• Journal of Microbiology
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• v.38 no.4
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• pp.255-259
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• 2000

For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral activities of three known compounds and the result was compared with results from the conventional plaque assay for measuring the production of infectious viruses. When human fibroblast cells were infected with HCMV/pp28-luc, luciferase activity was observed at 2 days after infection and reached maximum at 6 days after infection, whereas the production of infectious virus was maximal at 4 days after infection. The antiviral activities of ganciclovir, acyclovir, and papaverine were measured in HFF cells infected with HCMV/PP28-luc and the luciferase activity was compared with the infectious virus titers. Luciferase activity decreased as the concentration of ganciclovir or papaverine increased, while there was a slight decrease in luciferase activity with acyclovir. The level of the decrease in Luciferase activity was comparable to the level of decrease in the production of infectious virus. Therefore, the antiviral assay using recombinant virus HCMV/pp28-luc resulted in sensitivity similar to the conventional plaque assay with a significant reduction in assay time.

• The Journal of Korean Society of Virology
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• v.30 no.2
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• pp.125-130
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• 2000

Infection with human cytomegalovirus (HCMV) is of considerable clinical relevance after placental transmission and in immunosuppressed patients such as transplant recipients or patients with AIDS. The rapid detection method of HCMV has been required to overcome the time-consuming methods such as classical plaque assay or other immunological methods. This study was performed to establish the in situ ELISA, in which human lung fibroblasts infected with HCMV were fixed and used directly as antigen in 96 well culture plate. Expressed HCMV antigens were detected with HCMV-specific monoclonal antibodies. This method could detect HCMV dose-dependently upto $3{\times}10^2\;pfu/ml$. Antiviral activity of ganciclovir could be assayed within the known range of effective dose. This result showed that HCMV could be quantitated by in situ ELISA. The chemical, which was selected on the basis of component analysis in natural product, was tested to have the anti-HCMV activity by in situ ELISA, and three among five samples were found to have anti-HCMV activity with the dose-dependent manner. Conclusively in situ ELISA could be useful method for quantitation of HCMV and screening antiviral activity of samples to HCMV.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2010.10a
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• pp.660-662
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• 2010

To control replication of human cytomegalovirus (HCMV) effectively, inhibitors of peptide nucleic acids (PNA) with a gene delivery agent, PEI (polyethylenimine) against HCMV were applied. The transfection of these PNA inhibitors with PEI agent into host cells showed synergic inhibition effect of HCMV replication. These inhibition effect was confirmed by methods of RT-PCR, CPE, real-time-PCR, and Western blot.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2008.05a
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• pp.971-975
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• 2008

Human cytomegalovirus (HCMV) provokes fatal infections in AIDS patients that have deficient immune functions and patients that have cellular immune responses repressed after bone marrow transplantation. A new candidate for therapeutic against HCMV is needed because conventional treatments as ganciclovir, acyclovir, cidofovir, and foscarnet cytosine used currently are improper due to their side effects and advent of resistant HCMV. In this study, peptide nucleic acids (PNAs) against UL54 (DNA polymerase) and UL97 (phosphotransferase) that were essential in replication of HCMV were applied in inhibition of replication of HCMV. From the results of this study, 4 PNAs $_{PNA}UL97-1$, $_{PNA}UL97-2$, $_{PNA}UL54-3$, and $_{PNA}UL54-4$ showed 3.7, 3.1, 1.7, and 1.6 folds of inhibition effect against replication of HCMV in the human fibroblast cells. These PNA suggest a novel possibility as therapeutic against HCMV.

• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.261-269
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• 1999

The relationship between second messenger cGMP and human cytomegalovirus (HCMV) replication was investigated. First, the intracellular level of cGMP ([cGMP]i) in HCMV-infected cells was measured. The [cGMP]i increased at early times after HCMV infection, reached maximum level at 12 hr and returned to basal level at 24 hr after virus infection, while [cGMP]i in mock-infected cells remained relatively unchanged. Increasing [cGMP]i resulted in enhanced transcription of HCMV major immediate early gene. For early gene expression, cGMP had varying effect. Expression of 1.2 kb RNA decreased and 2.2 kb RNA increased with increasing cGMP, while 2.7 kb RNA gene expression was not affected. HCMV early genes are regulated by immediate early gene, and the effect of cGMP on the regulatory effect of major immediate early gene on early genes was investigated. In the absence of cGMP, major immediate early gene repressed 2.7 kb RNA gene expression, while 1.2 kb RNA and 2.2 kb RNA early genes were not significantly affected. In the presence of $1\;{\mu}M$ cGMP, however, major immediate early gene stimulated the expression of three early genes.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.255-255
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• 2002

For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral activities of three known compounds and the result was compared with results from the conventional plaque assay for measuring the production of infectious viruses. When human fibroblast cells were infected with HCMV/pp28-luc, luciferase activity was observed at 2 days after infection and reached maximum at 6 days after infection, whereas the production of infectious virus was maximal at 4 days after infection. The antiviral activities of ganciclovir, acyclovir, and papaverine were measured in HFF cells infected with HCMV/PP28-luc and the luciferase activity was compared with the infectious virus titers. Luciferase activity decreased as the concentration of ganciclovir or papaverine increased, while there was a slight decrease in luciferase activity with acyclovir. The level of the decrease in Luciferase activity was comparable to the level of decrease in the production of infectious virus. Therefore, the antiviral assay using recombinant virus HCMV/pp28-luc resulted in sensitivity similar to the conventional plaque assay with a significant reduction in assay time.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1800-1807
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• 2016

