The Cucumber vein yellowing virus silencing suppressor P1b can functionally replace HCPro in Plum pox virus infection in a host-specific manner

Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silencing suppressors (RSS) to counteract antiviral RNA silencing responses. HCPro is the RSS of Potyvirus spp., and its activity is enhanced by the upstream P1 protein. Distinctively, the ipomovirus Cucumber vein yellowing virus (CVYV) lacks HCPro but contains two P1 copies in tandem (P1aP1b), the second of which functions as RSS. Using chimeras based on the potyvirus Plum pox virus (PPV), we found that P1b can functionally replace HCPro in potyviral infections of Nicotiana plants. Interestingly, P1a, the CVYV protein homologous to potyviral P1, disrupted the silencing suppression activity of P1b and reduced the infection efficiency of PPV in Nicotiana benthamiana. Testing the influence of RSS in host specificity, we found that a P1b-expressing chimera poorly infected PPV's natural host, Prunus persica. Conversely, P1b conferred on PPV chimeras the ability to replicate locally in cucumber, CVYV's natural host. The deleterious effect of P1a on PPV infection is host dependent, because the P1aP1b-expressing PPV chimera accumulated in cucumber to higher levels than PPV expressing P1b alone. These results demonstrate that a potyvirus can use different RSS, and that particular RSS and upstream P1-like proteins contribute to defining the virus host range.     

The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication.

The plum pox potyvirus (PPV) protein CI is an RNA helicase whose function in the viral life cycle is still unknown. The CI protein contains seven conserved sequence motifs typical of RNA helicases of the superfamily SF2. We have introduced several individual point mutations into the region coding for motif V of the PPV CI protein and expressed these proteins in Escherichia coli as maltose binding protein fusions. Mutations that abolished RNA helicase activity also disturbed NTP hydrolysis. No mutations affected the RNA binding capacity of the CI protein. These mutations were also introduced in the PPV genome making use of a full-length cDNA clone. Mutant viruses carrying CI proteins with reduced RNA helicase activity replicated very poorly in protoplasts and were unable to infect whole plants without rapid pseudoreversion to wild-type. These results indicate that motif V is involved in the NTP hydrolysis step required for potyvirus RNA helicase activity, and that this activity plays an essential role in virus RNA replication inside the infected cell.     

Host-Specific Involvement of the HC Protein in the Long-Distance Movement of Potyviruses

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.     

Pathogenicity determinants in the complex virus population of a Plum pox virus isolate

Several subisolates were separated from a single Plum pox virus (PPV) isolate, PPV-PS. In spite of an extremely high sequence conservation (more than 99.9% similarity), different subisolates differed largely in pathogenicity in herbaceous hosts and infectivity in woody plants. The severity of symptomatology did not seem to correlate with virus accumulation. Sequence analysis and site-directed mutagenesis demonstrated that single amino acid changes in the helper component (HC) protein caused a drastic effect on virus symptoms in herbaceous hosts and notably modified virus infectivity in peach seedlings. These results indicate that HC variation might play an important role in virulence evolution of natural plant virus infections. Moreover, the analysis of Potato virus X (PVX)-HC chimeras showed that the identified HC amino acid changes had parallel effects on the severity of symptoms caused by PPV and on HC-induced enhancement of PVX pathogenicity, indicating that HC functions in potyvirus symptomatology and in synergism with other viruses have overlapping determinants.     

RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus.

Most positive strand RNA viruses infecting plants and animals encode proteins containing the so-called nucleotide binding motif (NTBM) (1) in their amino acid sequences (2). As suggested from the high level of sequence similarity of these viral proteins with the recently described superfamilies of helicase-like proteins (3-5), the NTBM-containing cylindrical inclusion (CI) protein from plum pox virus (PPV), which belongs to the potyvirus group of positive strand RNA viruses, is shown to be able to unwind RNA duplexes. This activity was found to be dependent on the hydrolysis of NTP to NDP and Pi, and thus it can be considered as an RNA helicase activity. In the in vitro assay used, the PPV CI protein was only able to unwind double strand RNA substrates with 3' single strand overhangs. This result indicates that the helicase activity of the PPV CI protein functions in the 3' to 5' direction (6). To our knowledge, this is the first report on a helicase activity associated with a protein encoded by an RNA virus.     

