HCMV에 감염된 TBP-1 세포에서 세포간 부착분자-1 발현 촉진물질의 방출

김미숙,이현아,이찬희,Kim, Mi-Suk,Yi, Hyun-Ah,Lee, Chan-Hee 한국미생물학회 2008 미생물학회지 Vol.44 No.2

다양한 염증 질환을 유발하는 사람세포거대바이러스(Human cytomegalovirus: HCMV)는 단핵구 세포주인 THP-1 세포에서 염증반응의 중요한 매개체인 세포간부착분자-1(intercellular adhesion molecule: ICAM-1) 발현을 촉진한다. ICAM-1 발현은 자외선으로 불활화시킨 HCMV (UV-HCMV)에 의해서도 촉진되므로 이 과정에 HCMV 유전자발현은 꼭 필요하지는 않은 것 같다. HCMV에 감염된 THP-1 세포 배양액을 감염되지 않은 THP-1 세포에 처리하거나 공유하게 하였을 시 감염되지 않은 세포에서도 ICAM-1 발현이 증가하였다. 감염된 세포 배양액에 의한 ICAM-1 발현 증가는 $NF-{\kappa}B$ 경로를 거친다. UV-HCMV에 감염된 세포의 배양액은 ICAM-1 발현을 촉진시키지 못하였다. 따라서 HCMV에 의한 THP-1 세포에서 ICAM-1 발현 증가는 바이러스 유전자 발현을 필요로 하지 않지만, 감염된 세포에서 ICAM-1 발현을 촉진하는 물질을 분비하는 과정에는 바이러스 유전자 발현이 필요한 것으로 생각된다. Human cytomegalovirus (HCMV) stimulates the expression of intercellular adhesion molecule (ICAM-l) on the surface of monocytic THP-1 cells. Stimulation of ICAM-l did not require HCMV gene expression since UV-inactivated HCMV (UV-HCMV) was able to induce ICAM-l expression. ICAM-l expression was also stimulated in uninfected THP-l cells which were fed with culture supernatant of HCMV-infected THP-l cells. Co-culture experiment using trans-well insert supported that HCMV-infected THP-l cells secreted some cytokine(s) stimulating ICAM-l expression. The stimulation of ICAM-l by HCMV-infected cell culture supernatant appears to involve $NF-{\kappa}B$ pathway. Culture supernatant from THP-l cells infected with UV-HCMV, whose gene expression was abrogated, failed to stimulate ICAM-l expression on naive THP-l cells. Thus, HCMV gene expression seems to be required in secretion of cytokine(s) stimulating ICAM-l expression.

Inhibitory effects of piceatannol on human cytomegalovirus (hCMV) in vitro

Wang San-Ying,Zhang Jing,Xu Xiao-Gang,Su Hui-Li,Xing Wen-Min,Zhang Zhong-Shan,Jin Wei-Hua,Dai Ji-Huan,Wang Ya-Zhen,He Xin-Yue,Sun Chuan,Yan Jing,Mao Gen-Xiang 한국미생물학회 2020 The journal of microbiology Vol.58 No.8

Human cytomegalovirus (hCMV) is a ubiquitous herpesvirus, which results in the establishment of a latent infection that persists throughout the life of the host and can be reactivated when the immunity is low. Currently, there is no vaccine for hCMV infection, and the licensed antiviral drugs mainly target the viral enzymes and have obvious adverse reactions. Thus, it is important to search for compounds with antihCMV properties. The present study aimed to investigate the suppressive effects of piceatannol on hCMV Towne strain infection and the putative underlying mechanisms using human diploid fibroblast WI-38 cells. Piceatannol supplementation prevented the lytic changes induced by hCMV infection in WI-38 cells. Furthermore, piceatannol suppressed the expression of hCMV immediate-early (IE) and early (E) proteins as well as the replication of hCMV DNA in a dose-dependent manner. Moreover, hCMV-induced cellular senescence was suppressed by piceatannol, as shown by a decline in the senescence-associated β-galactosidase (SA-β-Gal) activity and decreased production of intracellular reactive oxygen species (ROS). p16INK4a, a major senescence-associated molecule, was dramatically elevated by current hCMV infection that was attenuated by pre-incubation with piceatannol in a dose-dependent manner. These results demonstrated that piceatannol suppressed the hCMV infection via inhibition of the activation of p16INK4a and cellular senescence induced by hCMV. Together, these findings indicate piceatannol as a novel and potent anti-hCMV agent with the potential to be developed as an effective treatment for chronic hCMV infection.