To understand how human cytomegalovirus (HCMV) might change and evolve after reactivation, it is very important to understand how the nucleotide sequence of cultured HCMV changes after in vitro passaging in cell culture, and how these changes affect the genome of HCMV and the consequent variation in amino acid sequence. Strain JHC of HCMV was propagated in vitro for more than 40 passages and its biological and genetic changes were monitored. For each passage, real-time PCR was performed in order to determine the genome copy number, and a plaque assay was employed to get virus infection titers. The infectious virus titers gradually increased with passaging in cell culture, whereas the number of virus genome copies remained relatively unchanged. A linear correlation was observed between the passage number and the log₁₀ infectious virus titer per virus genome copy number. To understand the genetic basis underlying the increase in HCMV infectivity with increasing passage, the whole-genome DNA sequence of the high-passage strain was determined and compared with the genome sequence of the low-passage strain. Out of 100 mutations found in the high-passage strain, only two were located in an open reading frame. A G-T substitution in the RL13 gene resulted in a nonsense mutation and caused an early stop. A G-A substitution in the UL122 gene generated an S-F nonsynonymous mutation. The mutations in the RL13 and UL122 genes might be related to the increase in virus infectivity, although the role of the mutations found in noncoding regions could not be excluded.

• Journal of Microbiology
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• v.38 no.1
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• pp.24-30
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• 2000

US3 gene is a member of the human cytomegalovirus (HCMV) immediate early gene. Although the precise functions of the US3 gene in HCMV replication and pathogenesis are not known, it has been reported to play a role in inhibiting major histocompatibility class I antigen presentation. For further knowledge of US3 gene expression, rabbit polyclonal antiserum of the US3 gene product was used for indirect immunofluorescence assay. In permissive human foreskin fibroblast (HFF) cells, US3 gene expression was detectable as crescent or half-moon shape in the perinuclear region at immediate early times after virus infection. HFF cells infected with mutant HCMV lacking US3 open reading frames were negative for US3 immunofluorescence assay. Double immunofluorescence assay using monoclonal antibody to gamma adaptin (specific for the Golgi complex) and rabbit anti-US3 antiserum revealed that US3 gene product could be localized to the Golgi complex. At later time after HCMV infection, US3 gene products were detected as globular aggregates in the cytosol. These aggregates were positive for gamma adaptin and stained with preimmune serum, suggesting a nonspecific reaction to the Golgi complex. Northern blot analysis revealed that transcription of US3 was observed only during immediate early times after virus infection (until 6 h postinfection). Therefore US3 gene expression appears to be confined to immediate early time and its gene products are localized to the Golgi complex as crescent shaped forms in the perinuclear cytoplasm.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.544-550
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• 2005

To develop a highly efficient mammalian expression vector, a series of vectors were constructed based on the murine cytomegalovirus (MCMV) immediate-early (IE) promoter and human ${\beta}$-globin second intron. The resulting MCMV promoter was several-fold stronger than the HCMV promoter in various mammalian cell lines, such as the NIH3T3, Neuro-2a, 293T, and HT1080 cell lines, and was only slightly weaker than the HCMV promoter in HeLa and CHO cells. The inclusion of the human ${\beta}$-globin second intron behind the MCMV promoter or HCMV promoter markedly enhanced the promoter activity in various mammalian cell lines, and the resultant MCMV/Glo-I expression system was stronger than the HCMV promoter from 4.7- to 11.2-fold in every cell line tested. Also, the MCMV/Glo-I promoter induced a higher level of the VSV-G protein in a transiently transfected 293T cell line, which is useful for the production of recombinant retrovirus and lentivirus vectors.

• Biomolecules & Therapeutics
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• v.25 no.1
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• pp.69-79
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• 2017

Viruses continue to evolve a new strategy to take advantage of every aspect of host cells in order to maximize their survival. Due to their central roles in transducing a variety of transmembrane signals, GPCRs seem to be a prime target for viruses to pirate for their own use. Incorporation of GPCR functionality into the genome of herpesviruses has been demonstrated to be essential for pathogenesis of many herpesviruses-induced diseases. Here, we introduce US28 of human cytomegalovirus (HCMV) as the best-studied example of virally-encoded GPCRs to manipulate host GPCR signaling. In this review, we wish to summarize a number of US28-related topics including its regulation of host signaling pathways, its constitutive internalization, its structural and functional analysis, its roles in HCMV biology and pathogenesis, its proliferative activities and role in oncogenesis, and pharmacological modulation of its biological activities. This review will aid in our understanding of how pathogenic viruses usurp the host GPCR signaling for successful viral infection. This kind of knowledge will enable us to build a better strategy to control viral infection by normalizing the virally-dysregulated host GPCR signaling.