• 미생물학회지
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• 제31권1호
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• pp.72-78
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• 1993

Human cytomegalovirus (HCMV) 의 immediate early(IE) 유전자의 promoter/enhancer 부위에 cAMP response element가 있다는 것으부터 caMP 가 HCMV 의 증식에 관여할 것이라고 생각할 수 있다. 이러한 가능성을 알아보기 위해 8-bromoadenosine 3', 5'-cyclic monophosphate (BrA) 와 papaverine 같은 세포내 cAMP 농도를 변화시키는 약제를 사용하여 HCMV 의 증식, DNA 합성 및 IE 유전자 발현에 대한 영향을 알아보았다. HCMV 증식과 DNA 합성은 papaverine 에 의해 억제된 반면, BrA 는 HCMV 의 증식에는 큰 영향을 주지 않았고 DNA 합성은 오히려 촉진시키는 것을 알 수 있었다. HCMV IE promoter 에 의해 작동되는 CAT 유전자를 함유한 plasmid pCMVIE/CAT 을 세포내로 transfection 시켰을 때, papaverine 을 처리한 세포에서는 CAT 효소 활성도가 감소한 반면 BrA 를 처리한 세포에서는 증가하였다. HCMV 에 감수성이 없는 HeLa 세포에서는 CAT 활성도가 감소성 세포인 HEL 세포에서보다 높게 나타난 반면, Vero 세포에서는 낮게 나타났다. 이들 비감수성 세포인 HEL 세포에서 보다 높게 나타난 반면 Vero 세포에서는 낮게 나타났다. 이 들 비감수성 세포에서의 CAT 활성도는 BrA 를 처리하여 주었을 때 모두 증가하였다. 이상과 같은 결과로부터 cAMP 는 HCMV 증식과 IE 유전자 발현에 어느 정도 관여한다는 것을 알 수 있다.

• Biomolecules & Therapeutics
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• 제25권1호
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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.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1627-1635
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• 2013

Mitochondria often play central roles in apoptotic pathways, and disruption of the mitochondrial transmembrane potential (${\Delta}{\psi}m$) has been observed in various cells undergoing apoptosis. Human cytomegalovirus (HCMV) infection induces apoptosis in permissive cells; however, investigations of mitochondria-targeted apoptosis in HCMV-infected human foreskin fibroblast (HFF) cells have been limited. Here, we investigated the mitochondrial apoptosis pathway in HCMV-infected HFF cells. Flow cytometry analysis using JC-1 revealed that HCMV infection induces disruption of ${\Delta}{\psi}m$ in HFF cells when administered 24 h post-infection (hpi), and this disruption was maximized at 48 hpi. Moreover, cytochrome c, normally a mitochondrial inner membrane protein, was detected in cytoplasmic extracts of HCMV-infected cells, but not mock-infected cells, by western blot analysis at 24 hpi. A caspase activity assay based on fluorescence spectrophotometry using a fluorogenic substrate revealed an increase in caspase-3 activity at 48 hpi in HCMV-infected cells. Caspase-8 activity was increased at 72 hpi in HCMV-infected cells. These results imply that HCMV infection induces mitochondria-mediated apoptosis in HFF cells.

• Archives of Pharmacal Research
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• 제22권6호
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• pp.542-548
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• 1999

The UL47 gene (b 60390-b 60388) located in the unique long region of the human cytomegalovirus (HCMV) AD169 strain genome was analyzed RNA mapping. Northern blot analysis showed that the UL47 gene was expressed at late times after infection (72 h postinfection). The 9.7-kb transcript was expressed in the infected cells but not in phosphonoformate-treated cells at 72 hpi, indicating that the UL47 gene was only expressed at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer extension and RNase protection analysis were performed. Primer extension analysis revealed that the transcription initiation site of UL47 was located in 27 bp downstream (b 60323) of the TATA box motif. The sizes of UL47 ORF (approximately 2.9-kb) and UL48 ORF (approximately 6.7-kb) deduced from computer sequence analysis suggest that the expressed 9.7-kb transcript of UL47 uses the 3'-end polyadenylation signal of Ul48. The result of RNase protection determined that the 3'-end of UL47 RNA utilized the 3'-end polyadenylation signal of UL48, which is located in HCMV genome b 70082.

• 대한바이러스학회지
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• 제26권1호
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• pp.1-8
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• 1996

Human cytomegalovirus (HCMV) replication and $Ca^{2+}$ response in human cell lines of neuronal origin were investigated. SK-N-SH (neuroblastoma cells) and A172 cells (glioblastoma cells) were used. SK-N-SH cells were permissive for HCMV multiplication with a delay of one day compared to virus multiplication in human embryo lung (HEL) cells. The delay of HCMV multiplication in SK-N-SH cells appeared to be correlated with a delay in the $Ca^{2+}$ response. The cytoplasmic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) began to increase at 12 h p.i. in HCMV-infected SK-N-SH cells, while $[Ca^{2+}]_i$ increase in HCMV-infected HEL cells was observed as early as 3 h p.i. On the whole, the level of the increase in $[Ca^{2+}]_i$ in SK-N-SH cells was about 30% of that in HEL cells. On the other hand, in A172 cells infected with HCMV, neither production of infectious virus nor detectable increase in $[Ca^{2+}]_i$ was observed. Treatment with TPA of HCMV-infected SK-N-SH cells resulted in $[Ca^{2+}]_i$ increase at 6 h p.i. The stimulatory effect of TPA on HCMV- induced $[Ca^{2+}]_i$ increase continued until 12 h p.i., but TPA failed to stimulate the $Ca^{2+}$ response in SK-N-SH cells at 24 h p.i., suggesting that the effect of TPA had disappeared in SK-N-SH cells at that time point. In conclusion, SK-N-SH cells are permissive for HCMV replication and the delay in $Ca^{2+}$ response may be a consequence of the lower responsiveness of SK-N-SH cells than HEL cells to HCMV infection.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.294.1-294
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• 1995

No Abstract, See Full Text

• Journal of Microbiology
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• 제38권1호
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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.

• 미생물학회지
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• 제38권4호
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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.

• 대한바이러스학회지
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• 제30권2호
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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.

• 미생물학회지
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• 제44권4호
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• pp.299-304
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• 2008

마이엘로이드 계열의 단핵세포는 인체세포거대바이러스(human cytomegalovirus, HCMV)의 잠복 부위로 알려져 있다. 다양한 세포에서 HCMV에 의한 세포주기의 촉진 또는 억제에 관한 연구는 많이 있었지만, 단핵세포에서의 연구는 거의 없는 상태이다. 이에 본 연구에서는 단핵세포에 HCMV가 감염되면 세포주기에 어떤 변화가 나타나는지 알아보고자 하였다. Propidium iodide를 이용한 유세포 분석을 통한 세포주기 분석에서 HCMV에 감염된 THP-1 세포에서는 G0-G1기가 증가하고, 그만큼 S가 감소함을 보았다. 반면 HL-60 세포에서는G0-G1기와 S기의 상대적인 비율에 큰 변화가 없었다. BrdU 유입 실험에서 THP-1세포의 DNA 합성이 HCMV 감염에 의해 억제됨을 알 수 있었다. 세포주기의 G1기에서 S기로의 전환을 억제하는 p21 단백질은HCMV에 감염된 THP-1 세포에서는 발현이 유도되었지만 HL-60 세포에서는 거의 발현이 되지 않았다. 따라서 HCMV는 promocyte THP-1 세포에서 p21 단백질의 유도에 의해 세포주기를 G0-G1기에서 억류함에 따라 세포중식을 억제하는 것으로 생각된다.