• Journal of Microbiology and Biotechnology
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• v.20 no.8
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• pp.1163-1178
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• 2010

Plasmids play important roles in horizontal gene transfer among Vibrionaceae, but surprisingly little is known about their replication and incompatibility systems. In this study, we successfully developed a bioinformatics-assisted strategy of experimental identification of seven Vibrio plasmid replicons. Comparative sequences analysis of the seven Vibrio plasmid replicons obtained in this study together with eight published Vibrionaceae plasmid sequences revealed replication-participating elements involved in the ColE1 mode of replication initiation and regulation. Like plasmid ColE1, these Vibrionaceae plasmids encode two RNA species (the primer RNA and the antisense RNA) for replication initiation and regulation, and as a result, the 15 Vibrionaceae plasmids were designated as ColE1-like Vibrionaceae (CLV) plasmids. Two subgroups were obtained for the 15 CLV plasmids, based on comparison of replicon organization and phylogenetic analysis of replication regions. Coexistence of CLV plasmids were demonstrated by direct sequencing analysis and Southern hybridization, strongly suggesting that the incompatibility of CLV plasmids is determined mainly by the RNA I species like the ColE1-like plasmids. Sequences resembling the conserved Xer recombination sites were also identified on the CLV plasmids, indicating that the CLV plasmids probably use the host site-specific recombination system for multimer resolution like that used by ColE1-like plasmids. All the results indicated that the 15 plasmids form a new ColE1-like group, providing a basis for the rapid characterization and classification of Vibrionaceae plasmids.

• Korean Journal of Microbiology
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• v.39 no.4
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• pp.235-241
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• 2003

We have previously demonstrated that intracellular expression of an RNA aptamer termed RRE40, which was selected in vitro to bind HIV Rev 10-fold much tighter than wild-type RRE, efficiently protected human CD4+ T cell line, CEM, from HIV-1. In this study, to evaluate the efficacy of the RRE40 RNA in clinical settings, polyclonal CD4+ peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors expressing RRE40 decoy RNA and then challenged with clinical isolates of HIV-1. In contrast to the control cells transduced with vectors expressing control tRNA, intracellular expression of RRE40 RNA more effectively inhibited HIV-1 replication in CD4+ PBLs. However, transient and diminished inhibition, rather than complete inhibition, of HIV-1 replication in PBLs expressing RRE40 decoys have been observed. These results suggest that RRE40 decoy RNA would be useful to inhibit HIV-1 replication in cells. However, development of more efficient gene transfer protocols and/or more effective decoy RNAs would be needed to apply RNA decoy to modulate HIV-1 patient.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.14-14
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• 2003

Potato virus X (PVX), the type member of Potexvirus genus, is a flexuous rod-shaped virus containing a single-stranded (＋) RNA. Infection by PVX produces genomic plus- and minus-strand RNAs and two major subgenomic RNAs (sgRNAs). To understand the mechanism for PVX replication, we are studying the cis- and/or trans-acting elements required for RNA replication. Previous studies have shown that the conserved sequences located upstream of two major sgRNAs, as well as elements in the 5' non-translated region (NTR) affect accumulation of genomic and sg RNAs. Complementarity between sequences at the 5' NTR and those located upstream of two major sgRNAs and the binding of host protein(s) to the 5' NTR have shown to be important for PVX RNA replication. The 5 NTR of PVX contains single-stranded AC-rich sequence and stem-loop structure. The potential role(s) of these cis-elements on virus replication, assembly, and their interaction with viral and host protein(s) during virus infection will be discussed based on the data obtained by in vitro binding, in vitro assembly, gel shift mobility assay, host gene expression profiling using various mutants at these regions.

• Journal of Microbiology
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• v.41 no.4
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• pp.300-305
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• 2003

Intracellular expression of RNA decoys, such as TAR or RRE decoy, has been previously shown to protect immune cells from human immunodeficiency virus type 1 (HIV-1) replication by inhibiting the binding of the HIV-1 regulatory protein to the authentic HIV RNA sequence. However, HIV-1 challenge experiments of primary human T cells, which express the RNA decoy, demonstrated that the cells were only transiently protected, and hence, more improved protocols for HIV-1 inhibition with the RNA decoys need to be developed. In this report, in order to develop a more effective RNA decoy, we analyzed and compared the ability of a series of RNA decoy derivatives in inhibiting HIV-1 replication in CEM cells. Using an improved tRNA cassette to express high levels of RNA decoy transcripts in cells, we found that co-expression of both TAR and RRE decoys, in the form of an aligned sequence in a single transcription cassette, much more potently blocked cells from HIV-1 than the expression of only one kind of RNA decoy. This observation will have an important implication for experiments involving optimization of clinical applications in RNA decoy-based gene therapy against HIV-1.

