Nonvirion Protein Research Articles

Heterologous Exchanges of Glycoprotein and Non-Virion Protein in Novirhabdoviruses: Assessment of Virulence in Yellow Perch (Perca flavescens) and Rainbow Trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins.

Effect of the ati Gene Deletion on the Pathogenicity and Immunogenicity of the Vaccinia Virus.

Among the nonvirion proteins of the vaccinia virus (VACV), a 94-kDa long protein is most abundantly present; the protein is a truncated form of the 150-kDa A-type inclusion (ATI) protein of the cowpox virus encoded by the ati gene. This VACV protein does not form intracellular ATIs, being as it is a major immunogen upon infection/immunization of humans or animals with the VACV. Antibodies specific to this protein are not virus-neutralizing. The present study focused on the effect of the production of this nonstructural major immunogenic VACV protein on the manifestation of pathogenicity and immunogenicity of the virus in the BALB/c mouse model of infection. In order to introduce a targeted deletion into the VACV LIVP genome, the recombinant integration/deletion plasmid pΔati was constructed and further used to generate the recombinant virus LIVPΔati. The pathogenicity of the VACV LIVP and LIVPΔati strains was studied in 3-week-old mice. The mice were intranasally infected with the viruses at a dose of 107 pfu; 50% of the animals infected with the parent LIVP strain died, while infection with the LIVPΔati strain led to the death of only 20% of the mice. Intradermal vaccination of mice aged 6- weeks with the LIVPΔati virus statistically significantly increased the production of VACV-specific IgG, compared to that after intradermal vaccination with VACV LIVP. Meanwhile, no differences were noted in the cell-mediated immune response to the vaccination of mice with VACV LIVP or LIVPΔati, which was assessed by ELISpot according to the number of splenocytes producing IFN-γ in response to stimulation with virus-specific peptides. Intranasal infection of mice with lethal doses of the cowpox virus or the ectromelia virus on day 60 post-immunization with the studied VACV variants demonstrated that the mutant LIVPΔati elicits a stronger protective response compared to the parent LIVP.

Galectin 9 restricts viral replication in teleost via autophagy-antiviral pathway and polarizes M2 macrophages for anti-inflammatory response: New insights into functional properties of fish Galectin-9 from Planiliza haematocheilus

Galectin 9 (Gal9) is a tandem repeat type ß-galactoside-binding galectin that mediates various cellular biochemical and immunological functions. Many studies have investigated the functional properties of Gal9 in mammals; however, knowledge of fish Gal9 is limited to antibacterial studies. In this context, our aim was to clone Gal9 from Planiliza haematocheilus (PhGal9) and investigate its structural and functional characteristics. We discovered the PhGal9 open reading frame, which was 969 base pairs long and encoded a 322 amino acid protein. PhGal9 had a projected molecular weight of 35.385 kDa but no signal peptide sequence. PhGal9 mRNA was ubiquitously produced in all investigated tissues but was predominant in the intestine, spleen, and brain. Its mRNA expression was increased in response to stimulation by Poly(I:C), LPS, and L. garvieae. The rPhGal9 exhibited a dose-dependent agglutination potential toward gram-positive and gram-negative bacteria at a minimum concentration of 50 μg/mL. Overexpression of PhGal9 promoted M2-like phenotype changes in mouse macrophages, and RT-qPCR analysis of M1 and M2 marker genes confirmed M2 polarization with upregulation of M2 marker genes. In the antiviral assay, the expression levels of Viral Hemorrhagic Septicemia Virus (VHSV) glycoproteins, phosphoproteins, nucleoproteins, non-virion proteins, matrix proteins, and RNA polymerase were significantly reduced in PhGal9-overexpressed cells. Furthermore, the mRNA expression of autophagic genes (sqstm1, tax1bp1b, rnf13, lc3, and atg5) and antiviral genes (viperin) were upregulated in PhGal9 overexpressed cells. For the first time in teleosts, our study demonstrated that PhGal9 promotes M2 macrophage polarization by upregulating M2-associated genes (egr2 and cmyc) and suppressing M1-associated genes (iNOS and IL-6). Furthermore, our results show that exogenous and endogenous PhGal9 prevented VHSV attachment and replication by neutralizing virion and autophagy, respectively. Gal9 may be a potent modulator of the antimicrobial immune response in teleost fish.

