• 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.

• Journal of fish pathology
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• v.34 no.2
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• pp.177-184
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• 2021

Vaccines based on single-cycle viruses that are replication-incompetent due to knockout of replication-related structural gene(s) are more immunogenic than inactivated or subunit vaccines and can be used as delivery vehicles for foreign antigens without concerns on the reverting to virulent forms. The aim of this study was to develop a delivery vehicle for nervous necrosis virus (NNV)-like particles (VLPs) using G gene deleted single-cycle VHSV (rVHSV-𝚫G). Recombinant single-cycle VHSVs carrying NNV capsid protein gene between N and P gene of rVHSV-𝚫G genome (rVHSV-𝚫G-NNV_Cap) were rescued by reverse genetic technology. The successful expression of NNV capsid protein in cells infected with rVHSV-𝚫G-NNV_Cap was demonstrated by Western blot analysis, and the production of NNV VLPs in infected cells was confirmed using an electron microscopy. The results suggest that single-cycle VHSVs can be used as a safe delivery vehicle for NNV VLPs, and can be extended to other pathogens for the development of prophylactic vaccines.

• Journal of Veterinary Science
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• v.21 no.6
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• pp.85.1-85.6
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• 2020

A cold-adapted porcine reproductive and respiratory syndrome virus (CA-VR2332) was generated from the modified live virus strain VR2332. CA-VR2332 showed impaired growth when cultured at 37℃ with numerous mutations (^S731^F, ^E819^D, ^G975^E, and ^D1014^N) in the hypervariable region of the NSP2, in which the mutation ^S731^F might play a vital role in viral replication at 30℃. Conserved amino acid sequences of the GP5 protein suggests that CA-VR2332 is a promising candidate for producing an effective vaccine against PRRSV infection. Further studies on replication and immunogenicity in vivo are required to evaluate the properties of CA-VR2332.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.121-127
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• 2004

Amphotericin B (AmB), an amphipathic polyene macrolide, is an antifungal drug produced by Streptomyces nodosus. Recently, AmB has been shown to exert antiviral activity against rubella virus and human immunodeficiency virus by different mechanisms. In this study, we evaluated the antiviral effect of AmB against Japanese encephalitis virus (JEV) and investigated which step of the viral life cycle was inhibited by AmB to understand the mechanism of antiviral action of AmB. AmB reduced both plaque size and number in the infected cells in a dose-dependent manner. In addition, a 200-fold reduction of infectious virus titer was observed by treatment of infected cells with $5\mug/ml$ of AmB. AmB acted at the post virus-infection step, but not during adsorption of virus to host cells. Western blot analysis revealed that the accumulated level of JEV envelope protein dramatically decreased in the infected cells by treatment with $5-10\mug/ml$ of AmB. Our results indicate that AmB inhibits the replication of JEV at the postinfection step by interfering with viral replication and/or by inhibiting the synthesis of viral proteins.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.77-77
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• 1995

Human immunodeficiency virus type 1 (HIV-1) contains several nonstructural genes which are required for the viral replication and disease pathogenesis. Among them, tat and nef genes encode an essential transactivator of HIV-1 LTR and a pluripotent protein which seems to be essential for the in vivo but not in vitro viral replication, respectively. We constructed two tat and n of defective HIV-1 and tested for their ability to replicate in several T cells. The defective viruses did not replicate in CD4$\^$+/ T cells, but rescued in the recombinant Jurkat-tat cell which also contains tat gene. The replication of tat and nef defective HIV-1 which expresses chloramphenicol acetyltransferase(CAT) gene was easily detected by a sensitive CAT assay. No revertant was identified during the passages of the mutant viruses for more than two months in Jurkat-tat cells. tat and n of defective HIV-1 could be used instead of wild type viruse for several purposes such as inhibitor screening and development of attenuated AIDS vaccine.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1485-1490
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• 2006

In this study, comparative replicational properties of Hyphantria cunea nucleopolyhedrovirus (HycuNPV) in Lymantria dispar (IPLB-LdElta) and Spodoptera frugiperda (IPLB-Sf21) cell lines were investigated. Our microscopic observations showed that cytopathic effects (CPEs) in LdElta cells appeared 12 h later than those in Sf21 cells. Whereas polyhedral inclusion bodies (PIBs) formed at 48 h postinfection (p.i.) in LdElta cells, it formed at 36 h p.i. in Sf21 cells. Extracellular virus production determined according to the 50% tissue culture infective dose ($TCID_{50}$) method in LdElta cells started about 12 h later when compared with Sf21 cells. Titers of extracellular virus in LdElta and Sf21 cells were calculated as $1.77{\times}10^9$ plaque forming units (PFU)/ml and $5.6{\times}10^9PFU/ml$, respectively, at 72 h p.i. We also showed that viral DNA replication began at 12 h p.i. in both cell lines. Viral protein synthesis was determined by SDS-polyacrylamide gel electrophoresis (PAGE) and polyhedrin synthesis was observed at 12 h p.i. in both cell lines. The results indicate that while the synthesis of macromolecules is 12 h later and production of extracellular virus is almost 3-fold lower in LdElta cells compared with those in Sf21 cells, the LdElta cell line is still a productive cell line for infection of HycuNPV.

