Journal of Microbiology

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Phosphorylation of the nucleocapsid protein of Hantaan virus by casein kinase II: Jeong-Joong Yoon , Yun-Tai Lee , Hin Chu , Seung-yeol Son , Manbok Kim; J. Microbiol. 2015;53(5):343-347. Published online May 3, 2015; DOI: https://doi.org/10.1007/s12275-015-5095-3

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Hantaanvirus (HTNV) is the prototype of the genus Hantavirus, which belongs to the family Bunyaviridae. Hantaviruses are carried and transmitted by rodents and are known to cause two serious disease syndromes in humans i.e., hemorrhagic fever with renal syndrome (HFRS) and the hantavirus pulmonary syndrome (HPS). HTNV is an enveloped virus that contains a tripartite genome consisting of three negative-sense RNA segments (L, M, S), and the S and M segment of HTNV, respectively, encode the viral nucleocapsid protein (NP) and envelope glycoproteins. Possible phosphorylation motifs of casein kinase II (CKII) and protein kinase C (PKC) were identified in HTNV NP through bioinformatics searches. Sucrose gradient SDS-PAGE analysis indicated that dephosphorylated HTNV NP migrated faster than non-dephosphorylated NP, suggesting that HTNV NP is phosphorylated in infected Vero E6 cells. Immunoblot anaylsis of HTNV particles with anti-phosphoserine antibody and anti-phosphothreonine antibody after immunoprecipitation showed that viral particles are readily phosphorylated at threonine residues. In vitro kinase assay further showed that HTNV NP is phosphorylated by CK II, but not by PKC. Full length or truncated HTNV NPs expressed in E. coli were phosphorylated in vitro by CKII suggesting that phosphorylation may occur in vivo at multiple sites. Site specific mutagenesis studies suggest that HTNV NP phosphorylation might occur at unknown sites excluding the site-directly mutagenized locations. Taken together, HTNV NP can be phosphorylated mainly at threonine residues in vivo by CK II treatment.

Citations

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Protein kinase CK2: a potential therapeutic target for diverse human diseases
Christian Borgo, Claudio D’Amore, Stefania Sarno, Mauro Salvi, Maria Ruzzene
Signal Transduction and Targeted Therapy.2021;[Epub] CrossRef
Unique Interferon Pathway Regulation by the Andes Virus Nucleocapsid Protein Is Conferred by Phosphorylation of Serine 386
Matthew J. Simons, Elena E. Gorbunova, Erich R. Mackow, Susana López
Journal of Virology.2019;[Epub] CrossRef

Journal Article

RNA Interference Targeting Nucleocapsid Protein Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication in Marc-145 Cells: Minnan Yang , Qun Xiang , Xiaodong Zhang , Xiang Li , Seydou Sylla , Zhuang Ding; J. Microbiol. 2014;52(4):333-339. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-3419-3

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Abstract

Porcine reproductive and respiratory syndrome (PRRS) is an important disease, which leads to severe economic losses in swine-producing areas of the world. However, current antiviral strategies cannot provide highly effective protection. In this study, three theoretically effective interference target sites (71-91, 144-164, 218-238) targeting the nucleocapsid (N) gene of PRRSV were designed and selected, and then three siRNA-expressing plasmids were constructed, respectively named p2.1-N71, p2.1-N144, and p2.1-N218. The recombinant siRNA-expressing plasmids were transfected into Marc-145 cells; then the cells were infected with PRRSV (JL07SW strain); finally, after incubation for 48 h, the antiviral activity of those siRNA-expressing plasmids in Marc-145 cells was assessed by cytopathic effects, virus titers, indirect immunofluorescence, and quantitative real-time PCR. Experimental results demonstrated that these three siRNA-expressing plasmids could effectively and significantly inhibit the replication of PRRSV by 93.2%, 83.6%, and 89.2% in Marc-145 cells, respectively. Among these three siRNA-expressing plasmids, p2.1-N71 was found to be most effective, while p2.1-N144 and p2.1-N218 displayed relatively weak inhibition of virus replication. The results indicated that siRNA-expressing plasmids targeting the N gene of PRRSV could significantly inhibit PRRSV replication in Marc-145 cells. Based on our experimental results and previous reports, the 71-91, 179-197, and 234-252 sites of the N gene are good choices to effectively inhibit the replication of PRRSV, and this RNA interference technique can be a potential anti-PRRSV strategy.