Integrating Low-Order and High-Order Correlation Information for Identifying Phage Virion Proteins.

Phage virion proteins (PVPs) play an important role in the host cell. Fast and accurate identification of PVPs is beneficial for the discovery and development of related drugs. Although wet experimental approaches are the first choice to identify PVPs, they are costly and time-consuming. Thus, researchers have turned their attention to computational models, which can speed up related studies. Therefore, we proposed a novel machine-learning model to identify PVPs in the current study. First, 50 different types of physicochemical properties were used to denote protein sequences. Next, two different approaches, including Pearson's correlation coefficient (PCC) and maximal information coefficient (MIC), were employed to extract discriminative information. Further, to capture the high-order correlation information, we used PCC and MIC once again. After that, we adopted the least absolute shrinkage and selection operator algorithm to select the optimal feature subset. Finally, these chosen features were fed into a support vector machine to discriminate PVPs from phage non-virion proteins. We performed experiments on two different datasets to validate the effectiveness of our proposed method. Experimental results showed a significant improvement in performance compared with state-of-the-art approaches. It indicates that the proposed computational model may become a powerful predictor in identifying PVPs.

Expression characteristics of non-virion protein of Hirame novirhabdovirus and its transfection induced response in hirame natural embryo cells

The non-virion (NV) protein is the signature of genus Novirhabdovirus, which has been of considerable concern due to its potential role in viral pathogenicity. However, its expression characteristics and induced immune response remain limited. In the present work, it was demonstrated that Hirame novirhabdovirus (HIRRV) NV protein was only detected in the viral infected hirame natural embryo (HINAE) cells, but absent in the purified virions. Results showed that the transcription of NV gene could be stably detected in HIRRV-infected HINAE cells at 12 h post infection (hpi) and then reached the peak at 72 hpi. A similar expression trend of NV gene was also found in HIRRV-infected flounders. Subcellular localization analysis further exhibited that HIRRV-NV protein was predominantly localized in the cytoplasm. To elucidate the biological function of HIRRV-NV protein, NV eukaryotic plasmid was transfected into HINAE cells for RNA-seq. Compared to empty plasmid group, some key genes in RLR signaling pathway were significantly downregulated in NV-overexpressed HINAE cells, indicating that RLR signaling pathway was inhibited by HIRRV-NV protein. The interferon-associated genes were also significantly suppressed upon transfection of NV gene. This research would improve our understanding of expression characteristics and biological function of NV protein during HIRRV infection process.

RF_phage virion: Classification of phage virion proteins with a random forest model.

Introduction: Phages play essential roles in biological procession, and the virion proteins encoded by the phage genome constitute critical elements of the assembled phage particle. Methods: This study uses machine learning methods to classify phage virion proteins. We proposed a novel approach, RF_phage virion, for the effective classification of the virion and non-virion proteins. The model uses four protein sequence coding methods as features, and the random forest algorithm was employed to solve the classification problem. Results: The performance of the RF_phage virion model was analyzed by comparing the performance of this algorithm with that of classical machine learning methods. The proposed method achieved a specificity (Sp) of 93.37%%, sensitivity (Sn) of 90.30%, accuracy (Acc) of 91.84%, Matthews correlation coefficient (MCC) of .8371, and an F1 score of .9196.

Effects of Non-Virion Gene Expression Level and Viral Genome Length on the Replication and Pathogenicity of Viral Hemorrhagic Septicemia Virus.

