• The Plant Pathology Journal
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• v.23 no.4
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• pp.281-286
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• 2007

Interactions between viral proteins and host proteins are essential for virus replication. Especially, translation of viral genes completely depends on the host machinery. In potyviruses, interactions of genome-linked viral protein (VPg) with host translation factors including eIF4E, eIF(iso)4E, and poly(A)-binding protein (PABP) has previously been characterized. In this study, we investigated interactions between Soybean mosaic virus (SMV) viral proteins and host translation factors by yeast two-hybrid system. SMV VPg interacted with eIF4E, eIF(iso)4E, and PABP in yeast two-hybrid system, while SMV helper component proteinase (HC-pro) interacted with neither of those proteins. The interaction between SMV NIb and PABP was also detected. These results are consistent with those reported previously in other potyviruses. Interestingly, we found reproducible and specific interactions between SMV coat protein (CP) and PABP. Deletion analysis showed that the region of CP comprising amino acids 116 to 206 and the region of PABP comprising amino acids 520 to 580 are involved in CP/PABP interactions. Soybean library screening with SMV NIb by yeast two-hybrid assay also identified several soybean proteins including chlorophyll a/b binding preprotein, photo-system I-N subunit, ribulose 1,5-biphosphate carboxylase, ST-LSI protein, translation initiation factor 1, TIR-NBS type R protein, RNA binding protein, ubiquitin, and LRR protein kinase. Altogether, these results suggest that potyviral replicase may comprise a multi-protein complex with PABP, CP, and other host factors.

• Journal of Veterinary Science
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• v.22 no.4
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• pp.56.1-56.10
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• 2021

Background: Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. Objectives: We aimed to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). Methods: A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. Results: The virus propagated in VERO cells was confirmed as RABV expressing GFP. The ERAGS-GFP showed the highest titer (10^8.0 TCID₅₀/mL) in VERO cells at 5 days post-inoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN (r = 0.95). Conclusions: We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test.

• IMMUNE NETWORK
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• v.6 no.1
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• pp.20-26
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• 2006

Background: Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease that is characterized by invasive synovial hyperplasia, leading to progressive joint destruction. Recent studies have described that RA is caused by virus, bacteria or outside material. Approximately 2 to 20% of RA cases arc reported to be associated with infected hepatitis C virus (HCV). However, the mechanisms underlying virus-induced RA are still unknown. Moreover, few molecular studies have addressed the inflammatory aspects of HCV-associated autoimmune RA. In this study, we aimed to determine whe ther or not another HCV core protein transactivates the IL-8 gene expression, prototypic chemokine, in synovial cell. Methods: To establish the HCV core expressing stable synovial cell line, pCI-neo-core, a plasmid encoding HCV core protein, were transfected to HIG-82 cell line that is an established cell line from rabbit periaricular soft tissue. We examined the morphological changes and cell cycle distribution of HIG-82 cells with expression of HCV core protein by inverted microscopy and flow cytometry analysis, respectively. Also, we determined the mRNA levels of Interleukin (IL)-6 and IL-8 related to the inflammation by RT-PCR and then analyzed regulation of IL-8 expression by the NF-${\kappa}B$ pathway. Results: Our study showed no significant differences in morphology and cell cycle between HIG-82 control cell line and HIG-82 expressing HCV core protein. However, expression of HCV core protein induces the IL-8 mRNA expression in HIG-82 core cells via activated NF-${\kappa}B$ pathway. Conclusion: These results suggest that HCV core protein can lead to enhanced IL-8 expression. Such a proinflammatory role may contribute to the etiologic pathogenesis in RA patients with HCV infection.

• Genomics & Informatics
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• v.16 no.4
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• pp.23.1-23.5
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• 2018

Virus taxonomy was initially determined by clinical experiments based on phenotype. However, with the development of sequence analysis methods, genotype-based classification was also applied. With the development of genome sequence analysis technology, there is an increasing demand for virus taxonomy to be extended from in vivo and in vitro to in silico. In this study, we verified the consistency of the current International Committee on Taxonomy of Viruses taxonomy using an in silico approach, aiming to identify the specific sequence for each virus. We applied this approach to norovirus in Caliciviridae, which causes 90% of gastroenteritis cases worldwide. First, based on the dogma "protein structure determines its function," we hypothesized that the specific sequence can be identified by the specific structure. Firstly, we extracted the coding region (CDS). Secondly, the CDS protein sequences of each genus were annotated by the conserved domain database (CDD) search. Finally, the conserved domains of each genus in Caliciviridae are classified by RPS-BLAST with CDD. The analysis result is that Caliciviridae has sequences including RNA helicase in common. In case of Norovirus, Calicivirus coat protein C terminal and viral polyprotein N-terminal appears as a specific domain in Caliciviridae. It does not include in the other genera in Caliciviridae. If this method is utilized to detect specific conserved domains, it can be used as classification keywords based on protein functional structure. After determining the specific protein domains, the specific protein domain sequences would be converted to gene sequences. This sequences would be re-used one of viral bio-marks.

• Korean Journal of Veterinary Research
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• v.32 no.3
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• pp.399-402
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• 1992

The causative virus causing rabbit hepatitis has been further characterized by evaluating viral proteins and viral nucleic acids of purified viruses from the liver of the experimentally infected rabbits. Rabbit hepatitis virus has one major structural protein of 54 kilodaltons and some minor proteins. Vrial RNA was resistant to DNAse I. The size of viral nucleic acid of this virus was calculated to be about 7.5 kilobases. These findings indicate that rabbit hepatitis virus belongs to the family Caliciviridae.

