Pepper Ringspot Virus Research Articles

Production of antigens expressed in Nicotiana benthamiana plant and Escherichia coli for the SARS-CoV-2 IgG antibody detection by ELISA

The recent COVID-19 pandemic disclosed a critical shortage of diagnostic kits worldwide, emphasizing the urgency of utilizing all resources available for the development and production of diagnostic tests. Different heterologous protein expression systems can be employed for antigen production. This study assessed novel SARS-CoV-2 proteins produced by a transient expression system in Nicotiana benthamiana utilizing an infectious clone vector based on pepper ringspot virus (PepRSV). These proteins included the truncated S1-N protein (spike protein N-terminus residues 12–316) and antigen N (nucleocapsid residues 37–402). Two other distinct SARS-CoV-2 antigens expressed in Escherichia coli were evaluated: QCoV9 chimeric antigen protein (spike protein residues 449–711 and nucleocapsid protein residues 160–406) and QCoV7 truncated antigen (nucleocapsid residues 37–402). ELISAs using the four antigens individually and the same panel of samples were performed for the detection of anti-SARS-CoV-2 IgG antibodies. Sensitivity was evaluated using 816 samples from 351 COVID-19 patients hospitalized between 5 and 65 days after symptoms onset; specificity was tested using 195 samples collected before 2018, from domiciliary contacts of leprosy patients. Our findings demonstrated consistent test sensitivity, ranging from 85 % to 88 % with specificity of 97.5 %, regardless of the SARS-CoV2 antigen and the expression system used for production. Our results highlight the potential of plant expression systems as useful alternative platforms to produce recombinant antigens and for the development of diagnostic tests, particularly in resource-constrained settings.

Specific antibody production using recombinant proteins to elucidate seed transmission and nuclear localization of Coguvirus citrulli and Coguvirus henanense in radicles of watermelon crop

Watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2, both belonging to the genus Coguvirus (family Phenuiviridae), have been identified in watermelon plants in Brazil. To study tissue tropism and the potential for seed transmission of these viruses, we initially planned to produce specific antibodies. However, difficulties in isolating and propagating the virus in host plants hindered the purified virus preparations. To overcome this problem, the nucleocapsid (N) proteins of WCLaV-1 and −2 were produced using the pepper ringspot virus vector. The N protein genes and the vector backbone were prepared by (RT-)PCR and ligated by Gibson assembly. The constructs were agro-infiltrated in Nicotiana benthamiana plants. The expressed N proteins were purified and used for polyclonal antibody production. The specificity of both antibodies was confirmed by antigen-coating ELISA, tissue-blot immunobinding assay and Western blot. By antigen-coating ELISA demonstrated that WCLaV-1 showed 93.1% of seed-transmission, while WCLaV-2 showed only 17.8%. The N protein of WCLaV-1 was detected in the cytoplasm of the seed tissues. It was also found in the nuclei of the radicle, as confirmed by confocal microscopy. We concluded that the antibodies exhibited both a high titer and sufficient specificity for use in ELISA-based diagnostics and for subcellular localization study.

Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40–60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients’ sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

Development of a heterologous gene expression vector in plants based on an infectious clone of a tobravirus, pepper ringspot virus

AbstractHeterologous gene expression systems are important tools for biotechnology, especially due to the increasing demands for mass production of pharmaceutical proteins. In this study, we assessed the use of the infectious clone of a tobravirus, pepper ringspot virus (PepRSV), as a heterologous gene expression tool in Nicotiana benthamiana plants. The insert region of gene of interest was evaluated in the infectious clone of PepRSV using the reporter gene encoding the green fluorescent protein (GFP). The insertion of the GFP gene in the coat protein gene region resulted in a higher protein yield than the previous system. Then, the reporter gene was replaced by the xylanase gene, and enzymatically active xylanase was produced. This study provides an efficient tool for the production of heterologous proteins using a plant system.

Characterization of an infectious clone of pepper ringspot virus and its use as a viral vector.