Fish novirhabdoviruses, including viral hemorrhagic septicemia virus (VHSV), hirame rhabdovirus (HIRRV), and infectious hematopoietic necrosis virus (IHNV), harbor a unique non-virion (NV) gene that is crucial for efficient replication and pathogenicity. The effective levels and the function of the N-terminal region of the NV protein, however, remain poorly understood. In the present study, several recombinant VHSVs, which completely lack (rVHSV-ΔNV) or harbor an additional (rVHSV-dNV) NV gene, were generated using reverse genetics. To confirm the function of the N-terminal region of the NV protein, recombinant VHSVs with the NV gene that gradually mutated from the start codon (ATG) to the stop codon (TGA), expressed as N-terminally truncated NV proteins (rVHSV-NV1, -NV2, and -NV3), were generated. CPE progression and viral growth analyses showed that epithelioma papulosum cyprini (EPC) cells infected with rVHSV-ΔNV or rVHSV-NV3—which did not express NV protein—rarely showed CPE and viral replication as opposed to EPC cells infected with rVHSV-wild. Interestingly, regardless of the presence of two NV genes in the rVHSV-dNV genome, EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP (control) failed to induce CPE and viral replication. In EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP, which harbored a longer VHSV genome than the wild-type, Mx gene expression levels, which were detected by luciferase activity assay, were particularly high; Mx gene expression levels were higher in EPC cells infected with rVHSV-ΔNV, -NV2, or -NV3 than in those infected with rVHSV-wild or rVHSV-NV1. The total amount of NV transcript produced in EPC cells infected with rVHSV-wild was much higher than that in EPC cells infected with rVHSV-dNV. However, the expression levels of the NV gene per viral particle were significantly higher in EPC cells infected with rVHSV-dNV than in cells infected with rVHSV-wild. These results suggest that the NV protein is an essential component in the inhibition of host type-I interferon (IFN) and the induction of viral replication. Most importantly, viral genome length might affect viral replication efficiency to a greater extent than does NV gene expression. In in vivo pathogenicity experiments, the cumulative mortality rates of olive flounder fingerlings infected with rVHSV-dNV or rVHSV-wild were similar (60–70%), while those of fingerlings infected with rVHSV-A-EGFP were lower. Moreover, the virulence of rVHSV-ΔNV and rVHSV, both harboring a truncated NV gene (rVHSV-NV1, -NV2, and -NV3), was completely attenuated in the olive flounder. These results suggest that viral pathogenicity is affected by the viral replication rate and NV gene expression. In conclusion, the genome length and NV gene (particularly the N-terminal region) expression of VHSVs are closely associated with viral replication in host type-I IFN response and the viral pathogenicity.

A single amino acid variation of NV protein of viral hemorrhagic septicemia virus increases protein stability and decreases immune gene expression

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV–S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.

Modifications of the nucleoprotein of viral haemorrhagic septicaemia virus showed gain of virulence in intraperitoneally infected rainbow trout.

Viral haemorrhagic septicaemia virus (VHSV) is the cause of an important listed disease in European rainbow trout (Oncorhynchus mykiss) aquaculture and can be present in a wide range of fish species, including marine fish, which can act as viral reservoir. Recent studies revealed putative genetic virulence markers of VHSV to rainbow trout highlighting the roles of the nucleoprotein, phosphoprotein and non-virion protein. Using reverse genetics, we produced recombinant viruses by introducing parts of or the entire nucleoprotein from a high-virulent isolate VHSV into a low-virulent backbone. Furthermore, we also made recombinant viruses by introducing residue modifications in the nucleoprotein that seem to play a role in virulence. Rainbow trout challenged with these recombinant viruses (rVHSVs) by intraperitoneal injection (IP) developed clinical signs and showed lower survival when compared to the parental rVHSV whereas fish challenged by immersion did not show clinical signs except for the high-virulent control. The mutations did not influence the viral growth in cell culture. The recombinant viruses and parental recombinant were unable to replicate and show cytopathic effect in EPC cells whereas the high-virulent control was well adapted in all the fish cell lines tested. We showed evidence that corroborates with the hypothesis that the nucleoprotein has virulence motifs associated with VHSV virulence in rainbow trout.

A preliminary investigation on the mechanism of action of 4-(8-(2-ethylimidazole)octyloxy)-arctigenin against IHNV

Arctigenin derivatives form an elite class of naturally occurring compounds that possess promising antiviral therapeutic perspectives. In a previous study, we design and synthesize a arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), to evaluate its antiviral activity on infectious hematopoietic necrosis virus (IHNV). In this study, we find that the half maximal inhibitory concentrations (IC50) of EOA on IHNV nucleoprotein (N), phosphoprotein (P), matrix protein (M), nonvirion protein (NV) and polymerase (L) mRNA expression is 0.92, 0.80, 0.98, 0.89 and 0.87 μM, respectively. Mechanistically, our results show that EOA do not damage the viral particles directly, indicating EOA does not possess antiviral activity by destroying virions. Viral binding assays reveal that EOA do not interfere with IHNV adsorption. Because rapamycin has been shown to exhibit anti-IHNV activity by inducing autophagy of epithelioma papulosum cyprini (EPC) cells, we further investigate the relationship between EOA and autophagy in EPC cells. Autophagy fluorescence detection shows that EPC cells have a strong autophagy body after being treated with derivative EOA. The electron microscopy results show that EOA could induce typical autophagosomes which are representative structures of autophagy activation. Moreover, the punctate accumulation of green fluorescence-tagged microtubule-associate protein 1 light chain 3 (LC3) and the protein conversion from LC3-I to LC3-II are respectively confirmed by confocal fluorescence microscopy and western blotting. Overall, these findings demonstrate that EOA plays an anti-IHNV role via inducing autophagy in EPC cells.

