HC-Pro Protein Research Articles

Mutating the arginine residue within the FRNK motif of telosma mosaic virus (TelMV) HC-Pro protein attenuates viral infection and confers effective protection against TelMV in passion fruit (Passiflora edulis).

Telosma mosaic virus (TelMV, Potyvirus, Potyviridae) is an emerging viral pathogen that threatens passion fruit plantations worldwide. However, an efficient strategy for controlling such a virus is not yet available. Cross protection is a phenomenon in which pre-infection of a plant with one mild strain prevents or delays subsequent infection by the same or closely related virus. HC-Pro is the potyviral encoded multifunctional protein involved in several steps of viral infection, including multiplication, movement, transmission and RNA silencing suppression. In this study, we tested whether it is possible to generate attenuated viral strains capable of conferring protection against severe TelMV infection by manipulating the HC-Pro gene. By introducing point mutation into the conserved motif FRNK of HC-Pro that is essential for RNA silencing suppression, we have successfully obtained three attenuated mutants of TelMV (R181K, R181D, and R181E, respectively). These attenuated TelMV mutants could systemically infect passion fruit plants without noticeable symptoms. Pre-inoculation of one of these attenuated mutants confers efficient protection against subsequent infection by severe TelMV strain. Moreover, we demonstrated that the HC-Pros harbored by the attenuated mutants exhibit reduced RNA silencing suppression activity in Nicotiana benthamiana leaves. The attenuated TelMV mutants developed in this study that are suitable for cross protection offer a practical, powerful tool to fight against TelMV for sustainable passion fruit production. © 2024 Society of Chemical Industry.

A new point mutation in the HC-Pro of potato virus Y is involved in tobacco vein necrosis.

Tobacco vein necrosis (TVN) is a complex phenomenon regulated by different genetic determinants mapped in the HC-Pro protein (amino acids N330, K391 and E410) and in two regions of potato virus Y (PVY) genome, corresponding to the cytoplasmic inclusion (CI) protein and the nuclear inclusion protein a-protease (NIa-Pro), respectively. A new determinant of TVN was discovered in the MK isolate of PVY which, although carried the HC-Pro determinants associated to TVN, did not induce TVN. The HC-Pro open reading frame (ORF) of the necrotic infectious clone PVY N605 was replaced with that of the non-necrotic MK isolate, which differed only by one amino acid at position 392 (T392 instead of I392). The cDNA clone N605_MKHCPro inoculated in tobacco induced only weak mosaics at the systemic level, demostrating that the amino acid at position 392 is a new determinant for TVN. No significant difference in accumulation in tobacco was observed between N605 and N605_MKHCPro. Since phylogenetic analyses showed that the loss of necrosis in tobacco has occurred several times independently during PVY evolution, these repeated evolutions strongly suggest that tobacco necrosis is a costly trait in PVY.

The Fifth Residue of the Coat Protein of Turnip Mosaic Virus Is Responsible for Long-Distance Movement in a Local-Lesion Host and Aphid Transmissibility in a Systemic Host.

HC-Pro and coat protein (CP) genes of a potyvirus facilitate cell-to-cell movement and are involved in the systemic movement of the viruses. The interaction between HC-Pro and CP is mandatory for aphid transmission. Two turnip mosaic virus (TuMV) isolates, RC4 and YC5, were collected from calla lily plants in Taiwan. The virus derived from the infectious clone pYC5 cannot move systemically in Chenopodium quinoa plants and loses aphid transmissibility in Nicotiana benthamiana plants, like the initially isolated virus. Sequence analysis revealed that two amino acids, P5 and A206, of YC5 CP uniquely differ from RC4 and other TuMV strains. Recombination assay and site-directed mutagenesis revealed that the fifth residue of leucine (L) at the N-terminal region of the CP (TuMV-RC4), rather than proline (P) (TuMV-YC5), is critical to permit the systemic spread in C. quinoa plants. Moreover, the single substitution mutant YC5-CPP5L became aphid transmissible, similar to RC4. Fluorescence microscopy revealed that YC5-GFP was restricted in the petioles of inoculated leaves, whereas YC5-CPP5L-GFP translocated through the petioles of inoculated leaves, the main stem, and the petioles of the upper uninoculated leaves of C. quinoa plants. In addition, YC5-GUS was blocked at the basal part of the petiole connecting to the main stem of the inoculated C. quinoa plants, whereas YC5-CPP5L-GFP translocated to the upper leaves. Thus, a single amino acid, the residue L5 at the N-terminal region right before the 6DAG8 motif, is critical for the systemic translocation ability of TuMV in a local lesion host and for aphid transmissibility in a systemic host.