The genus Tobravirus comprises three species: Tobacco rattle virus, Pea early-browning virus and Pepper ringspot virus. The genomes of tobraviruses consist of two positive-sense single-stranded RNA segments (RNA1 and RNA2). Infectious clones of TRV are extensively used as virus-induced gene-silencing (VIGS) vectors for studies of virus-host interactions and functions of plant genes. Complete infectious clones of pepper ringspot virus (PepRSV), the only tobravirus present in Brazil, however, have not yet been reported. Infectious clones will help to identify unique features of PepRSV RNA2 and provide another option for development of VIGS vectors. We constructed infectious clones based on two PepRSV isolates, CAM (RNA1 and RNA2) and LAV (RNA2). The cDNA constructs for both homologous (RNA1 and RNA2 of the CAM isolate) and heterologous (RNA1/CAM and RNA2/LAV) combinations were infectious in Nicotiana benthamiana plants. VIGS vector constructs with green fluorescent protein or phytoene desaturase genes inserted in RNA2 silenced the target genes. The systemic translocation of the PepRSV RNA1 construct alone (nonmultiple infection) was also confirmed in an N. benthamiana plant. These results are similar to those reported for tobacco rattle virus.

Subcellular localization of p29, a putative movement protein of pepper ringspot virus.

Pepper ringspot virus (PepRSV) is a member of the genus Tobravirus. It possesses a bipartite single-strand RNA genome in a positive-sense polarity. The p29 protein is encoded by RNA 1 and is presumed to be the movement protein (MP) of this virus. In this study, the intracellular distribution of the p29 protein was analyzed by confocal microscopy. Transient expression of the PepRSV p29 protein fused to green fluorescent protein was observed as punctate spots localized next to the cell wall. This protein partially co-localized with the eCFP-tagged tobacco mosaic virus 30K MP, which is known to associate with plasmodesmata. This result suggests that the p29 protein is most probably the movement protein for PepRSV.

Unique RNA 2 sequences of two Brazilian isolates of Pepper ringspot virus, a tobravirus.

Pepper ringspot virus (PepRSV) is a tobravirus reported only in Brazil. Here, the sequences of the complete RNA 2 segments and the 3' end of the RNA 1 genomic regions of two new isolates from tomato plants were analyzed. The main ORF encodes the CP gene as other tobraviruses and termed ORF 1 of RNA 2. The second ORF was found only in one of the new isolates, although this gene was absent in the type isolate, CAM (collected in the 1960's). Interestingly, this ORF 2 gene did not show any nucleotide and amino acid sequence similarities with known 2b genes of tobraviruses, an essential gene of tobraviruses for nematodes-transmission. The 5'UTR sequence of RNA 2 segment of CAM isolate was previously reported showing two impaired direct repeats; however, the direct-repeats were absent in these new isolates. An additional ORF was predicted upstream of the CP gene. This putative protein possessed a transmembrane domain similar to the ORFN1 of RNA 2 of Tobacco rattle virus SYM isolate, although there was no sequence similarity. This is the first report on the diversity of the RNA 2 sequences of PepRSV.

RNA2 of TRV SYM breaks the rules for tobravirus genome structure

Currently, all of the RNA2 molecules described for all of the more than thirty sequenced isolates of the three tobraviruses, Tobacco rattle virus (TRV), Pea early-browning virus (PEBV) and Pepper ringspot virus (PepRSV), have the virus coat protein (CP) gene located in the 5′ proximal position. However, sequencing of the RNA2 of the SYM isolate of TRV revealed that this isolate has a unique genome structure in which the virus CP gene is located in the central region of RNA2 downstream of three completely novel open reading frames (ORFN1, ORFN2 and ORFN3). An infectious clone of SYM RNA2 was constructed and mutations were introduced separately into each of the novel genes to interrupt their translation. However, none of the mutations resulted in any noticeable change in the ability of TRV RNA1 or RNA2 to replicate and move systemically in the leaves or roots of infected plants. In addition, individual expression of the novel ORFs either from a Potato virus X (PVX) vector or from a binary plasmid in Agrobacterium tumefaciens did not reveal any potential function.

ARGONAUTE2 mediates RNA-silencing antiviral defenses against Potato virus X in Arabidopsis.

RNA-silencing mechanisms control many aspects of gene regulation including the detection and degradation of viral RNA through the action of, among others, Dicer-like and Argonaute (AGO) proteins. However, the extent to which RNA silencing restricts virus host range has been difficult to separate from other factors that can affect virus-plant compatibility. Here we show that Potato virus X (PVX) can infect Arabidopsis (Arabidopsis thaliana), which is normally a nonhost for PVX, if coinfected with a second virus, Pepper ringspot virus. Here we show that the pepper ringspot virus 12K protein functions as a suppressor of silencing that appears to enable PVX to infect Arabidopsis. We also show that PVX is able to infect Arabidopsis Dicer-like mutants, indicating that RNA silencing is responsible for Arabidopsis nonhost resistance to PVX. Furthermore, we find that restriction of PVX on Arabidopsis also depends on AGO2, suggesting that this AGO protein has evolved to specialize in antiviral defenses.