• BMB Reports
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• v.28 no.4
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• pp.336-341
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• 1995

Using a mutant M13 phage derivative lacking a great part of the complementary strand synthesis origin, we identified six single-strand initiation (ssi) signals for DNA replication in pACYC184, pLG214, pGKV21, and pDPT270 plasmids, and named them $ssiA_{YC}$, $ssiA_{LG}$, $ssiB_{LG}$, $ssiA_{KV}$, $ssiA_{PT}$, and $ssiB_{PT}$, respectively. Two of them were from pDPT270, one from downstream the on of pACYC184, two from pLG214, one from upstream the plus origin of pGKV21. Introduction of these ssi signals into the deleted $ori_c$ site of a mutant filamentous M13 phage ($M13{\Delta}lac182$) resulted in the restoration of growth activity of this phage. These ssi signals were classified into a number of groups on the basis of sequence similarity. $ssiA_{YC}$ and $ssiA_{LG}$ show extensive sequence homology to the n'-site (primosome assembly sites) of ColE1, whereas $ssiB_{PT}$ is homologous to the n'-site of ${\Phi}X174$. $ssiA_{PT}$ belongs to G4-type ssi signals which require only dnaG primase and SSB protein for the priming of replication. In addition, possible biological roles of these ssi signals are discussed.

• Molecules and Cells
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• v.26 no.3
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• pp.217-227
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• 2008

Heterochromatin protein 1 (HP1) was first described in Drosophila melanogaster as a heterochromatin associated protein with dose-dependent effect on gene silencing. The HP1 family is evolutionarily highly conserved and there are multiple members within the same species. The multi-functionality of HP1 reflects its ability to interact with diverse nuclear proteins, ranging from histones and transcriptional co-repressors to cohesion and DNA replication factors. As its name suggests, HP1 is well-known as a silencing protein found at pericentromeres and telomeres. In contrast to previous views that heterochromatin is transcriptionally inactive; noncoding RNAs transcribed from heterochromatic DNA repeats regulates the assembly and function of heterochromatin ranging from fission yeast to animals. Moreover, more recent progress has shed light on the paradoxical properties of HP1 in the nucleus and has revealed, unexpectedly, its existence in the euchromatin. Therefore, HP1 proteins might participate in both transcription repression in heterochromatin and euchromatin.

• BMB Reports
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• v.34 no.5
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• pp.452-456
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• 2001

Surface plasmon resonance has been used for a biospecific interaction analysis between two macromolecules in real time. Determination of an antibody that is capable of specifically interacting with the native form of antigen is very useful for many biological and medical applications. Twenty monoclonal antibodies against the $\alpha$ subunit of E. coli DNA polymerase III were screened for specifically recognizing the native form of protein using surface plasmon resonance. Only four monoclonal antibodies among them specifically recognized the native $\alpha$ protein, although all of the antibodies were able to specifically interact with the denatured $\alpha$ subunit. These antibodies failed to interfere with the interaction between the $\tau$ and $\alpha$ subunits that were required for dimerization of the two polymerases at the DNA replication fork. This real-time analysis using surface plasmon resonance provides an easy method to screen antibodies that are capable of binding to the native form of the antigen molecule and determine the biological interaction between the two molecules.

• BMB Reports
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• v.50 no.7
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• pp.379-383
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• 2017

We previously reported that p53 plays a role as a key regulator in the tetraploid G1 checkpoint, which is activated by actin damage-induced cytokinesis blockade and then prevents uncoupled DNA replication and nuclear division without cytokinesis. In this study, we investigated a role of Skp2, which targets CDK2 inhibitor p27/Kip1, in actin damage-induced tetraploid G1 arrest. Expression of Skp2 was reduced, but p27/Kip1 was increased, after actin damage-induced cytokinesis blockade. The role of Skp2 repression in tetraploid G1 arrest was investigated by analyzing the effects of ectopic expression of Skp2. After actin damage, ectopic expression of Skp2 resulted in DNA synthesis and accumulation of multinucleated cells, and ultimately, induction of apoptosis. These results suggest that Skp2 repression is important for sustaining tetraploid G1 arrest after cytokinesis blockade and is required to prevent uncoupled DNA replication and nuclear division without cytokinesis.