P3 and NIa-Pro of Turnip Mosaic Virus Are Independent Elicitors of Superinfection Exclusion.

Superinfection exclusion (SIE) is an antagonistic interaction between identical or closely related viruses in host cells. Previous studies by us and others led to the hypothesis that SIE was elicited by one or more proteins encoded in the genomes of primary viruses. Here, we tested this hypothesis using Turnip mosaic virus (TuMV), a member of the genus Potyvirus of the family Potyviridae, with significant economic consequences. To this end, individual TuMV-encoded proteins were transiently expressed in the cells of Nicotiana benthamiana leaves, followed by challenging them with a modified TuMV expressing the green fluorescent protein (TuMV-GFP). Three days after TuMV-GFP delivery, these cells were examined for the replication-dependent expression of GFP. Cells expressing TuMV P1, HC-Pro, 6K1, CI, 6K2, NIa-VPg, NIb, or CP proteins permitted an efficient expression of GFP, suggesting that these proteins failed to block the replication of a superinfecting TuMV-GFP. By contrast, N. benthamiana cells expressing TuMV P3 or NIa-Pro did not express visible GFP fluorescence, suggesting that both of them could elicit potent SIE against TuMV-GFP. The SIE elicitor activity of P3 and NIa-Pro was further confirmed by their heterologous expression from a different potyvirus, potato virus A (PVA). Plants systemically infected with PVA variants expressing TuMV P3 or NIa-Pro blocked subsequent infection by TuMV-GFP. A +1-frameshift mutation in P3 and NIa-Pro cistrons facilitated superinfection by TuMV-GFP, suggesting that the P3 and NIa-Pro proteins, but not the RNA, are involved in SIE activity. Additionally, deletion mutagenesis identified P3 amino acids 3 to 200 of 352 and NIa-Pro amino acids 3 to 40 and 181 to 242 of 242 as essential for SIE elicitation. Collectively, our study demonstrates that TuMV encodes two spatially separated proteins that act independently to exert SIE on superinfecting TuMV. These results lay the foundation for further mechanistic interrogations of SIE in this virus.

A Novel Isolate of Bean Common Mosaic Virus Isolated from Crownvetch (Securigera varia L. Lassen)

Bean common mosaic virus from the genus Potyvirus has a wide range of hosts and a very negative impact on cultivated crops from the genus Phaseolus. The risk of viral infection of economically important crops increases even if the carriers of the virus are related plant species growing on agroecological interfaces. Such plant species have emerged as new hosts for BCMV, usually harboring novel genetic variants of the virus. A novel genetic variant of BCMV was isolated from a symptomatic crownvetch plant, where the presence of this virus was confirmed via Western blot analysis and via amino acid identities in peptide fragments of CI, HC-pro, and CP proteins using the nanoLC-ESI-Q-TOF. The novel BCMV SVK isolate differed from the most genetically similar one in 0.91% of nucleotides and 1.55% of amino acids. The highest number of amino acid substitutions (8.8% of amino acids) was in the P1 protein, followed by CP (2.44% of amino acids). Minor substitutions were in Hc-pro, CI, and Nib proteins. The symptomatic crownvetch plant was confirmed as a new host and carrier of the novel BCMV isolate.

Leek Yellow Stripe Virus Can Adjust for Host Adaptation by Trimming the N-Terminal Domain to Allow the P1 Protein to Function as an RNA Silencing Suppressor.

In Japan, the P1 protein (S-type) encoded by leek yellow stripe virus (LYSV) isolates detected in Honshu and southward is shorter than the P1 (N-type) of LYSV isolates from garlic grown in Hokkaido due to a large deletion in the N-terminal half. In garlic fields in Hokkaido, two types of LYSV isolate with N- and S-type P1s are sometimes found in mixed infections. In this study, we confirmed that N- and S-type P1 sequences were present in the same plant and that they belong to different evolutionary phylogenetic groups. To investigate how LYSV with S-type P1 (LYSV-S) could have invaded LYSV with N-type P1 (LYSV-N)-infected garlic, we examined wild Allium spp. plants in Hokkaido and found that LYSV was almost undetectable. On the other hand, in Honshu, LYSV-S was detected at a high frequency in Allium spp. other than garlic, suggesting that the LYSV-S can infect a wider host range of Allium spp. compared to LYSV-N. Because P1 proteins of potyviruses have been reported to promote RNA silencing suppressor (RSS) activity of HC-Pro proteins, we analyzed whether the same was true for P1 of LYSV. In onion, contrary to expectation, the P1 protein itself had RSS activity. Moreover, the RSS activity of S-type P1 was considerably stronger than that of N-type P1, suggesting that LYSV P1 may be able to enhance its RSS activity when the deletion is in the N-terminal half and that acquiring S-type P1 may have enabled LYSV to expand its host range.