Whole-genome next-generation sequencing and phylogenetic characterization of viral haemorrhagic septicaemia virus in Korea.

Whole-genome next-generation sequencing was used to investigate the local evolution of viral haemorrhagic septicaemia virus, a serious pathogen affecting economically important fish such as rainbow trout and turbot in Europe and olive flounder in Asia. Sequence analysis showed that all isolates were genotype IVa, but could be classified further into four subgroups (K1-K4). In addition, genomic regions encompassing the nucleoprotein, phosphoprotein, matrix protein and non-virion protein genes, as well as the seven non-coding regions, were relatively conserved, whereas glycoprotein and RNA-dependent RNA polymerase genes were variable in the coding region. Taken together, the data demonstrate that whole-genome next-generation sequencing may be useful for future surveillance, prevention and control strategies against viral haemorrhagic septicaemia.

Identification of Phage Virion Proteins by Using the g-gap Tripeptide Composition

Phages are widely distributed in locations populated by bacterial hosts. Phage proteins can be divided into two main categories, that is, virion and non-virion proteins with different functions. In practice, people mainly use phage virion proteins to clarify the lysis mechanism of bacterial cells and develop new antibacterial drugs. Accurate identification of phage virion proteins is therefore essential to understanding the phage lysis mechanism. Although some computational methods have been focused on identifying virion proteins, the result is not satisfying which gives more room for improvement. In this study, a new sequence-based method was proposed to identify phage virion proteins using g-gap tripeptide composition. In this approach, the protein features were firstly extracted from the ggap tripeptide composition. Subsequently, we obtained an optimal feature subset by performing incremental feature selection (IFS) with information gain. Finally, the support vector machine (SVM) was used as the classifier to discriminate virion proteins from non-virion proteins. In 10-fold crossvalidation test, our proposed method achieved an accuracy of 97.40% with AUC of 0.9958, which outperforms state-of-the-art methods. The result reveals that our proposed method could be a promising method in the work of phage virion proteins identification.

The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout

BackgroundViral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus belonging to the Novirhabdovirus genus, causes severe disease and mortality in many marine and freshwater fish species worldwide. VHSV isolates are classified into four genotypes and each group is endemic to specific geographic regions in the north Atlantic and Pacific Oceans. Most viruses in the European VHSV genotype Ia are highly virulent for rainbow trout (Oncorhynchus mykiss), whereas, VHSV genotype IVb viruses from the Great Lakes region in the United States, which caused high mortality in wild freshwater fish species, are avirulent for trout. This study describes molecular characterization and construction of an infectious clone of the virulent VHSV-Ia strain DK-3592B from Denmark, and application of the clone in reverse genetics to investigate the role of selected VHSV protein(s) in host-specific virulence in rainbow trout (referred to as trout-virulence).MethodsOverlapping cDNA fragments of the DK-3592B genome were cloned after RT-PCR amplification, and their DNA sequenced by the di-deoxy chain termination method. A full-length cDNA copy (pVHSVdk) of the DK-3592B strain genome was constructed by assembling six overlapping cDNA fragments by using natural or artificially created unique restriction sites in the overlapping regions of the clones. Using an existing clone of the trout-avirulent VHSV-IVb strain MI03 (pVHSVmi), eight chimeric VHSV clones were constructed in which the coding region(s) of the glycoprotein (G), non-virion protein (NV), G and NV, or G, NV and L (polymerase) genes together, were exchanged between the two clones. Ten recombinant VHSVs (rVHSVs) were generated, including two parental rVHSVs, by transfecting fish cells with ten individual full-length plasmid constructs along with supporting plasmids using the established protocol. Recovered rVHSVs were characterized for viability and growth in vitro and used to challenge groups of juvenile rainbow trout by intraperitoneal injection.ResultsComplete sequence of the VHSV DK-3592B genome was determined from the cloned cDNA and deposited in GenBank under the accession no. KC778774. The trout-virulent DK-3592B genome (genotype Ia) is 11,159 nt in length and differs from the trout-avirulent MI03 genome (pVHSVmi) by 13% at the nucleotide level. When the rVHSVs were assessed for the trout-virulence phenotype in vivo, the parental rVHSVdk and rVHSVmi were virulent and avirulent, respectively, as expected. Four chimeric rVHSVdk viruses with the substitutions of the G, NV, G and NV, or G, NV and L genes from the avirulent pVHSVmi constructs were still highly virulent (100% mortality), while the reciprocal four chimeric rVHSVmi viruses with genes from pVHSVdk remained avirulent (0–10% mortality).ConclusionsWhen chimeric rVHSVs, containing all the G, NV, and L gene substitutions, were tested in vivo, they did not exhibit any change in trout-virulence relative to the background clones. These results demonstrate that the G, NV and L genes of VHSV are not, by themselves or in combination, major determinants of host-specific virulence in trout.