• KSBB Journal
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• v.13 no.6
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• pp.649-655
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• 1998

Hantaviruses are rodent hosts-borne viruses belonging to the family Bunyaviridae, and are etiologic agents for two acute diseases, i.e., Haemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS). There have been a lot of reports on prophylactic vaccines and diagnostics for the diseases, but most of viral antigens have been prepared by eukaryotic cell culture. Nucleocapsid proteins of Hantaviruses are known as the major viral antigens. Thereby, we prepared nucleocapsid genes of Hantaan virus and Seoul virus by RT-PCR and cloned into plasmid vectors, pET-3a and pKK223-3. Both genes were expressed in Escherichia coli with higher expression level of Seoul viral nucleocapsid protein compared to that of Hantaan in pET-3a. Hantaan viral gene was expressed much higher level in plasmid pET-3a that in pKK223-3. About 30% of expressed nucleocapsid protein was soluble and the rest was remained in insoluble fraction.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2004.10a
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• pp.25-25
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• 2004

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.844-850
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• 2000

The specific binding and internalization of viral particles is an essential step for the successful infection of viral pathogens. In the case of the hepatitis B virus (HBV), virions bind to the host cell via the preS domain of the viral surface antigen and are subsequently internalized by endocytosis. HBV-preS specific receptors are primarily expressed on hepatocytes, however, viral DNA and proteins have also been detected in extrahepatic sites, suggsting that celluar recepators for HBV may also exist on extrahepatic cells. Recently, an EBV-transformed B-cell line was identified onto which the preS region binds in a receptor-ligand specific manner. In this study, this specific interaction was further characterized, and the binding region within the preS protein was locaized. Also the internalization after host cell attachment was visualized and analyzed by fluorescence-labeled HBV-preS1 proteins using confocal microscopy. Energy depletion by sodium azide treatment effectively inhibited the internalization of the membrane-bound preS1 ligands, thereby indicating an energy-dependent receptor-mediated endocytotic pathway. Accordingly, the interaction of HBV-pres! with this specific B-cell line may serve as an effective model for an infection pathway in extrahepatic cells.

• The Plant Pathology Journal
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• v.21 no.4
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• pp.334-342
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• 2005

The prototype Chlorella virus PBCV-l encodes 11 tRNA genes and over 350 protein-encoding genes in its 330 kbp genome. Initial attempts to overexpress the recombinant A189/192R protein, a putative virus attachment protein, in E. coli strain BL21(DE3) SI were unsuccessful, and multiple protein bands were detected on Western blots. However, the full-length A189/192R recombinant protein or fragments derived from it were detected when they were expressed in E. coli BL21 CodonPlus (DE3) RIL, which contains extra tRNAs. Codon usage analysis of the a189/192r gene showed highly biased usage of the AGA and AVA codons compared to genes encoded by E. coli and Chlorella. In addition, there were biases of XXA/U($56\%$) and XXG/ C($44\%$) in the codons recognized by the viral tRNAs, which correspond to the codon usage bias in the PBCV-1 genome of XXA/U ($63\%$) over those ending in XXC/G ($37\%$). Analysis of the codon usage in the major capsid protein and DNA polymerase showed preferential usage of codons that can be recognized by the viral tRNAs. The Asn (AAC) and Lys (AAG) codons whose corresponding tRNA genes are duplicated in the tRNA gene cluster were the most abundant (i.e., preferred) codons in these two proteins. The tRNA genes encoded in the PBCV-l genome seem to play a very important role during the synthesis of viral proteins through supplementing the tRNAs that are frequently used in viral proteins, but are rare in the host cells. In addition, these tRNAs would help the virus to adapt to a wide range of hosts by providing tRNAs that are rare in the host cells.

• Korean Journal of Microbiology
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• v.21 no.2
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• pp.61-70
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• 1983

Synchronized cat kidney cells chronically infected with feline leukemia virus (FeLV) were used to study virus production, the synthesis of group specific antigen (gag) and envelope (env) proteins, the expression of env protein on the cell surface during the cell cycle, and the stability of viral RNA. As detecting method, we developed the radioimmunoassay (RIA) system using beta-emission of $^{131}I$ and demonstrated the validity of this system by comparison with routine RIA system using gamma-emission of $^{125}I$. The produced virus was analysed by developed RIA interval was determined by measuring reverse transcriptase activity. The results show that infected cells produce the complete virus particle containing products of gag, env and pol genes of FeLV, and maximum virus production occurs during mitosis of synchronized cells. Labeling of the cell surface of synchronized cells with $^{131}I$ shows that the amount of $gp70^{env}$ on the cell surface parallels cellular gorwth. Therefore, the cell cycle-dependent release of virus is not petition RIA of synchronized cells with $^{131}I$ labeled viral proteins synthesis during the cell cycle. The rate of synthesis of gag protein shows three peaks, corresponding to the $G_1,\;late\;S\;and\;late\;G_2$ phases of cell cycle. But the rate of synthesis of env protein dose not change, suggesting that in these cells the synthesis of these two gene products in controlled seperately. In Actionomycin D treated cells, the synthesis of viral proteins decreased sharply from 8 hours after treatment, and the late S and $G_2$ peaks of gag protein synthesis were disappeared. This shows the stability of viral RNA for about 6 hours in the absence of continuing viral RNA synthesis.