Tobraviruses—plant pathogens and tools for biotechnology

The tobraviruses, Tobacco rattle virus (TRV), Pea early-browning virus (PEBV) and Pepper ringspot virus (PepRSV), are positive-strand RNA viruses with rod-shaped virus particles that are transmitted between plants by trichodorid nematodes. As a group, these viruses infect many plant species, with TRV having the widest host range. Recent studies have begun to dissect the interaction of TRV with potato, currently the most commercially important crop disease caused by any of the tobraviruses. As well as being successful plant pathogens, these viruses have become widely used as vectors for expression in plants of nonviral proteins or, more frequently, as initiators of virus-induced gene silencing (VIGS). Precisely why tobraviruses should be so effective as VIGS vectors is not known; however, molecular studies of the mode of action of the tobravirus silencing suppressor protein are shedding some light on this process.

First Report of the Stubby Root Nematode, Paratrichodorus minor, in Mainland China.

The stubby root nematode, Paratrichodorus minor (Colbran, 1956) Siddiqi, 1974, is an economically important species. It not only causes direct damage to the meristemic tissues of plants, but also can be a vector of Tobacco rattle virus and Pepper ringspot virus (2). It has been documented in Afghanistan, Argentina, Australia, Brazil, Canary Islands, Cuba, Egypt, Fiji, India, Israel, Ivory Coast, Japan, Java, Mauritania, New Zealand, Nicaragua, Philippines, Puerto Rico, Senegal, South Africa, Taiwan, Upper Volta, the United States, the former USSR, and Venezuela (1). During a recent investigation on Trichodoridae occurring in China, eight stubby root nematode populations were detected in Hainan, Yunnan, and Fujian provinces in the rhizospheres of eggplant (Solanum melongena), Chinaberry (Melia azedarach), lettuce (Lactuca sativa), longan (Dimocarpus longan), peach (Prunus persica), pear (Pyrus sp.), and walnut (Juglans regia) in soils ranging from clay to sand. The eight populations had similar morphological and molecular characters. The females were cigar shaped with rod-shaped vaginal sclerotization parallel to the longitudinal body axis with a quadrangular vagina in side view having a short vulval slit. The key morphometrics of females were consistent with those of P. minor: body length, 631 to 757 μm; body diameter, 27 to 43 μm; onchiostyle, 35 to 36 μm; a, 18 to 24; b, 4.9 to 5.5; v%, 53 to 55; length of vagina, 10.8 to 11.1 μm; and size of vaginal pieces, 1.7 to 1.9 μm. Males in P. minor are rare and were not found in any of the eight populations. Partial sequences of the 18s RNA gene of each population were amplified with forward primer 5'-AAA GAT TAA GCC ATG CAT G-3' (2), and reverse primer 5'-AGT CAA ATT AAG CCG CAG-3' (3), which yielded a PCR fragment of 1,153 bp. PCR products from all eight populations were sequenced and submitted to GenBank and assigned Accession Nos. GQ995703, GQ995704, GQ995705, GQ995706, GQ995707, GQ995708, GQ995709, and GQ995710. The sequences exhibited 99.8 to 100% similarity with those of P. minor isolates DQ345526, AM269897, AJ438052, AJ438053, and AJ438058 from GenBank. Morphological and molecular identification confirmed that all populations were P. minor. To our knowledge, this is the first report of P. minor in mainland China.

Development of a generic PCR detection method for tobraviruses

A generic PCR assay was developed to detect all known members of the genus Tobravirus, using two forward and three reverse PCR primers with little or no degeneracy. These primers amplified a fragment of the 194K RNA polymerase gene in the RNA-1 section of the bipartite tobravirus genome from plant tissue infected with Tobacco rattle virus, Pea-early browning virus and Pepper ringspot virus. All six primer combinations produced a band of the expected size when used with cDNA derived from plant tissue infected with each of the three viruses, following reverse-transcription using either a poly-T or specific primer. A single primer pair was selected for further optimisation of annealing temperature and MgCl2 concentration, and the sensitivity was assessed using serial dilutions of either virus-infected tissue or a purified plasmid containing the 194K RNA polymerase gene from TRV. This primer pair was able to detect all three tobravirus species using a single PCR protocol.