Prokaryotic expression and subcellular localization of the matrix protein of the viral hemorrhagic septicemia virus in finfish

PDF HTML阅读 XML下载 导出引用 引用提醒 鱼类病毒性出血性败血症病毒基质蛋白的原核表达及其亚细胞定位 DOI: 作者: 作者单位: 安徽农业大学动物科技学院, 安徽 合肥 230036 作者简介: 朱若林(1987-),男,博士,研究方向为鱼类疾病防控.E-mail:jollinz@163.com 通讯作者: 中图分类号: S941 基金项目: 国家自然科学基金青年项目（31402331）；安徽省现代农业产业技术体系（2016-2020）水产产业体系（皖农科[2016]84号）. Prokaryotic expression and subcellular localization of the matrix protein of the viral hemorrhagic septicemia virus in finfish Author: Affiliation: School of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为了对鱼类病毒性出血性败血症病毒（viral hemorrhagic septicemia virus，VHSV）基质蛋白（matrix protein，M）进行功能研究，本实验通过PCR扩增了M基因全长序列，将其克隆至原核表达载体pET-32a（+），转化至大肠杆菌Rosetta（DE3）感受态细胞后进行IPTG诱导表达，将纯化后的重组蛋白免疫BALB/c小鼠制备多克隆抗体，采用间接ELISA检测抗体效价，并运用Western blot和间接免疫荧光检测抗体特异性。结果显示，M基因全长为606 bp，IPTG诱导得到的融合蛋白主要以包涵体的形式存在，大小约为36 kD，比预计略小。间接ELISA检测抗体效价大于1：102400，Western blot检测显示该抗体可以特异性识别纯化的融合蛋白和VHSV感染的鲤上皮瘤（epithelioma papulosum cyprini，EPC）细胞中的M蛋白。间接免疫荧光结果显示M蛋白多抗能识别感染VHSV的EPC细胞中的M蛋白，且M蛋白主要定位于细胞质和细胞膜。本研究中M蛋白多克隆抗体的制备将有助于开展M蛋白的功能研究及疾病的免疫学诊断。 Abstract:Viral hemorrhagic septicemia virus (VHSV) is one of the most serious pathogens of finfish that affects over 80 marine and freshwater species in North America, Europe, and Asia. The genome of VHSV is a negative-sense, single stranded RNA containing approximately 12000 base pairs that encode six proteins, which are the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), non-virion protein (NV), and RNA polymerase protein (L) in order from 3' to 5'. The M protein functions in a variety of rhabdovirus infection processes such as assembly and budding, inhibiting host-cell directed transcription and gene expression, inducing apoptosis of the host cell, et al., which were all thoroughly investigated in other rhabdoviruses such as vesicular stomatitis virus and rabies virus, while less so in VHSV. In order to investigate the function of the M protein of VHSV, the M gene was amplified by PCR and cloned into the prokaryotic expression vector pET-32a(+), which was transformed into Rosetta (DE3) competent cells. The recombinant protein was induced by IPTG, and the polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant protein. The titer of the antibody was detected by an indirect ELISA, and the antibody specificity was tested by western blot and indirect immunofluorescence. The results showed that the full-length M gene was 606 bp. The fusion protein induced by IPTG mainly existed in the form of an inclusion body, and the size was about 36 kDa, which was slightly smaller than expected. The indirect ELISA assay showed that the titer of the antibody was greater than 1:102400. The western blot showed that the antibody could specifically identify the purified fusion protein and M protein in VHSV infected (EPC) cells. The indirect immunofluorescence showed that the M protein antibody can recognize the M protein in VHSV infected EPC cells, and the M protein was mainly localized in the cytoplasm and cell membrane. These results suggest that the prepared polyclonal antibody can be used as an effective tool to study the function of the M protein and for the diagnosis of VHSV. 参考文献 相似文献 引证文献