Nitro oxide in human cytomegalovirus replication and gene expression

Lee, Jee-Yeon,Lee, Chan-Hee The Microbiological Society of Korea 1997 The journal of microbiology Vol.35 No.2

Infection of human fibroblast (HF) cells with human cytomegalovirus (HCMV) result in changes in the intracellular level of second messengers. Since nitric oxide (NO) production has been known to be related with other second messengers, it is probable that HCMV infection of HF cells may involve NO. To test this possibility, the amount of NO was measured following ogenous addition of NO generators such as sodium nitroprusside (SNP) or S-nitroso-N-a-cetylpenicillamine (SNAP) immediately after HCMV infection, however, inhibited virus multiplication. Furthermore, immunoblot experiment using monoclonal antibody to HCMV major immediate early (MIE) proteins or CAT assay using pCMVIE/CAT (plasmid containing CAT gene driven by HCMV MIE promoter) revealed that SNP or SNAP blocked the MIE gene expression. SNP was more effective than SNAP in hibiting HCMV multiplication or MIE gene expression. SNP produced more NO than SNAP in inhibiting HCMV multiplication or MIE gene expression. SNP produced more NO than SNAP. Although the mechanism for the inhibition of HCMV multiplication and MIE gene expression by NO is still elusive some correlation with NO-mediated inhibition of HCMV-induced increase in cytosolic free Ca$\^$2+/ concentration ([Ca$\^$2+/]) was observed. The increase of [Ca$\^$2+/] following HCMV infection was inhibited by SNP, and less effectively by SNAP. Raising [Ca$\^$2+/ with bromo-A23187 partially reversed the SNP block of MIE gene expression. Thus, there appear to e some relationships among NO. [Ca$\^$2+/], and HCMV MIE gene expression.

신경세포에서의 Human Cytomegalovirus 증식과 이에 따른 세포내 유리칼슘 농도 변화

강경희,이찬희 대한바이러스학회 1996 Journal of Bacteriology and Virology Vol.26 No.1

Human cytomegalovirus (HCMV) replication and Ca 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 response. The cytoplasmic free Ca concentration ([Ca];) began to increase at 12 h p.I. In HCMV-infected SK- N-SH cells, while [Ca]; 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]; 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]; was observed. Treatment with TPA of HCMV-in- fected SK-N-SH cells resulted in [Ca]; increase at 6 h. p.I. The stimulatory effect of TPA on HCMV- induced [Ca]; increase continued until 12 h p.I., but TPA failed to stimulate the Ca' 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 response may be a consequence of the lower responsiveness of SK-N-SH cells than HEL cells to HCMV infection.

Induction of Apoptosis in Human Monocytes by Human Cytomegalovirus is Related with Calcium Increase

MyungSookMoon,GyuCheolLee,ChanH.Lee 한국미생물학회 2002 The journal of microbiology Vol.40 No.3

The effect of human cytomegalovirus (HCMV) on three human monocyte cell lines at different stages of differentiation was investigated. While the viability of HL-60 cells or U-937 cells was not significantly affected by HCMV infection, the viability of THP-1 cells was reduced. Acridine orange/ethidium bromide staining revealed that the reduction of THP-1 cell viability was due to increased apoptotic death following HCMV infection. Apoptosis in HL-60 cells was not affected by HCMV infection, and induction of apoptosis of U-937 cells by HCMV was intermediate between HL-60 and THP-1 cells. Since HL- 60 cells are the least differentiated and THP-1 cells are the most differentiated, the induction of apoptosis of human monocytes appears to be related to the degree of cell differentiation. Flow cytometric and confocal microscopic studies using fluorescent calcium indicator Fluo-3 suggested a significant increase in intracellular free calcium concentration ([Ca2+]i) in THP-1 cells undergoing apoptosis by HCMV infection. Again [Ca2+]i in HCMV-infected HL-60 cells was not critically altered, and that in HCMV-infected U-937 cells was intermediate between THP-1 cells and HL-60 cells. Calcium influx blockers such as verapamil and nifedipine partially reversed HCMV-induced apoptosis in THP-1 cells.

