Coat Protein Readthrough Research Articles

Molecular characterization of a novel benyvirus infecting wheat in China.

In this study, a novel positive single-stranded RNA (+ ssRNA) virus named wheat yellow stripe associated virus (WYSAV) was identified in wheat plants in China. Molecular characterization revealed that the complete genome of WYSAV is divided into two segments, RNA1 and RNA2, which are 6,460 and 4,935 nucleotides (nt) in length, excluding their respective poly(A) tails. RNA1 contains one large opening reading frame (ORF), encoding a replication-associated protein. RNA2 contains six ORFs, encoding a coat protein (CP), a coat protein readthrough domain protein (CP-RTD), triple gene block protein 1 (TGB1), triple gene block protein 2 (TGB2), triple gene block protein 3 (TGB3), and a cysteine-rich protein (CRP). Phylogenetic analysis showed that WYSAV is related to members of the genus Benyvirus in the family Benyviridae. Thus, WYSAV is proposed to be a new member of the genus Benyvirus. Wheat (Triticum aestivum L.) is one of the most important food crops and ranked third in the world in terms of production, only behind rice and maize [1]. During its growth cycle, wheat faces several biotic and abiotic stresses. Wheat soil-borne virus disease is an important disease that is difficult to control and causes severe yield loss in China each year [2]. The main pathogens causing wheat soil-borne virus disease are Chinese wheat mosaic virus (CWMV) and wheat yellow mosaic virus (WYMV), and their transmission vector is Polymyxa graminis [3-5]. Members of the viral family Benyviridae usually have two to five genomic RNA segments and are transmitted by root-infecting vectors belonging to the family "Plasmodiophoridae". Although few members of the family Benyviridae, of which beet necrotic yellow vein virus is the type member, have been identified [6], several recently identified viruses have been found to be phylogenetically related to benyviruses but are not classified as members of the family Benyviridae. These "unclassified benyviruses" include red clover RNA virus 1, Arceuthobium sichuanense virus 3, Dactylorhiza hatagirea beny-like virus, goji berry chlorosis virus [7], Guiyang benyvirus 1, Guiyang benyvirus 2, Mangifera indica latent virus [8], Rhizoctonia solani beny-like virus 1 [9], Sanya benyvirus 1 [10], and Sclerotium rolfsii beny-like virus 1 [11].In this study, we identified a novel + ssRNA virus in symptomatic leaf samples collected from cultivated wheat in the city of Zhumadian, Henan Province, China. We propose to name this virus "wheat yellow stripe associated virus" (WYSAV), and we have deposited its full-length sequence in the GenBank database under the accession numbers OQ547804 (RNA1) and OQ547805 (RNA2).

Recombinase Polymerase Amplification (RPA) for the Rapid Isothermal Detection of Spongospora subterranea f. sp. subterranea and Potato Mop-Top Virus

The amplification of specific nucleic acid sequences with high specificity and sensitivity is an essential technique for pathogen detection. Recombinase polymerase amplification (RPA) is a rapid isothermal amplification method. Here, we demonstrate the end-point and real-time detection of Spongospora subterranea f. sp. subterranea (Sss) using RPA and potato mop-top virus (PMTV) using reverse transcription (RT)-RPA. Oligonucleotide primers were designed for amplification that target the internal transcribed spacer 1 (ITS1) region and the coat protein readthrough (CP-RT) domain for the detection of Sss and PMTV, respectively. Our data showed that real-time RPA can detect 100 of Sss sporosori per gram of soil, while real-time RT-RPA could detect in ~1 ng total RNA of the PMTV-infected tuber. For Sss detection, the R2 value for real-time RPA and real-time PCR was 98.0% by linear regression analysis in the concentration range of 100–34,000 sporosori per gram of soil. The developed RPA assay here may provide a useful alternative tool for the rapid, simple and reliable detection of Sss and PMTV in diagnostic laboratories and in-field testing.

Characterization of burdock mottle virus, a novel member of the genus Benyvirus, and the identification of benyvirus-related sequences in the plant and insect genomes

