Viruses in subgroup 2 of the genus Ilarvirus share both serological relationships and characteristics at the molecular level

Scott, S. W.; Zimmerman, M. T.; Ge, Xijin

doi:10.1007/s00705-003-0148-z

Cited by 35 publications

(13 citation statements)

References 24 publications

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“…Comparative sequence analysis demonstrates that the 3Ј-terminal AUGC sequence is highly conserved among AMV and ilarvirus RNAs (15), although 3Ј-terminal AAGC (31,32) and AUGA (PubMed nucleotide entry AF434922) have been reported.…”

Section: Discussionmentioning

confidence: 99%

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Rocheleau

Petrillo

Guogas

et al. 2004

J Virol

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The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5 hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3 RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3 hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.Alfalfa mosaic virus (AMV) has a number of exceptional features that make it a useful system for studying RNA-protein interactions (reviewed in references 6 and 17). The three positive-sense genomic AMV RNAs (RNAs 1 to 3) and the subgenomic coat protein mRNA (RNA 4) are packaged individually into four bacilliform, aphid-transmitted particles. The viral genomic RNAs are infectious only in the presence of the viral coat protein (7), which binds specifically to the RNA 3Ј termini. Unlike the arginine-rich RNA binding domains of the human immunodeficiency virus Tat and Rev proteins, the AMV coat protein RNA binding domain is lysine rich (3); however, it includes an arginine residue whose position and side chain identity are crucial for specific, high-affinity RNA binding (2). The AMV RNAs lack a poly(A) tail and are further distinguished from closely related plant viruses (e.g., brome mosaic virus) by the absence of a canonical CCA 3Ј terminus and the inability to be charged by aminoacyl synthetases, both of which are defining features of tRNA-like ends (12).The relationship between the formation of the AMV coat protein-RNA complex and functional activity in viral RNA translation, replication, and assembly is poorly understood. Previous biochemical characterization has defined specific binding determinants in both the RNA and coat protein (1,3,14,15,18,(26)(27)(28...

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Section: Discussionmentioning

confidence: 99%

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Rocheleau

Petrillo

Guogas

et al. 2004

J Virol

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“…Specific reverse transcription-polymerase chain reaction (RT-PCR) tests have been used widely for routine diagnosis of individual Ilarvirus species (Parakh et al, 1994; MacKenzie et al, 1997; Pallás et al, 1998; Scott et al, 2003). However, the infection of some plant hosts by several Ilarvirus species complicates species identification and necessitates the use of several different specific RT-PCR tests for detection.…”

Section: Introductionmentioning

confidence: 99%

Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus

et al. 2017

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The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV) was the most frequently detected Ilarvirus, occurring in 48 of the 61 Ilarvirus-positive trees and Prune dwarf virus (PDV) and Apple mosaic virus (ApMV) were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus-like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus-like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus-like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the need for a standardized approach to accurately determine what constitutes an active, viable virus infection after detection by molecular based methods.

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“…Ilarviruses have been divided into subgroups based on serological affinities (Fauquet et al , 2005), and the serological relationships have been shown to extend to the molecular properties of some of the viruses within these subgroups (Scott et al , 2003). There are instances in the literature where serological relationships between ilarviruses have not been clear‐cut or are confusing (Scott et al , 2003; Tzanetakis & Martin, 2005), and molecular comparisons have proven to be more reliable in distinguishing viruses than serological data. The phylogenetic trees based on sequence data show clearly that BCRV should be grouped within subgroup 1 of the genus Ilarvirus (Fig.…”

Section: Discussionmentioning

confidence: 99%

Purification and properties of blackberry chlorotic ringspot, a new virus species in subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK

Jones¹,

McGavin²,

Gepp³

et al. 2006

Annals of Applied Biology

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A mechanically transmissible virus was isolated from Bedford Giant blackberry plants showing chlorotic mottling and ringspot symptoms growing in Scotland. It infected several herbaceous test plants, many of them symptomlessly. This virus was also transmitted to several Rubus species and cultivars by graft inoculation with scions from the field-infected Bedford Giant plant. Most grafted plants were infected symptomlessly, but Himalaya Giant blackberry and the hybrid berry Tayberry developed symptoms similar to those in the infected Bedford Giant plant. In the sap of infected Chenopodium quinoa, the virus lost infectivity when diluted 10 24 but not 10 23 , after 6 h and 48 h when kept at 20°C and 4°C, respectively, but was infective for more than 8 days when kept at 215°C. Preparations of purified virus from infected C. quinoa or spinach sedimented as three major nucleoprotein components and consisted of quasiisometric particles that varied in size from 24 to 32 nm in diameter and that were not penetrated by negative stain. Such virus particle preparations contained a major polypeptide of ca 28 kDa and three single-stranded RNA species of estimated size 3.2, 2.8 and 2.1 kb. The complete sequence of the largest RNA (RNA 1, 3478 nt) and the partial sequence of the other RNAs (1863 and 2102 nt long, respectively) were determined and compared with sequences in databases. These findings, together with the biological and biochemical properties of this virus, indicate that it should be regarded as a distinct species in subgroup 1 of the genus Ilarvirus even though it was serologically unrelated to existing members of this subgroup. The virus showed a very distant serological relationship with prune dwarf virus (PDV) but differed significantly from it in the amino acid sequence of its coat protein, experimental host range and symptomatology and was unrelated to PDV at the molecular level. The virus, tentatively named blackberry chlorotic ringspot virus, is therefore a newly described virus and the first ilarvirus found naturally infecting Rubus in the UK.

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Viruses in subgroup 2 of the genus Ilarvirus share both serological relationships and characteristics at the molecular level

Cited by 35 publications

References 24 publications

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus

Purification and properties of blackberry chlorotic ringspot, a new virus species in subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK

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