Citations

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Role of microRNAs in host defense against porcine reproductive and respiratory syndrome virus infection: a hidden front line
Xuewei Huang, Weiye Liu
Frontiers in Immunology.2024;[Epub] CrossRef
Porcine reproductive and respiratory syndrome virus infection induces microRNA novel-216 production to facilitate viral-replication by targeting MAVS 3´UTR
Xuegang Luo, Sha Xie, Xingsheng Xu, Yao Zhang, Yun Huang, Dongmei Tan, Yi Tan
Veterinary Microbiology.2024; 292: 110061. CrossRef
Antiviral Strategies against PRRSV Infection
Taofeng Du, Yuchen Nan, Shuqi Xiao, Qin Zhao, En-Min Zhou
Trends in Microbiology.2017; 25(12): 968. CrossRef
Anti-PRRSV effect and mechanism of tetrahydroaltersolanol Cin vitro
Song-Lin Zhang, Yi-Chun Wu, Fan Cheng, Zhi-Yong Guo, Jian-Feng Chen
Journal of Asian Natural Products Research.2016; 18(3): 303. CrossRef
Cellular microRNA miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by activating innate antiviral immunity
Xiaojuan Jia, Yuhai Bi, Jing Li, Qing Xie, Hanchun Yang, Wenjun Liu
Scientific Reports.2015;[Epub] CrossRef
Inhibition of porcine reproductive and respiratory syndrome virus replication in vitro using DNA-based short antisense oligonucleotides
Longlong Zheng, Xiang Li, Lingyun Zhu, Wengui Li, Junlong Bi, Guishu Yang, Gefen Yin, Jianping Liu
BMC Veterinary Research.2015;[Epub] CrossRef

Research Support, Non-U.S. Gov't

Packaging of Porcine Reproductive and Respiratory Syndrome Virus Replicon RNA by a Stable Cell Line Expressing Its Nucleocapsid Protein: Byung-Hak Song , Jeong-Min Kim , Jin-Kyoung Kim , Han-Saem Jang , Gil-Nam Yun , Eun-Jin Choi , Jae-Young Song , Sang-Im Yun , Young-Min Lee; J. Microbiol. 2011;49(3):516-523. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1280-1

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Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, is one of the most common and economically important swine pathogens. Although both live-attenuated and killed-inactivated vaccines against the virus have been available for a decade, PRRSV is still a major problem in the swine industry worldwide. To explore the possibility of producing single-round infectious PRRSV replicon particles as a potential vaccine strategy, we have now generated two necessary components: 1) a stable cell line (BHK/Sinrep19/PRRSV-N) that constitutively expresses the viral nucleocapsid (N) protein localized to the cytoplasm and the nucleolus and 2) a PRRSV replicon vector (pBAC/PRRSV/Replicon-ΔN) with a 177-nucleotide deletion, removing the 3′-half portion of ORF7 in the viral genome, from which the self-replicating propagation-defective replicon RNAs were synthesized in vitro by SP6 polymerase run-off transcription. Transfection of this replicon RNA into N protein-expressing BHK-21 cells led to the secretion of infectious particles that packaged the replicon RNA, albeit with a low production efficiency of 0.4×102 to 1.1×102 infectious units/ml; the produced particles had only single-round infectivity with no cell-to-cell spread. This trans-complementation system for PRRSV provides a useful platform for studies to define the packaging signals and motifs present within the viral genome and N protein, respectively, and to develop viral replicon-based antiviral vaccines that will stop the infection and spread of this pathogen.

Validation Study

Development and Validation of a Recombinant Nucleocapsid Protein-Based ELISA for Detection of the Antibody to Porcine Reproductive and Respiratory Syndrome Virus: Jia-Qi Chu , Xu-Min Hu , Myung-Cheol Kim , Chang-Sik Park , Moo-Hyung Jun; J. Microbiol. 2009;47(5):582-588. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0033-x

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Abstract: Three indirect enzyme-linked immunosorbent assays (iELISA) based on the North American like (NA-like), European like (EU-like) and co-expressed NA- and EU-like recombinant nucleocapsid proteins (N-protein) of porcine reproductive and respiratory syndrome virus (PRRSV) were validated for the detection of the antibodies in porcine sera. A total of 422 serum samples from unvaccinated pigs were tested. The cut-off value was optimized by a two-graph receiver operating characteristics analysis at a 95% confidence level. This assay was validated with Western blot analysis and IDEXX HerdChek™ ELISA. Cross-reactivity results showed that iELISA was PRRSV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 10%. The results indicate that iELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PRRSV infection at low cost, and is potentially useful to evaluate the efficiency of various vaccines against PRRSV.