The complete nucleotide sequence of the burdock mottle virus (BdMoV) isolated from an edible burdock plant (Arctium lappa) in Japan has been determined. BdMoV has a bipartite genome, whose organization is similar to RNA1 and RNA2 of benyviruses, beet necrotic yellow vein virus (BNYVV), beet soil-borne mosaic virus (BSBMV), and rice stripe necrosis virus (RSNV). BdMoV RNA1 (7038 nt) contains a single open reading frame (ORF) encoding a 249-kDa polypeptide that consists of methyl-transferase, helicase, papain-like protease, AlkB-like, and RNA-dependent RNA polymerase domains. The AlkB-like domain sequence is not present in the proteins encoded by other known benyviruses, but is found in replication-associated proteins of viruses mainly belonging to the families Alfaflexiviridae and Betaflexiviridae. BdMoV RNA2 (4315 nt) contains six ORFs that are similar to those of benyviruses: these are coat protein (CP), CP readthrough, triple gene block movement and cysteine-rich proteins. Phylogenetic analyses showed that BdMoV is more closely related to BNYVV and BSBMV than to RSNV. Database searches showed that benyvirus replicase-related sequences are present in the chromosomes of a chickpea plant (Cicer arietinum) and a blood-sucking insect (Rhodnius prolixus). Some other benyvirus-related sequences are found in the transcriptome shotgun libraries of a few species of plants and a bark beetle. Our results show that BdMoV is a distinct species of the genus Benyvirus and that ancestral and extant benyviruses may have infected or currently infect a wide range of hosts, including plants and insects.

The beet virus Q coat protein readthrough domain is longer than previously reported, with two transmembrane domains

Ten beet virus Q (BVQ) strains from six different countries were sequenced to characterize the readthrough (RT) domain of the coat protein (CP). The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are FM244643-FM244652. With three nucleotide additions of 5, 285 and 1 nt, the common RT of 76 kDa was found to be longer than the single reference available to date (35 kDa). It is hypothesized that multiple inoculation cycles on Chenopodium quinoa were responsible for these three deletions in the C-terminal part of the BVQ RNA-2 previously described. Two putative transmembrane domains, TM1 and TM2, were predicted in the consensus amino acid sequence of the ten BVQ strains, and the putative BVQ TM2 was aligned with that of potato mop-top virus.

Evidence That RNA Silencing-Mediated Resistance to Beet necrotic yellow vein virus Is Less Effective in Roots Than in Leaves

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.

Cloning and expression of the Chinese wheat mosaic virus RNA2 coat protein readthrough and 19 ku cysteinerich domains and localization of these proteins

The 5′-terminal (RTn) and 3′-terminal (RTc) halves of the coat protein readthrough domain and the 19 ku cysteine-rich protein of Chinese wheat mosaic virus (CWMV) were amplified by RT-PCR, cloned and expressed inE. coli. Antisera and monoclonal antibodies against these proteins were prepared by immunising these purified proteins to mice. Detection of RTn, RTc and 19 ku proteins in CWMV infected wheat sap and leaf tissue indicated that the RTn and RTc proteins were distributed on the surface of virus particles whereas the 19 ku protein was in the cytoplasm of the infected wheat cells.

Cloning and expression of the Chinese wheat mosaic virus RNA2 coat protein readthrough and 19 ku cysteine-rich do-mains and localization of these proteins

Cloning and expression of the Chinese wheat mosaic virus RNA2 coat protein readthrough and 19 ku cysteine-rich do-mains and localization of these proteins

Plant virus transmission by plasmodiophorid fungi is associated with distinctive transmembrane regions of virus-encoded proteins.

Computer analysis of published sequence data has consistently identified two complementary transmembrane domains in the coat protein readthrough domains of benyviruses, furoviruses and pomoviruses and in the P2 proteins of bymoviruses. These viruses differ in genome organisation but are all transmitted by plasmodiophorid fungi. The second domain is absent or disrupted in naturally-occurring deletion mutants that cannot be fungally-transmitted. In a non-transmissible substitution mutant of Beet necrotic yellow vein virus [Tamada et al. (1996) J Gen Virol 77: 1359-1367], the alignment of the helices is disrupted. From conserved patterns detected in transmembrane helix sequences and calculated relative helix tilts, structural arrangements consistent with tight packing of transmembrane helices were identified. These included ridge/groove arrangements between the two helices and strong electrostatic associations at the interfacial regions of the membrane. The data strongly suggest that these transmembrane helices facilitate the movement of virus particles across the fungal membrane.

Sequence of a Second Isolate of Chinese Wheat Mosaic Furovirus

The nucleotide sequences of the two RNAs of an isolate of Chinese wheat mosaic furovirus (CWMV) from Rongcheng, Shandong province, China, where the infected wheat crop developed particularly severe symptoms, were determined. The two RNAs consisted of 7147 and 3564 nucleotides, and shared 95.5% identity to those of the Yantai CWMV isolate previously described. Differences were greatest in the coat protein readthrough domain and the 19 kDa cysteine‐rich protein (both on RNA2). A CGxxH amino acid sequence motif common to the cysteine‐rich proteins of the genera Furovirus, Hordeivirus, Tobravirus and Pecluvirus was detected.