Susceptibility factor StEXA1 interacts with StnCBP to facilitate potato virus Y accumulation through the stress granule-dependent RNA regulatory pathway in potato.

Plant viruses recruit multiple host factors for translation, replication, and movement in the infection process. The loss-of-function mutation of the susceptibility genes will lead to the loss of susceptibility to viruses, which is referred to as ‘recessive resistance’. Essential for potexvirus Accumulation 1 (EXA1) has been identified as a susceptibility gene required for potexvirus, lolavirus, and bacterial and oomycete pathogens. In this study, EXA1 knockdown in potato (StEXA1) was found to confer novel resistance to potato virus Y (PVY, potyvirus) in a strain-specific manner. It significantly compromised PVYO accumulation but not PVYN:O and PVYNTN. Further analysis revealed that StEXA1 is associated with the HC-Pro of PVY through a member of eIF4Es (StnCBP). HC-ProO and HC-ProN, two HC-Pro proteins from PVYO and PVYN, exhibited strong and weak interactions with StnCBP, respectively, due to their different spatial conformation. Moreover, the accumulation of PVYO was mainly dependent on the stress granules (SGs) induced by StEXA1 and StnCBP, whereas PVYN:O and PVYNTN could induce SGs by HC-ProN independently through an unknown mechanism. These results could explain why StEXA1 or StnCBP knockdown conferred resistance to PVYO but not to PVYN:O and PVYNTN. In summary, our results for the first time demonstrate that EXA1 can act as a susceptibility gene for PVY infection. Finally, a hypothetical model was proposed for understanding the mechanism by which StEXA1 interacts with StnCBP to facilitate PVY accumulation in potato through the SG-dependent RNA regulatory pathway.

Genetic diversity of potato virus Y potato isolates from Shandong province, China

Potato virus Y (PVY) is one of the most important pathogenic viruses affecting potato production. Despite the importance of the virus, little information on PVY genetic diversity on potato plants in the central plain of China is available. In this study, we tested 584 symptomatic leaf samples collected from the main potato growing areas in Shandong province. Results showed that 88.2% of them were PVY positive. Sequence analysis showed that 51 PVY isolates have N-type HCpro and O-type coat protein; five samples were infected with different genotypes. Analysis of nearly complete PVY genome sequences from six field isolates and one isolate from seed potato revealed that they were close to the PVYNTN-NW SYR II group; however, they formed a separate group PVYNTN-NW CN. Moreover, our results provided evidence that PVYNTN-NW CN group isolates CN:TZ1:17 and CN:DY1:16 have obtained the ability to infect pepper systemically.

The Potyviruses: An Evolutionary Synthesis Is Emerging.

In this review, encouraged by the dictum of Theodosius Dobzhansky that “Nothing in biology makes sense except in the light of evolution”, we outline the likely evolutionary pathways that have resulted in the observed similarities and differences of the extant molecules, biology, distribution, etc. of the potyvirids and, especially, its largest genus, the potyviruses. The potyvirids are a family of plant-infecting RNA-genome viruses. They had a single polyphyletic origin, and all share at least three of their genes (i.e., the helicase region of their CI protein, the RdRp region of their NIb protein and their coat protein) with other viruses which are otherwise unrelated. Potyvirids fall into 11 genera of which the potyviruses, the largest, include more than 150 distinct viruses found worldwide. The first potyvirus probably originated 15,000–30,000 years ago, in a Eurasian grass host, by acquiring crucial changes to its coat protein and HC-Pro protein, which enabled it to be transmitted by migrating host-seeking aphids. All potyviruses are aphid-borne and, in nature, infect discreet sets of monocotyledonous or eudicotyledonous angiosperms. All potyvirus genomes are under negative selection; the HC-Pro, CP, Nia, and NIb genes are most strongly selected, and the PIPO gene least, but there are overriding virus specific differences; for example, all turnip mosaic virus genes are more strongly conserved than those of potato virus Y. Estimates of dN/dS (ω) indicate whether potyvirus populations have been evolving as one or more subpopulations and could be used to help define species boundaries. Recombinants are common in many potyvirus populations (20%–64% in five examined), but recombination seems to be an uncommon speciation mechanism as, of 149 distinct potyviruses, only two were clear recombinants. Human activities, especially trade and farming, have fostered and spread both potyviruses and their aphid vectors throughout the world, especially over the past five centuries. The world distribution of potyviruses, especially those found on islands, indicates that potyviruses may be more frequently or effectively transmitted by seed than experimental tests suggest. Only two meta-genomic potyviruses have been recorded from animal samples, and both are probably contaminants.