Journal Article

Nucleocapsid Amino Acids 211 to 254, in Particular, Tetrad Glutamines, are Essential for the Interaction Between the Nucleocapsid and Membrane Proteins of SARS-Associated Coronavirus: Xiaonan Fang , Lin-Bai Ye , Yijuan Zhang , Baozong Li , Shanshan Li , Lingbao Kong , Yuhua Wang , Hong Zheng , Wei Wang , Zhenghui Wu; J. Microbiol. 2006;44(5):577-580.; DOI: https://doi.org/2437 [pii]

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Abstract: GST pull-down assays were used to characterize the SARS-CoV membrane (M) and nucleocapsid (N) interaction, and it was found that the amino acids 211-254 of N protein were essential for this interaction. When tetrad glutamines (Q) were replaced with glutamic acids (E) at positions of 240-243 of the N protein, the interaction was disrupted.

Research Support, Non-U.S. Gov't

Morphological, Phylogenetic and Biological Characteristics of Ectropis obliqua Single-Nucleocapsid Nucleopolyhedrovirus: Xiu-cui Ma , Hai-Jun Xu , Mei-Jun Tang , Qiang Xiao , Jian Hong , Chuan-Xi Zhang; J. Microbiol. 2006;44(1):77-82.; DOI: https://doi.org/2333 [pii]

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Abstract: The tea looper caterpillar, Ectropis obliqua, is one of the major pests of tea bushes. E. obliqua single-nucleocapsid nucleopolyhedrovirus (EcobSNPV) has been used as a commercial pesticide for biocontrol of this insect. However only limited genetic analysis for this important virus has been done up to now. EcobSNPV was characterized in this study. Electron microscopy analysis of the occlusion body showed polyhedra of 0.7 to 1.7 μm in diameter containing a single nucleocapsid per envelope of the virion. A 15.5 kb genomic fragment containing EcoRI-L, EcoRI-N and HindIII-F fragments, was sequenced. Analysis of the sequence revealed that the fragment contained eleven potential open reading frames (ORFs): lef-1, egt, 38.7k, rr1, polyhedrin, orf1629, pk-1, hoar and homologues to Spodoptera exigua multicapsid NPV (SeMNPV) ORFs 15, 28, and 29. Gene arrangement and phylogeny analysis suggest that EcobSNPV is closely related to the previously described Group II NPV. Bioassays on lethal concentration (LC50 and LC90) and lethal time (LT50 and LT90) were conducted to test the susceptibility of E. obliqua larvae to the virus.

Journal Article

Analysis of Immune Responses Against Nucleocapsid Protein of the Hantaan Virus Elicited by Virus Infection or DNA Vaccination: Gyu-Jin Woo , Eun-Young Chun , Keun Hee Kim , Wankee Kim; J. Microbiol. 2005;43(6):537-545.; DOI: https://doi.org/2292 [pii]

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Abstract: Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2Kb restricted T-cell epitopes of NP. The NP-specific CD8+ T cell response was analyzed using a 51Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8+ T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8+ T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 ~ 4 weeks after immunization and maximized at 6~8 weeks. NP-specific CD8+ T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.

Production of the Nucleocapsid Protein of Newcastle Disease Virus in Escherichia coli and its Assembly into Ring- and Nucleocapsid-like Particles: Chiew Ling Kho , Wen Siang Tan , Khatijah Yusoff; J. Microbiol. 2001;39(4):293-299.

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Abstract: The nucleocapsid (NP) protein of Newcastle disease virus (NDV) and its derivative (NP_cfus ) containing the myc region and six histidine residues fused to its C-terminus were expressed abundantly in Escherichia coli. The proteins were purified by sucrose gradient centrifugation. Both the NP and NP_cfus proteins self-assembled into ring-like particles with a diameter of 24 +- 2 nm around a central hole of 7 +- 1 nm. Some of these ring-like particles stacked together to form nucleocapsid-like structures which are heterogeneous in length with a diameter of 20 +- 2 nm and a central hollow of 5 +- 1 nm. Only a very small amount of the monomers in the particles was linked by inter-molecular disulfide bonds. Fusion of the C-terminal end to 29 amino acids inclusive of the myc epitope and His-tag did not impair ring assembly but inhibited the formation of the long herringbone structures. Immunogold labeling of the particles with the anti-myc antibody showed that the C-terminus of the NP_cfus protein is exposed on the surface of these ring-like particles.

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