• Microbiology and Biotechnology Letters
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• v.43 no.1
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• pp.1-8
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• 2015

In this study, a DNA-launched reverse genetics system was developed from a type 2 porcine reproductive and respiratory syndrome virus (PRRSV) strain, KNU-12. The complete genome of 15,412 nucleotides was assembled as a single cDNA clone and placed under the eukaryotic CMV promoter. Upon transfection of BHK-tailless pCD163 cells with a full-length cDNA clone, viable and infectious type 2 progeny PRRSV were rescued. The reconstituted virus was found to maintain growth properties similar to those of the parental virus in porcine alveolar macrophage (PAM) cells. With the availability of this type 2 PRRSV infectious clone, we first explored the biological relevance of ORF5a in the PRRSV replication cycle. Therefore, we used a PRRSV reverse genetics system to generate an ORF5a knockout mutant clone by changing the ORF5a translation start codon and introducing a stop codon at the 7^th codon of ORF5a. The ORF5a knockout mutant was found to exhibit a lack of infectivity in both BHK-tailless pCD163 and PAM-pCD163 cells, suggesting that inactivation of ORF5a expression is lethal for infectious virus production. In order to restore the ORF5a gene-deleted PRRSV, complementing cell lines were established to stably express the ORF5a protein of PRRSV. ORF5a-expressing cells were capable of supporting the production of the replicationdefective virus, indicating complementation of the impaired ORF5a gene function of PRRSV in trans.

• Korean Journal of Veterinary Research
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• v.48 no.4
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• pp.441-449
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• 2008

The genus Lactobacillus is the largest of the genera included in lactic acid bacteria and is associated with mucosal membranes of human and animal. Only a few Lactobacillus plasmid-encoded functions have been discovered and used. In this study, a novel plasmid (pILR091) was isolated from a wild L. reuteri isolated from pig and described the characteristics of its replicons, genetic organization, and relationship with other plasmids. After digestion of the plasmid, pILR091, with SalI, plasmid DNA was cloned into the pQE-30Xa vector and sequenced. The complete sequence was confirmed by the sequencing of PCR products and analyzed with the Genbank database. The isolate copy number and stability were determined by quantitative-PCR. The complete sequence of L. reuteri contained 7,185 nucleotides with 39% G-C content and one cut site by two enzymes, SalI and HindIII. The similar ori sequence of the pC194- rolling circle replication family (TTTATATTGAT) was located 63 bp upstream of the protein replication sequence, ORF 1. Total of five ORFs was identified and the coding sequence represented 4,966 nucleotides (70.4%). ORF1 of pILR091 had a low similarity with the sequence of pTE44. Other ORFs also showed low homology and E-values. The average G-C content of pILR091 was 39%, similar with that of genomic DNA. The copy number of pILR091 was determined at approximately 24 to 25 molecules per genomic DNA. These results suggested that pILR091 might be a good candidate to construct a new vector, which could be used for cloning and expression of foreign genes in lactobacilli.

• Journal of Microbiology and Biotechnology
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• v.28 no.1
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• pp.109-114
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• 2018

Coxsackievirus Type B3 (CVB3) is an enterovirus that belongs to the Picornaviridae and causes various diseases such as myocarditis and hand-foot-mouth disease. However, an effective antiviral drug is still not developed. In this study, we looked for potential inhibitors of CVB3 replication by examining the survival of CVB3-infected HeLa cells. We detected an antiviral effect by cholic acid and identified it as a candidate inhibitor of CVB3 replication. Cholic acid circulates in the liver and intestines, and it helps the digestion and absorption of lipids in the small intestine. HeLa cells were cultured in 12-well plates and treated with cholic acid (1 and $10{\mu}g/ml$) and $10^6PFU/ml$ of CVB3. After 16 h post-infection, the cells were lysed and subjected to western blot analysis and RT-PCR. The production of the viral capsid protein VP1 was dramatically decreased, and translation initiation factor eIF4G1 cleavage was significantly inhibited by treatment with $10{\mu}g/ml$ cholic acid. Moreover, cholic acid inhibited ERK signaling in CVB3-infected HeLa cells. RT-PCR showed that the amounts of the CVB3 RNA genome and mRNA for the ER stress-related transcription factor ATF4 were significantly reduced. These results showed that cholic acid strongly reduced ER stress and CVB3 proliferation. This compound can be developed as a safe natural therapeutic agent for enterovirus infections.