Genomic sequence variability of an Italian Zucchini yellow mosaic virus isolate

Zucchini yellow mosaic virus (ZYMV) was first reported in Italy, but no Italian isolates have been sequenced so far. A ZYMV isolate, designated Z104, was isolated from a zucchini plant grown in the open field in Piedmont, Italy, and showing severe disease symptoms. We report the full-length genome sequence of Z104 as determined by Sanger sequencing and small (s)RNA deep sequencing. The latter technique was useful to confirm the isolate sequence, that was deposited in the NCBI database with the accession number MK956829 and to investigate the sequence variability of Z104 isolate. The genome of 9611 nucleotides (nt) consisted of an open reading frame of 9243 nt encoding a polyprotein of 3080 amino acids (349.88 kDa), flanked by the 5′- and 3′-untranslated regions, and an overlapping ORF of 74 amino acids, the P3N-PIPO. As in previously sequenced ZYMV isolates, Z104 polyprotein consists of 10 putative functional proteins. Several domains in the HC-Pro and P3 proteins related to the transmissibility by aphids and virus aggressiveness are conserved in Z104. Symptoms induced both in the field and laboratory by Z104 together with its molecular features indicate that it is an aggressive strain. Deep sequencing of sRNAs revealed a particularly high variability in the genome sequence at eight nucleotide positions, three of which resulted in missense mutations in the P3, 6K1, and NIb proteins. This may be indicative of the natural ZYMV genome variability within the host. The phylogenetic analysis of the whole genome or the sole coat protein of several ZYMV isolates did not provide evidence for a close relationship of Z104 with other isolates from European countries. To the best of our knowledge, this is the first-reported complete genome of a ZYMV isolate from Italy.

Phenotypic, molecular and phytohormonal evidence of Plum pox virus silencing in susceptible apricot genotypes

Plum pox virus (PPV; sharka disease) is the most important limiting factor for apricot production in affected areas. The generation of new resistant cultivars is the definitive solution to this problem. For this reason, resistance to PPV is one of the main objectives of breeding programmes. During the evaluation of resistance in these programmes, a reduction of sharka symptoms has been observed after the infection of virus in some susceptible cultivars. In this study, we present the results of phenotypic, gene expression and phytohormonal profile changes induced by PPV infection in susceptible apricot cultivars. At the phenotypic level, a reduction in the severity of symptoms during the second infection cycle of the study was observed. This reduction of symptoms could be explained by a silencing effect on the PPV. In line with this idea, the transcriptomic analyses revealed that the early response to PPV is associated with an induction of genes involved in pathogen resistance such as those encoding jasmonic acid, resistance proteins, chitinases, etc. However, the overexpression of the Dicer protein 2a gene in leaves with strong symptoms may indicate the suppression of a gene silencing mechanism in the plant by PPV HCPro and P1 PPV proteins. Salicylic acid appeared to be involved in susceptible infected genotypes. This could also be related to the gene silencing response elicited by the virus. The successful silencing of the virus could be the origin of the reduction in sharka symptoms observed after the infection of some susceptible cultivars.

Transient Transactivation Studies in Nicotiana benthamiana Leaves.

Transitory gene expression systems in Nicotiana benthamiana leaves, in combination with the use of gene silencing suppressors as the p19 or HC-pro proteins that allow for elevated levels of gene expression, have proven to be a highly versatile tool to analyze transcriptional function of DNA binding factors in the activated or repressed expression of their gene targets. This experimental setup uses Agrobacterium-mediated infection to deliver the various DNA constructs into the cell, and offers the advantage with respect to mesophyll protoplast transfection procedures that it entails a much easier protocol, in addition to preserving the intact leaf tissue, thus being more amenable to the study of wound and stress signaling pathways or to the functional analyses of regulators that respond to Ca+2 signatures. Furthermore, by using reporter constructs based on the LUCIFERASE (LUC) gene, which does not require a destructive determination assay, this expression system can be used to test for changes in gene activity over time or in response to various treatments, thus providing a comprehensive understanding of the signaling pathways that modulate activity of the expressed regulators and therefore their in vivo function in the control of the analyzed promoter.