Comparative expression profiling of Nicotiana benthamiana leaves systemically infected with three fruit tree viruses

Plant viruses cause a wide array of disease symptoms and cytopathic effects. Although some of these changes are virus specific, many appear to be common even among diverse viruses. Currently, little is known about the underlying molecular determinants. To identify gene expression changes that are concomitant with virus symptoms, we performed comparative expression profiling experiments on Nicotiana benthamiana leaves infected with one of three different fruit tree viruses that produce distinct symptoms: Plum pox potyvirus (PPV; leaf distortion and mosaic), Tomato ringspot nepovirus (ToRSV; tissue necrosis and general chlorosis), and Prunus necrotic ringspot ilarvirus (PNRSV; subtle chlorotic mottling). The numbers of statistically significant genes identified were consistent with the severity of the observed symptoms: 1,082 (ToRSV), 744 (PPV), and 89 (PNRSV). In all, 56% of the gene expression changes found in PPV-infected leaves also were altered by ToRSV, 87% of which changed in the same direction. Both PPV- and ToRSV-infected leaves showed widespread repression of genes associated with plastid functions. PPV uniquely induced the expression of large numbers of cytosolic ribosomal genes whereas ToRSV repressed the expression of plastidic ribosomal genes. How these and other observed expression changes might be associated with symptom development are discussed.     

Plum pox potyvirus resistance associated to transgene silencing that can be stabilized after different number of plant generations

Nicotiana benthamiana plants were transformed with a fragment of the plum pox potyvirus (PPV) genome that encodes the nuclear inclusion a (NIa) and b (NIb) proteins and the N-terminus of the capsid protein (NIa–NIb–CP*). Lines transformed with this PPV genomic fragment harboring mutations in the GDD replicase-motif were also obtained. Plants of NIaΔV lines that carry a GDD to VDD mutation in the PPV transgene, were immune to PPV infection. The resistance was highly specific, since it was only partially overcome by a PPV strain different to that from which the transgene was derived, and no resistance was observed after inoculation with a second potyvirus. PPV was not able to replicate in protoplasts isolated from NIaΔV transgenic plants, indicating that the resistance was functional at the single cell level. Only a fraction of plants from lines transformed with the NIa–NIb–CP* fragment harboring a GDD to ADD mutation (NIaΔA lines), were resistant to PPV infection. This same phenotype was observed in plants expressing the wild-type construction (NIaΔ), although the progeny of some non-infected plants seemed to be completely resistant to PPV, independently of the allelic status of the parental plant. In all cases, the resistance phenotype correlated positively with low levels of transgene mRNA accumulation, suggesting that it was mainly due to a gene silencing mechanism. Our results show that, although the transgene was not silenced in all R1 plants from some individual lines, a stable silenced status could be reached in the following generations.     

Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana

Twelve Arabidopsis accessions were challenged with Plum pox potyvirus (PPV) isolates representative of the four PPV strains. Each accession supported local and systemic infection by at least some of the PPV isolates, but high variability was observed in the behavior of the five PPV isolates or the 12 Arabidopsis accessions. Resistance to local infection or long-distance movement occurred in about 40% of all the accession-isolate combinations analyzed. Except for Nd-1, all accessions showed resistance to local infection by PPV-SoC; in the Landsberg erecta (Ler) accession, this resistance was compromised by sgt1 and rar1 mutations, suggesting that it could be controlled by an R gene-mediated resistance pathway. While most of the susceptible accessions were symptomless, PPV induced severe symptoms on inflorescences in C24, Ler, and Bay-0 as early as 15 days after inoculation. Genetic analyses indicated that these interaction phenotypes are controlled by different genetic systems. The restriction of long-distance movement of PPV-El Amar and of another member of genus Potyvirus, Lettuce mosaic virus, in Col-0 requires the RTM genes, indicating for the first time that the RTM system may provide a broad range, potyvirus-specific protection against systemic infection. The restriction to PPV-PS long-distance movement in Cvi-1 is controlled by a single recessive gene, designated rpv1, which was mapped to chromosome 1. The nuclear inclusion polymerase b-capsid protein region of the viral genome appears to be responsible for the ability of PPV-R to overcome rpv1-mediated resistance.     

Translationally controlled tumour protein: A protein necessary for potyvirus intracellular multiplication that supports plant infection by unrelated viruses

The multifunctional protein translationally controlled tumour protein (TCTP) was previously identified as necessary for infection by the potyvirus pepper yellow mosaic virus. Using turnip mosaic virus (TuMV) as a model to study potyvirus biology, we confirmed that TCTP has a positive effect on virus infection. Living cell confocal microscopy demonstrated that TCTP colocalises with 6K2-tagged replication vesicles and with a perinuclear globular structure typically observed during potyvirus infection. Also, TCTP silenced protoplasts showed reduced virus accumulation, quantified by qRT-PCR, which suggests an effect on virus replication, translation or other intracellular process. Finally, TCTP silencing in plants reduced the accumulation of two species belonging to Orthotospovirus and a Begomovirus genus, which are not closely related to potyviruses. The results suggest that TCTP is a general susceptibility factor to several unrelated viruses.     