A Distinct, Non-Virion Plant Virus Movement Protein Encoded by a Crinivirus Essential for Systemic Infection

Plant-infecting viruses utilize various strategies involving multiple viral and host factors to achieve successful systemic infections of their compatible hosts. Lettuce infectious yellows virus (LIYV), genus Crinivirus, family Closteroviridae, has long, filamentous flexuous virions and causes phloem-limited infections in its plant hosts. The LIYV-encoded P26 is a distinct non-virion protein that shows no similarities to proteins in current databases: it induces plasmalemma deposits over plasmadesmata (PD) pit fields and is speculated to have roles in LIYV virion transport within infected plants. In this study, P26 was demonstrated to be a PD-localized protein, and its biological significance was tested in planta by mutagenesis analysis. An LIYV P26 knockout mutant (P26X) showed viral RNA replication and virion formation in inoculated leaves of Nicotiana benthamiana plants, but failed to give systemic infection. Confirmation by using a modified green fluorescent protein (GFP)-tagged LIYV P26X showed GFP accumulation only in infiltrated leaf tissues, while wild-type LIYV GFP readily spread systemically in the phloem. Attempts to rescue P26X by complementation in trans were negative. However a translocated LIYV P26 gene in the LIYV genome rescued systemic infection, but P26 orthologs from other criniviruses did not. Mutagenesis in planta assays showed that deletions in P26, as well as 2 of 11 specific alanine-scanning mutants, abolished the ability to systemically infect N. benthamiana IMPORTANCE Plant viruses encode specific proteins that facilitate their ability to establish multicellular/systemic infections in their host plants. Relatively little is known of the transport mechanisms for plant viruses whose infections are phloem limited, including those of the family Closteroviridae. These viruses have complex, long filamentous virions that spread through the phloem. Lettuce infectious yellows virus (LIYV) encodes a non-virion protein, P26, which forms plasmalemma deposits over plasmodesmata pit fields, and LIYV virions are consistently found attached to those deposits. Here we demonstrate that P26 is a unique movement protein required for LIYV systemic infection in plants. LIYV P26 shows no sequence similarities to other proteins, but other criniviruses encode P26 orthologs. However, these failed to complement movement of LIYV P26 mutants.

Inspirations on Virus Replication and Cell-to-Cell Movement from Studies Examining the Cytopathology Induced by Lettuce infectious yellows virus in Plant Cells.

Lettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus in the family Closteroviridae. Like many other positive-strand RNA viruses, LIYV infections induce a number of cytopathic changes in plant cells, of which the two most characteristic are: Beet yellows virus-type inclusion bodies composed of vesicles derived from cytoplasmic membranes; and conical plasmalemma deposits (PLDs) located at the plasmalemma over plasmodesmata pit fields. The former are not only found in various closterovirus infections, but similar structures are known as ‘viral factories’ or viroplasms in cells infected with diverse types of animal and plant viruses. These are generally sites of virus replication, virion assembly and in some cases are involved in cell-to-cell transport. By contrast, PLDs induced by the LIYV-encoded P26 non-virion protein are not involved in replication but are speculated to have roles in virus intercellular movement. These deposits often harbor LIYV virions arranged to be perpendicular to the plasma membrane over plasmodesmata, and our recent studies show that P26 is required for LIYV systemic plant infection. The functional mechanism of how LIYV P26 facilitates intercellular movement remains unclear, however, research on other plant viruses provides some insights on the possible ways of viral intercellular movement through targeting and modifying plasmodesmata via interactions between plant cellular components and viral-encoded factors. In summary, beginning with LIYV, we review the studies that have uncovered the biological determinants giving rise to these cytopathological effects and their importance in viral replication, virion assembly and intercellular movement during the plant infection by closteroviruses, and compare these findings with those for other positive-strand RNA viruses.