Human Cytomegalovirus 감염에 의한 세포내 pH 변화

유정희,이찬희 대한바이러스학회 1995 Journal of Bacteriology and Virology Vol.25 No.2

Infection of human embroy lung (HEL) cells with human cytomegalovirus (HCMV) resulted in change of intracellular pH ([pH]I). A maximal increase in [pH]I was observed at 12 hr postinfection (p.I.) in HCMV-infected HEL cells. Inactivation of HCMV with heat treatment or UV irradiation effectively abolished the HCMV-induced increase in [pH]I observed at 12 hr p.I. In HEL cells. And the degree of the increase in [pH]I was dependent on the amount of infectious virus. Treatment of HCMV-infected HEL cells with amiloride, an inhibitor of Na'/H' exchanger, partially blocked the increase in [pH]I. These results suggest a possible relationship between the change of intracellular pH and HCMV replication.

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

Park, Jung-Sun,Park, Soo-Young,Cho, Hyun-Il,Sohn, Hyun-Jung,Kim, Tai-Gyu The Korean Association of Immunobiologists 2011 Immune Network Vol.11 No.3

Background: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-${\gamma}$ ELISPOT assay, cytotoxicity assay and tetramer staining. Results: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of $CD8^+$ and $CD4^+$ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

박정선,김태규,박수영,조현일,손현정 대한면역학회 2011 Immune Network Vol.11 No.3

Background: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs)pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-γ ELISPOT assay,cytotoxicity assay and tetramer staining. Results: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65antigen. Conclusion: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Allelic MHC Class I Chain Related B (MICB) Molecules Affect the Binding to the Human Cytomegalovirus (HCMV) Unique Long 16 (UL16) Protein: Implications for Immune Surveillance

Kanya Klumkrathok,Amonrat Jumnainsong,Chanvit Leelayuwat 한국미생물학회 2013 The journal of microbiology Vol.51 No.2

Unique long 16 (UL16) is a viral glycoprotein produced in a host cell infected with human cytomegalovirus (HCMV). It down regulates surface expression of MICB, one of the NKG2D ligands, by forming stable intracellular complexes and retained in the endoplasmic reticulum. Down expression of MICB renders cells less susceptible to NK cell lysis via the NKG2D receptor. Diverse UL16 sequences were identified from different strains of HCMV. MICB is known to be polymorphic. It is not known whether these polymorphisms affect the interactions between these molecules leading to alteration of the immune surveillance of HCMV. The soluble Fc fusion variant UL16 proteins from four laboratory and clinical isolates (AD169, Toledo, PH, and TR) were produced. Four allelic MICB alleles (008, 003, 004, and 00502) were cloned and stable cell lines expressing these MICB alleles were produced. The binding activities of variant UL16 to allelic MICB proteins were determined by flow cytometry. The variants of UL16 proteins did not affect the binding activities to allelic MICB proteins. However, diverse MICB alleles differentially bound UL16. We found that MICB*008which contains methionine and asparagine at the amino acid positions 98 and 113, respectively, in the alpha 2 domain showed decreased binding activities to UL16 when compared to MICB*003, 004, and MICB*00502 containing isoleucine and aspartic acid, respectively. This finding may imply that MICB*008 is a protective allele and involved in the immune surveillance of HCMV infected patients.

Human Cytomegalovirus (HCMV) US2 Protein Interacts with Human CD1d (hCD1d) and Down-Regulates Invariant NKT (iNKT) Cell Activity

한지혜,전태훈,노승배,이재연,배준범,박세호,이석준,이상열,안규리,김재영 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.5

To avoid host immune surveillance, human cytomegalovirus (HCMV) encoded endoplasmic reticulum (ER)-mem-brane glycoprotein US2, which interferes with antigen presenting mechanism of major histocompatibility complex (MHC) class Ia and class II molecules. However, not many attempts have been made to study the effect of HCMV US2 on the expression of MHC class Ib molecules. In this study, we examined the effect of HCMV US2 on the expression and function of human CD1d (hCD1d), which presents glycolipid antigens to invariant NKT (iNKT) cells. Our results clearly showed that the physiological interaction between ER lumenal domain of HCMV US2 and 3 domain of hCD1d was observed within ER. Compared with mature form of hCD1d, immature form of hCD1d is more susceptible to ubiquitin-dependent proteasomal degradation mediated by HCMV US2. Moreover, the ectopic expression of HCMV US2 leads to the down-modulation of iNKT cell activity without significant change of hCD1d expression. These results will advance our understanding of the function of HCMV US2 in immune evasive mechanisms against anti-viral immunity of iNKT cells.