Innovative RNAi Strategies and Tactics to Tackle Plum Pox Virus (PPV) Genome in Prunus domestica-Plum

We developed an innovative RNAi concept based on two gene constructs built from the capsid gene (CP) cistron of the Plum pox virus (PPV) genome. First, designated as amiCPRNA, a potential molecule interfering with PPV genome translation and the second one is the ami-siCPRNA to target viral genome translation and PPV RNA replication. Following the previous engineering of these constructs in an experimental herbaceous host, they were introduced into Prunus domestica (plum tree) genome. Previously propagated onto a susceptible rootstock, these clones were graft-inoculated with PPV. After four dormancy cycles, and consistent with our experience of PPV infection, some clones showed a common phenomenon of silencing that can differ between the detailed plant phenotypes. Three different phenotypes were developed by the amisiCPRNA clones. First, the high resistance character shown by the amisiCPRNA plum-7 that was similar to the resistance expressed by HoneySweet plum. Secondly, a recovery reaction was developed by the two other amisiCPRNA plum-3 and plum-4 that differed from the rest, characterized as susceptible clones, among these were the amiCPRNA plums. Having assessed the behavior of these plums versus the herbaceous host accumulating the similar form of RNAi: ami-, si-, and ami-siRNA, challenging assays in perennials consistently reflect the natural context of viral genome targeting.

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Resistance of Transgenic Prunus domestica to Plum Pox Virus Infection

Plant Disease ◽

10.1094/pdis.1997.81.11.1231 ◽

1997 ◽

Vol 81 (11) ◽

pp. 1231-1235 ◽

Cited By ~ 64

Author(s):

M. Ravelonandro ◽

R. Scorza ◽

J. C. Bachelier ◽

G. Labonne ◽

L. Levy ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Coat Protein ◽

Plum Pox Virus ◽

Prunus Domestica ◽

Chain Reaction ◽

Cp Gene ◽

Aphid Feeding ◽

Polymerase Chain ◽

Dormancy Cycles ◽

Das Elisa

Transgenic plum trees (Prunus domestica) containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV by aphid feeding or chip budding. Infection was monitored by evaluation of virus symptoms, DAS-ELISA, and immunoblot assays. Based on observations and analyses over 3 years including two dormancy cycles, one out of five transgenic clones (C-5), was found to be resistant to infection whether inoculated by aphids or by chip budding. PPV could not be detected in any inoculated plants of the C-5 clone by immunoblot or immunocap-ture-reverse transcriptase-polymerase chain reaction assays. To our knowledge, this is the first P. domestica clone resistant to PPV infection produced by genetic engineering.

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BothcisandtransActivities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

Journal of Virology ◽

10.1128/jvi.00469-16 ◽

2016 ◽

Vol 90 (15) ◽

pp. 6864-6883 ◽

Cited By ~ 6

Author(s):

Morgan R. Herod ◽

Cristina Ferrer-Orta ◽

Eleni-Anna Loundras ◽

Joseph C. Ward ◽

Nuria Verdaguer ◽

...

Keyword(s):

Disease Virus ◽

Viral Genome ◽

Foot And Mouth Disease ◽

Rna Replication ◽

Mouth Disease ◽

Viral Rna ◽

Genome Replication ◽

Replication Complex ◽

Mouth Disease Virus ◽

Foot And Mouth

ABSTRACTThePicornaviridaeis a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided intrans(i.e., via expression from a separate RNA molecule), while others are required incis(i.e., expressed from the template RNA molecule).In vitrostudies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymaticcis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that thiscis-acting role of 3D is distinct from the catalytic activity, which is predominantlytransacting. Immunofluorescence studies suggest that bothcis- andtrans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts inciswith RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.IMPORTANCEFoot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., intrans) while others must originate from the template (i.e., incis). Here, we present an analysis ofcisandtransactivities of the RNA-dependent RNA polymerase 3D. We demonstrate a novelcis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

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Transition from Acute to Persistent Theiler's Virus Infection Requires Active Viral Replication That Drives Proinflammatory Cytokine Expression and Chronic Demyelinating Disease

Journal of Virology ◽

10.1128/jvi.78.22.12480-12488.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12480-12488 ◽

Cited By ~ 25

Author(s):

Mark Trottier ◽

Brian P. Schlitt ◽

Aisha Y. Kung ◽

Howard L. Lipton

Keyword(s):

Spinal Cord ◽

Viral Replication ◽

Proinflammatory Cytokine ◽

Viral Genome ◽

Demyelinating Disease ◽

Rna Replication ◽

Viral Rna ◽

Mrna Levels ◽

Active Viral Replication ◽

The Brain

ABSTRACT The dynamics of Theiler's murine encephalomyelitis virus (TMEV) RNA replication in the central nervous systems of susceptible and resistant strains of mice were examined by quantitative real-time reverse transcription-PCR and were found to correlate with host immune responses. During the acute phase of infection in both susceptible and resistant mice, levels of viral replication were high in the brain and brain stem, while levels of viral genome equivalents were 10- to 100-fold lower in the spinal cord. In the brain, viral RNA replication decreased after a peak at 5 days postinfection (p.i.), in parallel with the appearance of virus-specific antibody responses; however, by 15 days p.i., viral RNA levels began to increase in the spinal cords of susceptible mice. During the transition to and the persistent phase of infection, the numbers of viral genome equivalents in the spinal cord varied substantially for individual mice, but high levels were consistently associated with high levels of proinflammatory Th1 cytokine and chemokine mRNAs. Moreover, a large number of viral genome equivalents and high proinflammatory cytokine mRNA levels in spinal cords were only observed for susceptible SJL/J mice who developed demyelinating disease. These results suggest that TMEV persistence requires active viral replication beginning about day 11 p.i. and that active viral replication with high viral genome loads leads to increased levels of Th1 cytokines that drive disease progression in infected mice.