• BMB Reports
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• v.43 no.4
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• pp.284-290
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• 2010

Replication of positive-strand RNA virus is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Ectropis obliqua virus (EoV), a newly identified insect virus belonging to the family Iflaviradae, we expressed the RNA polymerase domain in Escherichia coli and purified it on a Ni-chelating HisTrap affinity column. It is demonstrated that EoV RdRp initiated RNA synthesis in a primer and poly (A)-dependent manner in vitro. Furthermore, the effect of primer concentration, temperature, metal ions ($Mg^{2+}$, $Mn^{2+}$, and $K^+$) on enzymatic activity were determined. Our study represented a first step towards understanding the mechanism of EoV replication.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.188-193
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• 2011

As a specific and effective therapeutic genetic material against hepatitis C virus (HCV) multiplication, HCV internal ribosome entry site (IRES)-targeting hammerhead ribozyme which activity is allosterically regulated by HCV regulatory protein, NS5B RNA replicase, was constructed. The allosteric ribozyme was composed of sequence of RNA aptamer to HCV NS5B, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding NS5B to the aptamer, and sequence of ribozyme targeting +382 nucleotide of HCV IRES. With real-time PCR analysis, the ribozyme was found to efficiently inhibit HCV replicon replication in cells. Of note, the allosteric ribozyme was shown to inhibit HCV replicon replication more efficiently than either HCV genome-targeting ribozyme or NS5B aptamer only. This allosteric ribozyme can be used as a lead genetic agent for the specific and effective suppression of HCV replication.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.10a
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• pp.66-77
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• 2000

For a variety of viruses, the primary virus infection has been shown to prevent superinfection with a homologous secondary virus; however, the mechanism of exclusion has not been clearly understood. In this work, we demonstrated that BVDV -infected MDBK cells were protected from superinfection with a homologous superinfecting BVDV, one of the positive-sense RNA pestiviruses, but not with an unrelated rhabdovirus, such as vesicular stomatitis virus. Once superinfection exclusion was established by a primary infection with BVDV, the transfected infectious BVD viral RNA genome was shown to be competent for viral translation, but not viral replication. In addition, our results also demonstrated that upon superinfection, the. viral RNA genome of viral particles was not transferred into the cytoplasm of BVDV -infected cells. Using newly developed system involving rapid generation of the MDBK cells expressing BVD viral proteins, we subsequently found that expression of the viral structural proteins was dispensable for the block occurring at the level of viral RNA replication, but required for the exclusion at the level of viral entry step. Altogether, these findings provide evidence that the superinfection exclusion of BVDV occurs not only at the level of viral replication in which the viral replicase are involved, but also at the level of viral entry with which the viral structural proteins are associated, and that a cellular factor(s) play an essential role in this process.

• Journal of Life Science
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• v.30 no.11
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• pp.947-955
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• 2020

The foot-and-mouth disease virus (FMDV), a member of the Aphthovirus genus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. During replication of the FMDV RNA (ribonucleic acid) genome, FMDV-encoding RNA polymerase 3D acts in a highly location-specific manner. This suggests that specific RNA structures recognized by 3D polymerase within non-coding regions of the FMDV genome assist with binding during replication. One such region is the cis-acting replication element (CRE), which functions as a template for RNA replication. The FMDV CRE adopts a stem-loop conformation with an extended duplex stem, supporting a novel 15-17 nucleotide loop that derives stability from base-stacking interactions, with the exact RNA nucleotide sequence of the CRE producing different RNA secondary structures. Here, we show that CRE sequences of FMDVs isolated in Korea from 2010 to 2017 exhibit A and O genotypes. Interestingly, variations in the RNA secondary structure of the Korean FMDVs are consistent with the phylogenetic relationships between these viruses and reveal the specificity of FMDV infections for particular host species. Therefore, we conclude that each genetic clade of Korean FMDV is characterized by a unique functional CRE and that the evolutionary success of new genetic lineages may be associated with the invention of a novel CRE motif. Therefore, we propose that the specific RNA structure of a CRE is an additional criterion for FMDV classification dependent on the host species. These findings will help correctly analyze CRE sequences and indicate the specificity of host species for future FMDV epidemics.

• Applied Biological Chemistry
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• v.48 no.4
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• pp.301-310
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• 2005

Foot-and-mouth disease (FMD) is a highly contagious disease of mammals and has a great potential for causing severe economic loss in susceptible cloven-hoofed animals, such as cattle, pigs, sheep, goats and buffalo. FMDV, a member of the Aphthovirus genus in the Picornaviridae family, is a non-enveloped icosahedral virus that contains a positive sense RNA of about 8.2 kb in size. The genome carries one open reading frame consisting of 3 regions: capsid protein coding region P1, replication related protein coding region P2, and RNA-dependent RNA polymerase coding region P3. FMDV infects pharynx epithelial cell in the respiratory tract and viral replication is active in lung epithelial cell. Morbidity is extremely high. A FMD outbreak in Korea in 2002 caused severe economic loss. Although intense research is undergoing to develop appropriate drugs to treat FMDV infection, there is no specific therapeutic for controlling FMDV infection. Moreover, there is an increasing demand for the development of vaccine strategies against FMDV infection in many countries. In this report, more effective prevention strategies against FMDV infection were reviewed.

• Molecules and Cells
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• v.21 no.3
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• pp.330-336
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• 2006

Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, ${\alpha}$-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.