Molecular characterization of a new furovirus mainly infecting rye.

The complete or almost complete nucleotide sequences were determined for the two RNAs of three different sources of a new furovirus which mainly infects rye and for which the name Soil-borne rye mosaic furovirus (SBRMV) is proposed. The genome organization of this virus is virtually identical to that of Soil-borne wheat mosaic furovirus (SBWMV). However, SBRMV and SBWMV differ considerably in the nucleotide sequences of their genomes and in the derived amino acid sequences of their putative gene products. The sequences of the three sources of SBRMV (two from rye and one from wheat) differ between each other in various parts of their genomes by 1% to 10%. Larger differences were found in the readthrough part of the coat protein readthrough protein. There are no indications that the relationship of the C source from wheat to the other two sources from rye is more distant than the relationship between the two sources from rye. The 3'UTRs of both SBRMV RNAs are remarkable in having a predicted upstream pseudoknot domain (UPD) with seven pseudoknots. The same number was also predicted for the UPD of SBWMV RNA 1 whereas the much shorter UDP of SBWMV RNA 2 may form only four pseudoknots.

Sequences of European Wheat Mosaic Virus and Oat Golden Stripe Virus and Genome Analysis of the Genus Furovirus

The complete nucleotide sequences of both RNAs of oat golden stripe virus (OGSV) and a wheat-infecting furovirus isolate from France, previously thought to be soil-borne wheat mosaic virus (SBWMV), have been determined. Both viruses had a similar genomic organisation to SBWMV and Chinese wheat mosaic virus, the two other furoviruses previously sequenced but had <70% nucleotides identical to them. The French isolate has been named European wheat mosaic virus (EWMV). Phylogenetic analyses supported the recognition of these isolates as distinct viruses in the genus Furovirus. Analysis of the coat protein readthrough domain on RNA2 of all furoviruses strongly predicts two mutually compatible conserved transmembrane domains that may be significant for fungus transmission. The second of these regions is eliminated by a deletion in the isolate of OGSV studied. Leaky opal (UGA) stop codons occur on both RNAs of all four furoviruses characterised and, in common with most other leaky opal codons identified in plant viruses, they are followed by a CGG codon.

Aphid Transmission and Systemic Plant Infection Determinants of Barley Yellow Dwarf Luteovirus-PAV are Contained in the Coat Protein Readthrough Domain and 17-kDa Protein, Respectively

Proteins encoded by open reading frames (ORF) 3, 4, and 5 of the barley yellow dwarf luteovirus genome are translated from a single subgenomic RNA. The structural proteins are encoded by ORF 3 (coat protein) and ORF 5 (readthrough domain) and contain undefined domains that regulate the movement of virus through aphid vectors. The biological function of the nonstructural 17-kDa protein encoded by ORF 4 is unknown. A complementation method was employed to test the ability of barley yellow dwarf virions carrying mutations within the readthrough domain and the 17-kDa protein to be transmitted by aphids and to cause systemic infections in plants. We show that the readthrough domain is required for aphid transmission; however, it is not required for virus to be taken up by aphid hindgut cells and released into the hemocoel. The circulative pathway of luteoviruses in aphid vectors requires that virus be actively transported from the hemolymph into the salivary system. Thus, it appears that the readthrough domain is required for transport of virus through membranes of the aphid salivary glands. Furthermore, the readthrough domain was not required for systemic infection of plants, but did influence the accumulation of virus in infected plants. The 17-kDa protein is required for the systemic infection of plants.

Sequence analysis and gene content of potato mop-top virus RNA 3: Further evidence of heterogeneityin the genome organization of furoviruses

The complete sequence of the 2315 nucleotides in RNA 3 of potato mop-top furovirus (PMTV) isolate T was obtained by analysis of cDNA clones and by direct RNA sequencing. The sequence contains an open reading frame for the coat protein (20K) terminated by an amber codon, followed by an in-phase coding region for an additional 47K. PMTV therefore resembles soil-borne wheat mosaic (SBWMV) and beet necrotic yellow vein (BNYVV) viruses (two other fungus-transmitted viruses with rod-shaped particles) in having a coat protein-readthrough product. Comparison of the 3′ untranslated regions of PMTV RNA 2 and RNA 3 reveals a long conserved block of 150 nucleotides, which contains two repeated sequences and has the potential to form consecutive pseudoknot structures. PMTV RNA 3 ends a few nucleotides downstream of this conserved block, but RNA 2 extends for a further 140 nucleotides, which can potentially form a 3′-terminal tRNA-like structure similar to those in the RNA species of SBWMV, tymoviruses, and some tobamoviruses. PMTV coat protein has amino acid sequence identities of 30 and 28% with SBWMV and BNYVV coat proteins, respectively, and apparent structural similarities with tobacco mosaic virus coat protein. The coat protein readthrough domains of PMTV, SBWMV, and BNYVV have shared residues throughout their length but no extended sequences are conserved. The presence in RNA 3 of coding sequences for only the. coat protein and readthrough domain distinguishes PMTV from SBWMV and BNYVV, both of which have them in RNA 2 along with one or more other genes. Comparison of the genomes of PMTV, BNYVV, and SBWMV shows that furoviruses exhibit considerable heterogeneity in genome organization.