Development and detection application of monoclonal antibodies against Zucchini yellow mosaic virus

Aphid-borne Zucchini yellow mosaic virus (ZYMV) is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection technique. Using purified ZYMV virion and the conventional hybridoma technology, three hybridoma cell lines (16A11, 5A7 and 3B8) secreting monoclonal antibodies (MAbs) against ZYMV Zhejiang isolate were obtained. The working titers of the ascitic fluids secreted by the three hybridoma cell lines were up to 10−7 by indirect enzyme-linked immunosorbent assay (ELISA). All MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that the MAb 3B8 could specifically react with the coat protein of ZYMV while MAbs 5A7 and 16A11 reacted strongly with a protein of approximately 51 kDa from the ZYMV-infected leaf tissues. According to this molecular weight, we consider this reactive protein is likely to be the HC-Pro protein. Using these three MAbs, we have now developed five detection assays, i.e., antigen-coated-plate ELISA (ACP-ELISA), dot-ELISA, tissue blot-ELISA, double-antibody sandwich ELISA (DAS-ELISA), and immunocapture-RT-PCR (IC-RT-PCR), for the sensitive, specific, and easy detection of ZYMV. The sensitivity test revealed that ZYMV could be readily detected respectively by ACP-ELISA, dot-ELISA, DAS-ELISA and IC-RT-PCR in 1:163 840, 1:2 560, 1:327 680 and 1:1 310 720 (w/v, g mL−1) diluted crude extracts from the ZYMV-infected plants. We demonstrated in this study that the dot-ELISA could also be used to detect ZYMV in individual viruliferous aphids. A total of 275 cucurbitaceous plant samples collected from the Zhejiang, Jiangsu, Shandong and Hainan provinces, China, were screened for the presence of ZYMV with the described assays. Our results showed that 163 of the 275 samples (59%) were infected with ZYMV. This finding indicates that ZYMV is now widely present in cucurbitaceous crops in China. RT-PCR followed by DNA sequencing and sequence analyses confirmed the accuracy of the five assays. We consider that these detection assays can significantly benefit the control of ZYMV in China.

Gene Expression Analysis of Plum pox virus (Sharka) Susceptibility/Resistance in Apricot (Prunus armeniaca L.).

RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, “Rojo Pasión” and “Z506-7”, resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.

Identification of Soybean mosaic virus in Poland.

Soybean mosaic virus (SMV), the causal agent of a commonly occurring disease of soybean (Glycine max), is seed-borne and readily transmitted mechanically. Soybean plants and seeds in Poland were surveyed by ELISA to evaluate the occurence of SMV and characterize its isolates by RT-PCR using primers designed in the P1, HC-Pro and coat protein (CP) genes followed by sequencing. Two isolates designed A and M differed biologically with SMV-A infecting soybean plants without apparent symptoms and SMV-M causing foliar rugosity and curling, and stunted growth. The CP gene of the two isolates had 92% and 99% identity at the nucleotide and amino acid level, respectively. Phylogenetic analyses showed that the two Polish isolates grouped in two distinct clusters. This is the first report on SMV identification in Poland.

The amino acid 419 in HC-Pro is involved in the ability of PVY isolate N605 to induce necrotic symptoms on potato tubers

The ability to induce the potato tuber necrosis ringspot disease (PTNRD) is a property shared by PVY isolates belonging to different groups (e.g. PVYN and PVYO) and variants (e.g. PVYNTN and PVYN-W). The identification of viral molecular determinant(s) involved in the expression of PTNRD symptoms is essential for (i) an easier detection of tuber necrosis isolates and (ii) an improvement of our knowledge on the epidemiology of this potato disease. A reverse genetic approach associated with a biological typing of a collection of PVY chimeras and mutants indicated that residue E419 of the HC-Pro protein is linked to the ability of PVY to induce tuber necrosis on four PTNRD-susceptible potato cultivars. Indeed, the substitution of the N-type glutamic acid (E) in O-type aspartic acid (D) at position 419 in the HC-Pro cistron prevents the expression of tuber necrosis on infected tubers without reducing the virulence of the corresponding E/D419 mutant. This result opens opportunities for the future studies on potato/PVY interactions.