• Journal of fish pathology
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• v.36 no.2
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• pp.213-228
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• 2023

We evaluated the transcriptional response of interferon (IFN)-related genes in rock bream iridovirus (RBIV)-infected rock bream under high-, low-, or no-mortality conditions induced by different stocking water temperatures. Under the high susceptibility condition (group A, water temperature 26℃, 100% mortality), only the Mx gene was expressed early, with prolonged expression, and with heavy viral loads of approximately 10⁶~10⁷ major capsid protein gene copies/μL from 4 to 10 days post infection (dpi). However, IRF1, IRF3, IRF8, STAT1, ISG15, PKR, Viperin, GVIN1, IFI44, and ISG56 were activated at later time points (8 dpi) and then quickly decreased (10 dpi). For the low susceptibility condition, the water temperature was set at 23℃ for 7 days (group B) and then reduced to 17℃. Group B exhibited a 28% mortality rate, in which persistent and effective antiviral responses were observed for long periods of time. In particular, at 20 and 22 dpi, when virus replication was peaked at approximately 10⁷/μL, the expressions of most of the IFN-related genes (IRF1, IRF3, IRF8, Mx, STAT1, ISG15, PKR, Viperin, GVIN1, IFI44, and ISG56) were significantly higher in group B than in the control group. Moreover, prolonged and higher levels of IRF3 (at least 30 dpi), IRF8 (at least 30 dpi), ISG15 (at least 30 dpi), PKR (at least 28 dpi), Viperin (at least 30 dpi), and IFI44 (at least 30 dpi) were also observed in the recovery stage of infection. Under the no-susceptibility condition at 17℃ (0% mortality), significantly elevated levels of IRF3, Mx, ISG15, and PKR were observed mostly until 20 dpi. The findings indicate that RBIV infection can induce an efficient IFN-mediated antiviral immune response in low- and no-susceptibility conditions. The findings could be valuable for effective control of viral pathogens in fish.

• Journal of Life Science
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• v.32 no.6
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• pp.463-467
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• 2022

As a result of applying the COG (Cluster of Orthologous Groups of Protein) algorithm to 1,309 species to confirm the conserved genes of prokaryotes, ribosomal protein S11 (COG0100) was identified. The numbers of conservative genes were 2, 5, 5, and 6 in 1,308, 1,307, 1,306, and 1,305 species, respectively. Twenty-nine genes were conserved in over 1,302 species, and they encoded 23 ribosomal proteins, 3 tRNA synthetases, 2 translation factors, and 1 RNA polymerase subunit. Most of them were related to protein production, suggesting the importance of protein expression in prokaryotes. The highest conservative COG was COG0048 (ribosomal protein S12) among the 29 COGs. The 29 conserved genes usually have one protein for each prokaryote. COG0090 (ribosomal protein L2) had not only the lowest conservation value but also the largest standard deviation of phylogenetic distance value. As COG0090 is not only a member of the ribosome, but also a regulator of replication and transcription, it could be inferred that prokaryotes have large variations in COG0090 to survive in various environments. This study could provide data necessary for basic science, tumor control, and development of antibacterial agents.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.387-390
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• 2011

Nude mice (BALB/c) were grafted with human 293 cells and PERV (porcine endogenous retrovirus)-IRES-EGFP (a packageable retroviral vector plasmid containing an internal ribosome entry site-enhanced green fluorescent protein)-producing pig PK15 cells in order to determine whether the pig cells could transmit PERV-IRES-EGFP to mice and human 293 cells in vivo. None of the transplanted human 293 cell lines were infected by PERV, but PCR analysis identified PERV-B provirus integration into both the heart and salivary gland of the inoculated nude mice. Our data indicate that hearts and salivary glands can be used to identify PERV-B receptors.

• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.38-43
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• 2020

Hand, foot, and mouth disease (HFMD) is caused by enterovirus 71 (EV71) in infants and children under six years of age. HFMD is characterized by fever, mouth ulcers, and vesicular rashes on the palms and feet. EV71 also causes severe neurological manifestations, such as brainstem encephalitis and aseptic meningitis. Recently, frequent outbreaks of EV71 have occurred in the Asia-Pacific region, but currently, no effective antiviral drugs have been developed to treat the disease. In this study, we investigated the antiviral effect of salvianolic acid B (SalB) on EV71. SalB is a major component of the Salvia miltiorrhiza root and has been shown to be an effective treatment for subarachnoid hemorrhages and myocardial infarctions. HeLa cells were cultured in 12-well plates and treated with SalB (100 or 10 ㎍/ml) and 10⁶ PFU/ml of EV71. SalB treatment (100 ㎍/ml) significantly decreased the cleavage of the eukaryotic eIF4G1 protein and reduced the expression of the EV71 capsid protein VP1. In addition, SalB treatment showed a dramatic decrease in viral infection, measured by immunofluorescence staining. The Akt signaling pathway, a key component of cell survival and proliferation, was significantly increased in EV71-infected HeLa cells treated with 100 ㎍/ml SalB. RT-PCR results showed that the mRNA for anti-apoptotic protein Bcl-2 and the cell cycle regulator Cyclin-D1 were significantly increased by SalB treatment. These results indicate that SalB activates Akt/PKB signaling and inhibits apoptosis in infected HeLa cells. Taken together, these results suggest that SalB could be used to develop a new therapeutic drug for EV71-induced HFMD.

• Molecules and Cells
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• v.45 no.12
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• pp.911-922
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• 2022

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.189-196
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• 2017

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with formation of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Replication and transcription activator (RTA) genes are expressed upon reactivation of KSHV, which displays a biphasic life cycle consisting of latent and lytic replication phases. RTA protein expression results in KSHV genome amplification and successive viral lytic gene expression. Transcriptional activity of viral lytic genes is regulated through epigenetic modifications. In Raji cells latently infected with Epstein-Barr virus, various modifications, such as acetylation and methylation, have been identified at specific lysine residues in histone H4 during viral reactivation, supporting the theory that expression of specific lytic genes is controlled by histone modification processes. Data obtained from chromatin immunoprecipitation and quantitative real-time PCR analyses revealed alterations in the H4K8ac and H4K20me3 levels at lytic gene promoters during reactivation. Our results indicate that H4K20me3 is associated with the maintenance of latency, while H4K8ac contributes to KSHV reactivation in infected TREx BCBL-1 RTA cells.

• Journal of Microbiology and Biotechnology
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• v.28 no.10
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• pp.1736-1748
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• 2018

Brucella abortus can survive and replicate within host macrophages, and great efforts have been made to demonstrate the genes involved in pathogenicity, such as internalization, in Brucella research. Here, intracellular responses were compared between THP-1 macrophage cells stimulated with B. abortus wild-type and four mutants (C1, C10, C27, and C32) using microarray to demonstrate the role of genes related to intracellular survival and replication. These mutants were generated by deleting genes encoding BAB_RS13225 (4-hydrobenzoate 3-monooxygenase, PHBH), BAB_RS00455 (heme exporter protein cytochrome C, CcmC), BAB_RS03675 (exopolyphosphatase, PPX), and BAB_RS13225 (peptidase M24). The results showed that mutants C1 and C10 induced significant suppression of survival levels and cytokine expression relative to wild-type in the THP-1 macrophage cells. These findings suggest that the BAB_RS13225 and BAB_RS00455 genes play important roles in survival within human macrophages. Conversely, mutants C27 and C32 induced significantly higher survival level than wild-type in the cells inhibiting cellular signal transduction. It is assumed that the BAB_RS03675 and BAB_RS13225 genes play a role in cellular resistance to B. abortus. Therefore, the disrupted genes are involved in B. abortus intracellular growth, and especially in its survival, and they could be effective targets for understanding the intracellular bacterium, B. abortus.