Sorghum chlorotic spot virus binds to potyvirus cylindrical inclusions in tobacco leaf cells

Nicotiana benthamiana can be doubly infected with either potato virus Y or tobacco etch virus and sorghum chlorotic spot virus (SCSV). Immunogold labeling showed that cylindrical inclusions of either potyvirus bind virions of the unrelated rod-shaped furovirus SCSV. Not all cells in doubly infected N. benthamiana plants contained both viruses. In cells infected by the potyviruses but not by SCSV, cylindrical inclusions did not label with the antiserum to SCSV. Numbers of cells infected with SCSV did not increase in doubly infected plants compared to those in plants infected with SCSV alone. Systemic infection of N. benthamiana by either potyvirus was not prevented by SCSV infections. This provides further evidence that unrelated rod-shaped viruses can bind to potyvirus cylindrical inclusion bodies, and that this phenomenon is not limited to graminaceous hosts.     

Identification of immunogenic hot spots within plum pox potyvirus capsid protein for efficient antigen presentation

PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-gamma, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. III. Cosegregation of phenotypically similar dominant responses to nine potyviruses

We have identified monogenic dominant resistance to azuki bean mosaic poty virus (AzMV), passionfruit woodiness potyvirus-K (PWV-K), zucchini yellow mosaic potyvirus (ZYMV), and a dominant factor that conditioned lethal necrosis to Thailand Passiflora potyvirus (ThPV), in Phaseolus vulgaris Black Turtle Soup 1. Resistance to AzMV, PWV-K, ZYMV, watermelon mosaic potyvirus, cowpea aphid-borne mosaic potyvirus, blackeye cowpea mosaic potyvirus, and lethal necrosis to soybean mosaic potyvirus and ThPV cosegregated as a unit with the I gene for resistance to bean common mosaic potyvirus.     

Association of a potyvirus with mosaic disease of gherkin (<Emphasis Type="Italic">Cucumis anguria</Emphasis> L.) in India

A virus associated with severe mosaic disease of gherkin (Cucumis anguria L.) in south India was identified. The infected plants showed mosaic, vein banding, blistering on malformed leaves and fruits. Host range, transmission, serological and electron microscopic studies were carried out to identify the virus. The virus was readily transmitted by Sap inoculation and by aphids in a non-persistent manner. The host range of the virus was mainly limited to cucurbitaceous and chenopodium species. The virus showed positive serological relationships with members of potyvirus genus but not with cucumo, ilar and taspoviruses. Electron microscopy of leaf dip preparation of infected leaves revealed long flexuous filamentous virus particles measuring 750 × 12 nm. On the basis of symptomotology, host range, transmission, serology and particle morphology the virus associated with mosaic disease of gherkin might be the member of potyvirus genus.     

Some Hosts and Properties of a Potyvirus Infecting Vanilla fragrans (Orchidaceae) in the Kingdom of Tonga

Abstract A potyvirus was isolated from Vanilla fragrans exhibiting leaf distortion, chlorotic and necrotic lesions and vine die-back. The virus can be propagated in Nicotiana clevelandii and N. benthamiana and induces local lesions in Chenopodium amaranticolor and C. quinoa. The flexuous particles have a mean length of 765 nm, pinwheel inclusions are found in systemically infected N. benthamiana leaves and SDS-polyacrylamide gel electrophoresis indicates a single polypeptide of M 32.7 × 10³. The virus is transmissible from N. clevelandii to N. clevelandii by the aphid species Myzus persicae and Aphis gossypii. A polyclonal antiserum raised to the Tongan potyvirus failed to react, with the potyvirus associated with mosaic symptoms of V. tahitensis in French Polynesia and ISEM failed to detect any close relationship to 22 other potyviruses.     

Inhibitory effects of human cystatin C on plum pox potyvirus proteases

The effect of different protease inhibitors on the proteolytic processing of the plum pox potyvirus (PPV) polyprotein has been analyzed. Human cystatin C, an inhibitor of cysteine proteases, interfered with the outoprocessing of the viral papain-like cysteine protease HCPro. Unexpectedly, it also had an inhibitory effect on the autocatalytic cleavage of the Nla protease which, although it has a Cys residue in its active center, has been described as structurally related to serine proteases. Other protease inhibitors tested had no effect on any of the cleavage events analyzed.