Analysis of gene expression changes in peach leaves in response to Plum pox virus infection using RNA-Seq.

Differences in gene expression were studied after Plum pox virus (PPV, sharka disease) infection in peach GF305 leaves with and without sharka symptoms using RNA-Seq. For each sample, more than 80% of 100-nucleotide paired-end (PE) Illumina reads were aligned on the peach reference genome. In the symptomatic sample, a significant proportion of reads were mapped to PPV reference genomes (1.04% compared with 0.00002% in non-symptomatic leaves), allowing for the ultra-deep assembly of the complete genome of the PPV isolate used (9775 nucleotides, missing only 11 nucleotides at the 5' genome end). In addition, significant alternative splicing events were detected in 359 genes and 12 990 single nucleotide polymorphisms (SNPs) were identified, 425 of which could be annotated. Gene ontology annotation revealed that the high-ranking mRNA target genes associated with the expression of sharka symptoms are mainly related to the response to biotic stimuli, to lipid and carbohydrate metabolism and to the negative regulation of catalytic activity. A greater number of differentially expressed genes were observed in the early asymptomatic phase of PPV infection in comparison with the symptomatic phase. These early infection events were associated with the induction of genes related to pathogen resistance, such as jasmonic acid, chitinases, cytokinin glucosyl transferases and Lys-M proteins. Once the virus had accumulated, the overexpression of Dicer protein 2a genes suggested a gene silencing plant response that was suppressed by the virus HCPro and P1 proteins. These results illustrate the dynamic nature of the peach-PPV interaction at the transcriptome level and confirm that sharka symptom expression is a complex process that can be understood on the basis of changes in plant gene expression.

Mutation of a Short Variable Region in HCpro Protein of Potato virus A Affects Interactions with a Microtubule-Associated Protein and Induces Necrotic Responses in Tobacco

Helper component proteinase (HCpro) is a multifunctional protein of potyviruses (genus Potyvirus). HCpro of Potato virus A (PVA) interacts with the microtubule-associated protein HIP2 in host cells, and depletion of HIP2 reduces virus accumulation. This study shows that HCpro of Potato virus Y and Tobacco etch virus also interact with HIP2. The C-proximal portion of PVA HCpro determines the interaction with HIP2 and was found to contain a stretch of six residues comprising a highly variable region (HVR) in potyviruses. Mutations in HVR reduced PVA accumulation in tobacco plants and induced necrotic symptoms novel to PVA. Microarray and quantitative reverse transcription polymerase chain reaction analyses revealed induction of many defense-related genes including ethylene- and jasmonic acid-inducible pathways in systemically infected leaves at necrosis onset. Salicylic acid-mediated signaling was dispensable for the response. Genes related to microtubule functions were down-regulated. Structural modeling of HCpro suggested that all mutations in HVR caused conformational changes in adjacent regions containing functionally important motifs conserved in potyviruses. Those mutations, which also caused conformational changes in HVR, led to the greatest reduction of fitness. Our results implicate HVR in the regulation of HCpro conformation and virus-host interactions and suggest that mutation of HVR induces host defense.

Expression of a recombinant Human papillomavirus 16 E6GT oncoprotein fused to N- and C-termini of Potato virus X coat protein in Nicotiana benthamiana

Infection with Human papillomaviruses (HPVs) is linked to cervical cancer, which is one of the most common cancers found among women worldwide. Despite preventive immunization, a therapeutic vaccine, targeting infected individuals, is required. Vaccine strategies for treatment of HPV—induced cervical cancer are based on E7 and E6 oncoproteins. In this work we report the transient expression of chimeric particles containing the E6 oncoprotein from Human papillomavirus type 16 (HPV-16) in plants. We fused a mutagenized coding sequence of the E6 oncoprotein (E6GT) with the coding sequence of Potato virus X coat protein (PVX CP) both with the 5′- and 3′-terminus using linkers of different length (0, 4 or 15 amino acids). The expression in E. coli was performed to assess the characteristics of the recombinant protein prior to plant expression. The yield and immunological reactivity of the expressed proteins were screened with anti-PVX CP and E6 antibodies. The highest yields of chimeric particles were observed in the transgenic N. benthamiana expressing Potato virus A HC-Pro protein and the Tobacco mosaic virus movement protein. When inoculated on host plants, these recombinant viruses were not able to spread systemically. The obtained results revealed the new relations for design of expression cassettes for plant-based vaccine production.

The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein.

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.