Efficient Expression of Foreign Proteins in Roots from Tobravirus Vectors

Viral vectors were constructed from infectious cDNA clones of each of the three tobraviruses, tobacco rattle virus (TRV), pea early-browning virus (PEBV), and pepper ringspot virus (PepRSV). RNA2 of each of the three viruses was modified to carry an additional coat protein subgenomic promoter and was used to express green fluorescent protein (GFP) when inoculated to plants. The tobravirus expression vectors have a wide host range and were able to express GFP in, for example, Nicotiana species, tomato, pea, arabidopsis, and sugar beet. The TRV vector was able to invade and express GFP very efficiently in roots, whereas the widely used PVX vector was not.

Tobravirus isolation and RNA extraction.

AbstractThe tobraviruses have a genome consisting of two positive-sense, single-stranded RNA molecules. The two genomic RNAs, RNA-1 and RNA-2, are encapsidated separately in rod-shaped particles with lengths of 180–215 nm (L particles) and 46–115 nm (S particles), respectively (Fig. 1) (1,2). Both the RNAs are encapsidated by a single species of coat protein (CP) with a mol wt of approx 23 kDa. Tobraviruses can be divided into three serologically distinct subgroups: tobacco rattle virus (TRV), which is the type member, pea early browning virus (PEBV), and pepper ringspot virus (PRV). In the field, these viruses are transmitted by soil-inhabiting nematodes of the family Trichodoridae, and certain isolates are also transmitted through the seed. Tobraviruses can infect a wide range of plant species including economically important crops such as potatoes and ornamental bulbs. Tobravirus structure and genome organization. (ai) Genome organization of RNA-1 of TRV isolate SYM. (aii) Genome organization of RNA-2 of various TRV isolates. (bi and bii) Genome organization of RNA-1 and RNA-2 of PEBV isolate SP5. RNA is shown as a horizontal line and the positions of the open reading frames (ORFs) are indicated by boxes. The numbers inside the boxes are the approximate mol wt in kDa of the proteins encoded by each ORF. The coat protein ORF is denoted by CP. The position of the readthrough termination codon on RNA-1 is indicated by an asterisk. KeywordsCoat ProteinSodium Phosphate BufferTobacco Mosaic VirusTobacco Rattle VirusThioglycollic AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The leaky UGA termination codon of tobacco rattle virus RNA is suppressed by tobacco chloroplast and cytoplasmic tRNAs(Trp) with CmCA anticodon.

RNA-1 molecules from tobacco rattle virus (TRV) and pea early-browning virus (PEBV), two members of the tobravirus group, have recently been shown to contain internal, in-frame UGA termination codons which are suppressed in vitro. Our results suggest that a UGA stop codon also exists in RNA-1 of pepper ringspot virus (PRV), another tobravirus. UGA suppression may therefore be a universal feature of the expression of tobravirus genomes. We have isolated two natural suppressor tRNAs from uninfected tobacco plants on the basis of their ability to promote readthrough over the leaky UGA codon of TRV RNA-1 in a wheat germ extract depleted of endogenous mRNAs and tRNAs. Their amino acid acceptance and nucleotide sequences identify the two UGA-suppressor tRNAs as chloroplast (chl) and cytoplasmic (cyt) tryptophan-specific tRNAs with the anticodon CmCA. These are the first UGA suppressor tRNAs to be identified in plants. They have several interesting features. (i) Chl tRNA(Trp) suppresses the UGA stop codon more efficiently than cyt tRNA(Trp). (ii) Chl tRNA(Trp) contains an A24:U11 pair in the D-stem as does the mutated Escherichia coli UGA-suppressor tRNA(Trp) which is a more active suppressor than wild-type tRNA(Trp). (iii) The suppressor activity of chl tRNA(Trp) is dependent on the nucleotides surrounding the stop codon because it recognizes UGA in the TRV context but not the UGA in the beta-globin context.

Plants transformed with a region of the 201-kilodalton replicase gene from pea early browning virus RNA1 are resistant to virus infection.

The 3' proximal portion of the gene encoding the 201-kDa putative replicase protein from the Tobravirus pea early browning virus (PEBV) can potentially be expressed separately as a 54-kDa protein. Nicotiana benthamiana plants transformed with the open reading frame (ORF) encoding the 54-kDa protein, designated 54K ORF, were resistant to infection by purified PEBV at inoculum doses of up to 1 mg/ml, the highest concentration tested. However, resistance was abolished by the introduction into the 54K ORF of mutations that would cause premature termination of translation. This suggests that the resistance mechanism requires the involvement of an intact 54-kDa protein. The 54K ORF-transformed plants were also resistant to infection by broad bean yellow band virus and an uncharacterized isolate of British PEBV (PGRO R) but were not resistant to infection by two other tobraviruses, pepper ringspot virus and the I6 isolate of tobacco rattle virus. Additionally, two variants of PEBV which overcame 54K ORF-mediated resistance have been isolated, the analysis of which might provide important information about both the resistance mechanism itself and the process of normal virus replication.

