most of the nucleocapsids formed by CPm were short, but a few were full-length and infectious. Mutations within the 5' nontranslated region demonstrated that the CPm origin of assembly overlaps the previously described conserved stem-and-loop structures that function as a cis-acting element required for RNA synthesis. Thus, in the absence of CP, the CPm encapsidation is initiated from the 5' end of the genomic RNA. Coexpression of HSP70h and the p61 protein with CPm in protoplasts restricted encapsidation to the 5' approximately 630 nucleotides, which is close to the normal boundary of the bipolar virion, whereas the presence of either HSP70h or the p61 protein alone did not limit encapsidation by CPm.

Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly.

Assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. Closteroviridae virions are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. The Closteroviridae is the only virus family that encodes a protein with similarity to cellular chaperones, a 70-kDa heat-shock protein homolog (HSP70h). We examined the involvement of gene products of Citrus tristeza virus (CTV) in virion formation and found that the chaperone-like protein plus the p61 and both coat proteins were required for efficient virion assembly. Competency of virion assembly of different CTV mutants was assayed by their ability to be serially passaged in Nicotiana benthamiana protoplasts using crude sap as inoculum, and complete and partial virus particles were analyzed by serologically specific electron microscopy. Deletion mutagenesis revealed that p33, p6, p18, p13, p20, and p23 genes were not needed for virion formation. However, deletion of either minor- or major-coat protein resulted in formation of short particles which failed to be serially transferred in protoplasts, suggesting that both coat proteins are required for efficient virion assembly. Deletion or mutation of HSP70h and/or p61 dramatically reduced passage and formation of full-length virions. Frameshift mutations suggested that the HSP70h and p61 proteins, not the RNA sequences, were needed for virion assembly. Substitution of the key amino acid residues in the ATPase domain of HSP70h, Asp(7) to Lys or Glu(180) to Arg, reduced assembly, suggesting that the chaperone-like ATPase activity is involved in assembly. Both HSP70h and p61 proteins appeared to contribute equally to assembly, consistent with coordinate functions of these proteins in closterovirus virion formation. The requirement of two accessory proteins in addition to both coat proteins for efficient assembly is uniquely complex for helical virions.

The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies.

Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of approximately 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF expressed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20-GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein-p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells.

"Rattlesnake" structure of a filamentous plant RNA virus built of two capsid proteins.

Elongated particles of simple RNA viruses of plants are composed of an RNA molecule coated with numerous identical capsid protein subunits to form a regular helical structure, of which tobacco mosaic virus is the archetype. Filamentous particles of the closterovirus beet yellow virus (BYV) reportedly contain approximately 4000 identical 22-kDa (p22) capsid protein subunits. The BYV genome encodes a 24-kDa protein (p24) that is structurally related to the p22. We searched for the p24 in BYV particles by using immunoelectron microscopy with specific antibodies against the recombinant p24 protein and its N-terminal peptide. A 75-nm segment at one end of the 1370-nm filamentous viral particle was found to be consistently labeled with both types of antibodies, thus indicating that p24 is indeed the second capsid protein and that the closterovirus particle, unlike those of other plant viruses with helical symmetry, has a "rattlesnake" rather than uniform structure.

Relation of beet yellows virus to the phloem and to movement in the sieve tube.

In minor veins of leaves of Beta vulgaris L. (sugar beet) yellows virus particles were found both in parenchyma cells and in mature sieve elements. In parenchyma cells the particles were usually confined to the cytoplasm, that is, they were absent from the vacuoles. In the sieve elements, which at maturity have no vacuoles, the particles were scattered throughout the cell. In dense aggregations the particles tended to assume an orderly arrangement in both parenchyma cells and sieve elements. Most of the sieve elements containing virus particles had mitochondria, plastids, endoplasmic reticulum, and plasma membrane normal for mature sieve elements. Some sieve elements, however, showed evidence of degeneration. Virus particles were present also in the pores of the sieve plates, the plasmodesmata connecting the sieve elements with parenchyma cells, and the plasmodesmata between parenchyma cells. The distribution of the virus particles in the phloem of Beta is compatible with the concept that plant viruses move through the phloem in the sieve tubes and that this movement is a passive transport by mass flow. The observations also indicate that the beet yellows virus moves from cell to cell and in the sieve tube in the form of complete particles, and that this movement may occur through sieve-plate pores in the sieve tube and through plasmodesmata elsewhere.