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The phenotypically quantitative nature of hypersensitivity of European plum (Prunus domestica L.) against the Plum pox virus and its description using the hypersensitivity index

Horticultural Science ◽

10.17221/639-hortsci ◽

2008 ◽

Vol 35 (No. 2) ◽

pp. 50-64 ◽

Cited By ~ 14

Author(s):

M. Neumüller ◽

W. Hartmann

Keyword(s):

Quantitative Trait ◽

Leaf Blade ◽

Test System ◽

Artificial Inoculation ◽

Plum Pox Virus ◽

Prunus Domestica ◽

Individual Genotype ◽

Selection Tool ◽

Quantitative Nature

More than 1,300 seedlings of European plum originating from crossing combinations with at least one parent showing hypersensitivity resistance against PPV were analyzed for their reaction to artificial inoculation with PPV using the double grafting method with virus infected interstem. It was shown that the hypersensitivity resistance against the virus is a phenotypically quantitative trait. The different kinds of symptoms observed in the test system, which contribute to the hypersensitivity resistance, range from weak necrosis on the leaf blade and on the stem to the death of the whole young shoots. A hypersensitivity index was developed which helps to determine the degree of hypersensitivity resistance of an individual genotype. Its use is strongly recommended as selection tool in breeding for hypersensitivity resistance.

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Interactions of Plum pox virus strain Rec with Apple chlorotic leafspot virus and Prune dwarf viruses in field-grown transgenic plum Prunus domestica L., clone C5

Plant Protection Science ◽

10.17221/535-pps ◽

2008 ◽

Vol 44 (No. 1) ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

J. Polák ◽

M. Ravelonandro ◽

J. Kumar-Kundu ◽

J. Pívalová ◽

R. Scorza

Keyword(s):

Sequence Analysis ◽

Transgenic Plants ◽

Open Field ◽

Recombinant Strain ◽

Dwarf Virus ◽

Plum Pox Virus ◽

Prunus Domestica ◽

Rt Pcr ◽

Antagonistic Effects ◽

Das Elisa

Transgenic plums, Prunus domestica L. clone C5, were inoculated by bud grafting with Plum pox virus (PPV-Rec, recombinant strain originated from plum), PPV-Rec + Apple chlorotic leafspot virus (ACLSV), PPV-Rec + Prune dwarf virus (PDV), and PPV-Rec + ACLSV + PDV. Non-inoculated transgenic plums served as controls. Plants were grown in an open field for 5 years. They were evaluated by visible symptoms, by DAS-ELISA and RT-PCR. Mild PPV symptoms, diffuse spots or rings appeared two years after inoculation in some leaves of plants artificially inoculated with PPV-Rec, PPV-Rec + ACLSV, PPV-Rec + PDV, and PPV-Rec + ACLSV + PDV. Severe PPV symptoms appeared in leaves of shoots growing from infected buds used for inoculation. During the following three years, further weakening of PPV symptoms was observed in transgenic plants. In 2007, very mild PPV symptoms were found in only a few leaves, and over 60%, resp. 70% of the C5 trees showed no PPV symptoms. The presence of PPV was confirmed by ELISA, ISEM and RT-PCR. No difference in PPV symptoms was observed between PPV-Rec and combinations PPV-Rec + ACLSV, PPV-Rec + PDV, PPV-Rec + ACLSV + PDV. No symptoms of ACLSV appeared in combinations of ACLSV with PPV-Rec and PPV-Rec + PDV during 2004–2007, but the presence of ACLSV in leaves of transgenic plants clone C5 was proved by ELISA and RT-PCR. Neither synergistic nor antagonistic effects of ACLSV on PPV-Rec were observed. No symptoms of PDV appeared in combinations of viruses with PDV during 2004–2007. PDV was not detected by ELISA, and the presence of PDV was uncertain by RT-PCR in most of inoculated trees in 2006 and 2007. The results of RT-PCR will be further confirmed by sequence analysis and discussed. These results suggest a possible antagonistic interaction between PPV-Rec and PDV in plum clone C5.

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Stability of gene silencing-based resistance to Plum pox virus in transgenic plum (Prunus domestica L.) under field conditions

Transgenic Research ◽

10.1007/s11248-004-8702-3 ◽

2004 ◽

Vol 13 (5) ◽

pp. 427-436 ◽

Cited By ~ 57

Author(s):

Jean-Michel Hily ◽

Ralph Scorza ◽

Tadeusz Malinowski ◽

Barbara Zawadzka ◽

Michel Ravelonandro

Keyword(s):

Gene Silencing ◽

Field Conditions ◽

Plum Pox Virus ◽

Prunus Domestica

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Efficient Interaction between Arenavirus Nucleoprotein (NP) and RNA-Dependent RNA Polymerase (L) Is Mediated by the Virus Nucleocapsid (NP-RNA) Template

Journal of Virology ◽

10.1128/jvi.00103-15 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5734-5738 ◽

Cited By ~ 3

Author(s):

Masaharu Iwasaki ◽

Nhi Ngo ◽

Beatrice Cubitt ◽

Juan C. de la Torre

Keyword(s):

Rna Polymerase ◽

Gene Transcription ◽

Viral Genome ◽

Rna Replication ◽

Rna Dependent Rna Polymerase ◽

Rna Sequences ◽

L Protein ◽

Virus Rna

In this study, we document that efficient interaction between arenavirus nucleoprotein (NP) and RNA-dependent RNA polymerase (L protein), the twotrans-acting viral factors required for both virus RNA replication and gene transcription, requires the presence of virus-specific RNA sequences located within the untranslated 5′ and 3′ termini of the viral genome.

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