A model for the generation of tobacco rattle virus (TRV) anomalous isolates: pea early browning virus RNA-2 acquires TRV sequences from both RNA-1 and RNA-2

Comparison of the 5'-terminal sequences of several tobraviruses suggests that the RNA-2 molecule of the tobacco rattle virus (TRV) anomalous isolate TCM arose from pea early browning virus (PEBV) RNA-2 by acquisition of 3' and 5' sequences from TRV RNA-1 and RNA-2 molecules, respectively. We have identified a region of homology in the RNA-2 molecules of PEBV, TRV and pepper ringspot virus which could have facilitated this recombination.

Interactions between pepper ringspot virus and cylindrical inclusions of two potyviruses

Immunoelectron microscopy showed that cylindrical inclusions (CI) of the potyvirus potato virus Y (PVY) bound in addition to their homologous virions those of a co-infecting rod-shaped virus, pepper ringspot virus (PRV), in infected Nicotiana benthamiana leaf cells. The latter virus does not code for cylindrical inclusions and is cl assified as a Tobravirus. Virions of PRV were scattered throughout the cell cytoplasm and not associated with mitochondria in PVY + PRV double infections. Binding of PRV to mitochondria was disrupted in PVY + PRV infected cells. In double infections with a second potyvirus, tobacco etch virus (TEV), and PRV in N. benthamiana cells, TEV-CI bound homologous TEV virions but did not bind PRV. In contrast to PVY + PRV infections, virions of PRV attached end-on to mitochondrial limiting membranes in PRV-only and in TEV + PRV double infections. The results are interpreted to mean that there are differences in the PRV virion binding sites of PVY-CI and TEV-CI. In previous reports, potyviral CI have been nondiscriminating in binding virions or capsid proteins of other co-infecting rod-shaped viruses.

Intraviral homology and subgenomic RNAs of pepper ringspot virus

The Tobraviruses constitute a group of rod-shaped, bipartite, plus-stranded RNA viruses. We report on homologies between the two viral genomic RNA molecules of pepper ringspot virus (PRV) and on the subgenomic components generated from them during infection. It has previously been shown that the 3′-terminal 459 nucleotides of PRV RNA-1 and RNA-2 are identical. Here it is shown that there is a second homology between RNA-1 and a region of RNA-2, located at least 240 nucleotides downstream from its 5′ terminus. In agreement with strains in another Tobravirus group, we observe three subgenomic components in extracts of plants infected with PRV. Two of these, RNAs-1 a (1.6 kb) and −1 b (0.8 kb), are derived from RNA-1 (the larger genomic RNA). They are probably mRNAs for 29- and 16-kDa nonstructural virus proteins. The smaller genomic RNA, RNA-2, generates the subgenomic component RNA-2a (1.3–1.45 kb) which is probably an efficient capsid protein mRNA. Previously reported subgenomic components of 2.8 and 1.1 kb are shown to be electrophoretic artifacts. RNA-2a, but not 1a or 1b, is present in virion RNA preparations, indicating that it is the only subgenomic species to be encapsidated.

Unequal Variation in the Two Genome Parts of Tobraviruses and Evidence for the Existence of Three Separate Viruses

SUMMARY Hybridization experiments using complementary DNA copies showed that 14 tobravirus isolates could be divided into three groups, comprising ten of tobacco rattle virus (TRV) serotype I-II, one of TRV serotype III, and three of pea early-browning virus (PEBV), respectively. RNA from members of each group has much nucleotide sequence homology with RNA of other members of the same group but little or none with RNA from members of the two other groups. The ten TRV serotype I-II isolates share extensive sequences in their RNA-1 species, but show much diversity in their RNA-2 species and in the antigenic relatedness of their particles. However, homology was found between the RNA-2 sequences of isolates that are serologically closely related. Because strain CAM (TRV serotype III) is as different from isolates of TRV serotype I-II in nucleotide sequence, antigenic specificity and biological properties as is PEBV, it is now considered to be a separate virus and a return to its original name, pepper ringspot virus, is recommended.