In Vivo Expression and Mutational Analysis of the Barley Yellow Dwarf Virus Readthrough Gene

The barley yellow dwarf virus (BYDV) coat protein gene is separated from an adjacent downstream open reading frame (ORF) by a single termination codon. Immunological analysis of this downstream "readthrough" region reveals multiple coat protein readthrough products. A full-length 72-kDa (P72) coat protein-readthrough fusion product is detected in total lysates from infected cells. However, purified aphid transmissible virions contain only a 50-kDa (P50) coat protein-readthrough product. Virion-associated P50 lacks the C-terminal domain predicted by its ORF sequence. A separate 33-kDa polypeptide (P33) corresponding to the readthrough C-terminus domain is detected in the crude cellular membrane fraction. Site-directed and deletion mutational analysis demonstrate that the readthrough ORF is dispensable for BYDV replication and virion accumulation in protoplasts. In contrast, a mutant which results in a continuous fusion product of coat and readthrough sequences is not viable. Point mutations were used to map regions required for P50 and P72 synthesis. A model explaining the relationships between the three forms of the readthrough polypeptides is proposed.

Changes in the Amino Acid Sequence of the Coat Protein Readthrough Domain of Potato Leafroll Luteovirus Affect the Formation of an Epitope and Aphid Transmission

Potato leafroll luteovirus (PLRV) is transmitted naturally by aphids, but two isolates (15 and V) are known to be only poorly transmissible (PAT); these isolates are also distinct in that their particles lack an epitope present in transmissible (HAT) isolates of PLRV (Tamada et al., Ann. Appl. Biol. 104, 107-116, 1984; Massalski and Harrison, J. Gen. Virol. 68, 1813-1821, 1987). Virus cultures propagated vegetatively for several years in potato plants from the source of isolate V were shown still to be poorly transmissible by Myzus parsicae. Moreover, when isolate V was transmitted by aphids, the subisolates obtained (V4 and V31) were no more readily transmissible than was the stock culture PLRV-V. The subisolates were also unchanged antigenically by the transmission. Thus the inefficient transmission had not restored the properties of HAT isolates. Sequence comparisons among the coat proteins and the readthrough domains of HAT and PAT Scottish isolates suggested that either or both of two amino acid changes in the C-terminal part of the readthrough protein are responsible for the poor transrmission and the loss of an epitope. The readthrough protein is therefore thought to play a role in the circulative aphid transmission of PLRV.

Evidence that the 75K readthrough protein of beet necrotic yellow vein virus RNA-2 is essential for transmission by the fungus Polymyxa betae

Two mutant strains of beet necrotic yellow vein virus (BNYVV) containing deletion mutants of RNA-2 were produced during serial passage in mechanically inoculated Tetragonia expansa leaves. The mutant strains were referred to as S-0a (RNA-1 + 2a) and G-0b (RNA-1 + 2b). RNA-2a and RNA-2b were about 4.3 kb and 4.2 kb in length, respectively, whereas normal sized RNA-2 was about 4.8 kb in length. In vitro translation and immunoblot analysis showed that RNA-2, RNA-2a and RNA-2b all directed synthesis of the coat protein (Mr 22K). However, whereas wild-type RNA-2 also directed the synthesis of a coat protein readthrough protein with an Mr of 83K (predicted Mr 75K), RNA-2a and RNA-2b directed the production of readthrough proteins with MrS of 67K and 58K, respectively. This suggests that the deleted regions of RNA-2a and RNA-2b occur within the second open reading frame, which encodes a polypeptide of Mr 54K, which is translated by readthrough of the coat protein cistron. After the addition of wild-type RNA-3 and RNA-4 to all the strains, the mutant strains could not be transmitted by Polymyxa betae zoospores produced from either zoosporangia or resting spores, whereas the wild-type strains were readily transmitted. These results indicate that the 75K readthrough protein encoded by RNA-2 is essential for the transmission of BNYVV by P. betae.