Identification of the functional regions of the viral haemorrhagic septicaemia virus (VHSV) NV protein: Variants that improve function

Non-virion (NV) protein is essential for an efficient replication increasing the pathogenicity of the Salmonid novirhabdovirus (formerly IHNV), Piscine novirhabdovirus (formerly VHSV), and Hirame novirhabdovirus (HIRV). The interferon system, apoptosis, and other immune-related genes are modulated by NV to finally induce a deficient antiviral state in the cell. However, little is known about the VHSV NV regions involved in function and location. Here, eight different NV 07.71 fragments and eleven NV 07.71 mutants derived from the region between the two first α-helices have been studied in order to establish the mx and il8 transcript levels in ZF4 cells and the subcellular location. As a result, we determined that the N-terminal part of NV preserves the same ability as the wild-type (wt) NV in mx/il8 modulation and it also shares the subcellular location. Among NV mutants, some induced mx upregulation (N34A, C35A, D38A, and S40A) but maintained the il8 levels stable when compared to wt-NV in ZF4. Four NV mutants (D28A, N31A, L33A, and F37A) were not affected by the mutation and showed mx and il8 transcript levels similar to wt-NV. Surprisingly, mutants D36A, R39A, and D41A induced a stronger downregulation of both mx and il8 transcript levels than wt-NV, suggesting that a more stable structure and an improved interaction with ligands could be achieved through these mutations. Amino acids at positions 36 and 39 are conserved among known VHSV NV proteins whereas at position 41 two different amino acids have been described. To date, no natural NV proteins with alanine at positions 36, 39, and 41 have been found. In addition, wt-NV, all NV mutants, and one N-terminal NV fragment were located at cytoplasm with a characteristic pattern, which might support that cytoplasm is the site for interaction with candidate ligands such as PPM1Bb. Taken together, the data presented in this work indicated that NV function relies on the first part of the molecule and is dependent on tertiary structure rather than on the linear one. This study could lead to a better knowledge of VHSV escape from fish antiviral mechanisms as well as to future studies on immune targets.

A single amino acid change in the non-structural NV protein impacts the virulence phenotype of Viral hemorrhagic septicemia virus in trout.

Novirhabdoviruses like the Viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses infecting fish. In the current study, RNA genomes of different VHSV field isolates classified as high, medium or low virulent phenotypes have been sequenced by next-generation sequencing and compared. Various amino acid changes, depending on the VHSV phenotype, have been identified in all the VHSV proteins. As a starting point, we focused our study on the non-virion (NV) non-structural protein in which an arginine residue (R116) is present in all the virulent isolates and replaced by a serine/asparagine residue S/N116 in the attenuated isolates. A recombinant virus derived from a virulent VHSV strain in which the NV R116 residue has been replaced by a serine, rVHSVNVR116S, was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSVNVR116S was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.

NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction

Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.

Complete sequences of 4 viral hemorrhagic septicemia virus IVb isolates and their virulence in northern pike fry.

Four viral hemorrhagic septicemia virus (VHSV) genotype IVb isolates were sequenced, their genetic variation explored, and comparative virulence assayed with experimental infections of northern pike Esox lucius fry. In addition to the type strain MI03, the complete 11183 bp genome of the first round goby Neogobius melanostomus isolate from the St. Lawrence River, and the 2013 and 2014 isolates from gizzard shad Dorosoma cepedianum die-offs in Irondequoit Bay, Lake Ontario and Dunkirk Harbor, Lake Erie were all deep sequenced on an Illumina platform. Mutations documented in the 11 yr since the MI03 index case from Lake St. Clair muskellunge Esox masquinongy showed 87 polymorphisms among the 4 isolates. Twenty-six mutations were non-synonymous and located at 18 different positions within the matrix protein, glycoprotein, non-virion protein, and RNA polymerase genes. The same 4 isolates were used to infect northern pike fry by a single 1 h bath exposure. Cumulative percent mortality varied from 42.5 to 62.5%. VHSV was detected in 57% (41/72) of the survivors at the end of the 21-d trial, suggesting that the virus was not rapidly cleared. Lesions were observed in many of the moribund and dead northern pike, such as hemorrhaging in the skin and fins, as well as hydrocephalus. Mean viral load measured from the trunk and visceral tissues of MI03-infected pike was significantly higher than the quantities detected in fish infected with the most recent isolates of genotype IVb, but there were no differences in cumulative mortality observed.