• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.221-233
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• 1999

Prospect Hill Virus (PHV) is the well known serotype of hantavirus, a newly established genus in family Bunyaviridae. Extensive studies have upheld the original view of PHV genetics with three genes such as nucleocapsid (N) protein, envelope proteins (G1, G2) and RNA dependent RNA polymerase. In this study, we report the existence of additional gene that is encoded in an overlapping reading frame of the N protein gene within S genome segment of PHV. This gene is expected to encode a nonstructural small (NSs) protein and it seems to be only found in PHV infected cell. The presence and synthesis of NSs protein could be demonstrated in the cell infected with PHV using anti-peptide sera specific to the predicted amino acid sequence deduced from the second open reading frame. Ribosomal synthesis of this protein appears to occur at AUG codon at the 83rd base of S genome segment, downstream of N protein initiation codon. This protein is small in size (10.4 KDa) and highly basic in nature. The expression strategy of NSs protein appears that a signal mRNA is used to translate both N and NSs protein in PHV infected cell. 10 KDa protein in virus infected cell lysates can bind to mimic dsRNA. This fact strongly suggests that NSs protein may be involved in virus replication on late phase of viral life cycle.

• Journal of Microbiology
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• v.41 no.3
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• pp.239-247
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• 2003

The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single site-specific cleavage in the 5' untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5' 249-342) and several synthetic RNA transcripts encoding point-mutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5' 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg$\^$2＋/ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg$\^$2＋/ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca$\^$2＋/ and Mn$\^$2＋/ ions, among the reagents tested, could change the mobility of the substrate RNA 5' 249-342 on a gel similarly to that of Mg$\^$2＋/ ions, but only Ca$\^$2＋/ ions identically showed the stabilizing effect of Mg$\^$2＋/ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg$\^$2＋/ or Ca$\^$2＋/) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

• The Korean Journal of Blood Transfusion
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• v.23 no.2
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• pp.152-161
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• 2012

Background: Since Jan. 2012, for performance evaluation of viral reagents, analysis of domestic samples has been recommended in order to obtain approval from the KFDA when they are first introduced to Korea. This regulation requires the standard domestic materials driven from locally infected samples. We tried manufacturing the plasma working standards of HBV, HCV, and HIV NAT using a mixed titer of viral loads. Methods: Forty three HBV DNA positive plasmas, 25 HCV RNA positive plasmas, and 26 HIV RNA positive plasmas were evaluated according to viral load and genotype. Several plasma units, which had high-titer viral loads and the common viral genotypes in Korea, were selected as the source materials for each viral standard. To adjust the appropriate concentration based on the detectable range of variable viral reagents, the source plasma was diluted to several concentrations, divided into small vials, and analyzed for quantification. Results: The 13 plasma working standards, which had variable viral loads for the mixed titer performance panel of HIV, HCV, and HBV NAT, were produced. Conclusion: These national standard materials were first produced in order to supply the mixed titer performance panel for the viral NAT reagent of the level IV transfusion related high-risk group in Korea.

• Bulletin of the Korean Chemical Society
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• v.26 no.2
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• pp.285-291
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• 2005

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is the viral RNA-dependent RNA polymerase (RdRp), which is the essential catalytic enzyme for the viral replication and is an appealing target for the development of new therapeutic agents against HCV infection. A small amount of serum from a single patient with hepatitis C was used to get the genome of a Korean HCV isolate. Sequence analysis of NS5B 1701 nucleotides showed the genotype of a Korean isolate to be subtype 1b. The soluble recombinant HCV NS5B polymerase lacking the C-terminal 24 amino acids was expressed and purified to homogeneity. With the highly purified NS5B protein, we established in vitro systems for RdRp activity to identify potential polymerase inhibitors. The rhodanine family compounds were found to be potent and specific inhibitors of NS5B from high throughput screening (HTS) assay utilizing the scintillation proximity assay (SPA) system. The binding mode of an inhibitor was analyzed by measuring various kinetic parameters. Lineweaver-Burk plots of the inhibitor suggested it binds not to the active site of NS5B polymerase, but to an allosteric site of the enzyme. The activity of NS5B in in vitro polymerase reactions with homopolymeric RNA requires interaction with multiple substrates that include a template/primer and ribonucleotide triphosphate. Steady-state kinetic parameter, such as Km, was determined for the ribonucleotide triphosphate. One of compounds found interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitively with respect to UTP. Furthermore, we also investigated the ability of the compound to inhibit NS5B-directed viral RNA replication using the Huh7 cell-based HCV replicon system. The investigation is potentially very useful for the utility of such compounds as anti-hepatitic agents.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.529-539
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• 2021

The beet armyworm, Spodoptera exigua, is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Double-stranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua.

• BMB Reports
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• v.48 no.3
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• pp.147-152
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• 2015

Small interfering RNA (siRNA) functions through pairing with specific mRNA sequences and results in the mRNA's degradation. It is a potential therapeutic approach for many diseases caused by altered gene expression. The delivery of siRNA is still a major problem due to its rapid degradation in the circulation. Various strategies have been proposed to help with the cellular uptake of siRNA and short or small hairpin RNA (shRNA). Here, we reviewed recently published data regarding local applications of siRNA. Compared with systemic delivery methods, local delivery of siRNA/shRNA has many advantages, such as targeting the specific tissues or organs, mimicking a gene knockout effect, or developing certain diseases models. The eye, brain, and tumor tissues are 'hot' target tissues/organs for local siRNA delivery. The siRNA can be delivered locally, in naked form, with chemical modifications, or in formulations with viral or non-viral vectors, such as liposomes and nanoparticles. This review provides a comprehensive overview of RNAi local administration and potential future applications in clinical treatment.

• Biomedical Science Letters
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• v.17 no.3
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• 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.

• BMB Reports
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• v.30 no.3
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• pp.234-236
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• 1997

Chronic hepatitis C virus (HCV) infection is associated with the rapid development of cirrhosis and hepatocellular carcinoma. It has been reported that the amount of HCV RNA may be correlated with the progression of hepatitis and may be a prognostic marker for treatment of HCV patients. The direct detection of HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR) is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. In the present study, we developed the method for HCV quantitation using competitive reverse transcription (CRT)-PCR using the deleted HCV standard. The serially diluted standard was added in titrated amounts to the target HCV RNA. The mixture was then reverse transcribed and amplified in the same reaction tube. The methods were evaluated using over 110 HCV-PCR positive samples in Koreans. About 59% of the samples were judged to contain $10^{5}-10^{6}$ copies of HCV RNA in 1 ml of serum.

• BMB Reports
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• v.39 no.5
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• pp.571-577
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• 2006

Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis virus ($N{\omega}V$). To establish the function of DpTV RNA genome and to better understand the mechanism of viral replication, the putative RNA-dependent RNA polymerase (RdRp) domain has been cloned and expressed in Escherichia coli. The recombinant protein was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate viral RNA synthesis in a primer-independent manner but not by terminal nucleotidyle transferase activity in the presence of $Mg^{2+}$ and RNA template. Mutation of the GDD to GAA interferes with the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.