( 您好!臺灣時間:2021/07/28 20:03
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::


研究生(外文):Chia-Lin Lee
論文名稱(外文):The effect of cytomegalovirus pp71 driven by the late temporal UL94 promoter
指導教授(外文):Szu-Hao Kung
外文關鍵詞:HCMVtegument protein
  • 被引用被引用:0
  • 點閱點閱:75
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
Human cytomegalovirus (CMV) infection is a major cause of morbidity and mortality among immunocompromised patients, especially recipients of transplants and patients with acquired immunodeficiency syndrome (AIDS). Anti-CMV chemotherapeutic agents have been developed; however, prolonged administration of the drugs has prompted the emergence of drug-resistant CMV strains, compromising the efficacy of the therapy. Therefore, we propose to develop a genetically modified cell line to hopefully monitor sensitivity to anti-CMV drugs.
An HCMV tegument protein (encoded by the UL82 gene), has been shown to enhance the yield of progeny virus. The underlying mechanism has partially been its ability to transactivation of the immediate early (IE) promoter, resulting in the increased level of the virus lytic cycle. However, the role that the pp71 plays in the late phase of the viral cycle is largely elusive although the expression of pp71 is upregulated at this stage. Thus a stable cell line, designated U373MG-UL94p-FlagUL82-IRES-EGFP, was established to permit the pp71 and the green fluorescent protein (GFP) to be expressed late in the replication cycle of CMV. The cell line was developed by stable transfection of U373MG cell line with a plasmid encoding the UL82 and the GFP genes driven by a CMV true late promoter, the UL94 promoter. A stable line that only allows GFP expression under the control of the UL94 promoter (devoid of expression of ectopic pp71) was also established as a negative control.
The susceptibility of the U373MG-UL94p-FlagUL82-IRES-EGFP stable line to CMV infection appears to be similar to that of the MRC5 diploid cells. The U373MG-UL94p-FlagUL82-IRES-EGFP cell line exhibited detectable GFP as early as one day postinfection, while the control cells did not display GFP signal until 3 days postinfection. As the infection proceeds, GFP foci were seen on U373MG-UL94p-FlagUL82-IRES-EGFP cells 6-8 days postinfection whereas GFP foci were not seen on the infected control cells. In the presence of anti-CMV drug (ganciclovir), the GFP foci were not seen with the U373MG-UL94p-FlagUL82-IRES-EGFP cells infected with drug-sensitive CMV strain while the GFP foci were intact when infected by drug-resistant isolates. Stable cell lines expressing the short interfering RNA targeting pp71 transcript significantly reduced the number and intensity of GFP (+) cells of the infected U373MG-UL94p-FlagUL82-IRES-EGFP cells, indicating that the pp71 would serve as an alternative molecular target for anti-CMV agents.
The U373MG-UL94p-FlagUL82-IRES-EGFP cells could be used for rapid diagnosis, determination of antiviral susceptibility, and assessment of potential compounds against pp71 protein.
Baldanti F, Sarasini A, Silini E, Barbi M, Lazzarin A, Biron KK, and Gerna G.. 1995. Four dually resistant human cytomegalovirus strains from AIDS patients: single mutations in UL97 and UL54 open reading frames are responsible for ganciclovir- and foscarnet-specific resistance, respectively. Scand. J. Infect. Dis. Suppl. 99: 103-104.
Baldick CJ Jr, Marchini A, Patterson CE, and Shenk T. 1997. Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. J. Virol. 71: 4400-4408.
Boeckh M, Boivin G.. 1998. Quantization of cytomegalovirus: methodologic aspects and clinical applications. Clin. Microbiol. Rev. 11: 533-554.
Boivin G, Olson CA., Quirk MR, St-Cyr SM, Jordon MC. 1995. Quantization of human cytomegalovirus glycoprotein H gene in cell using competitive PCR and rapid fluorescence-based detection system. J. Virol. Methods. 51: 329-342.
Bresnahan WA and Shenk TE. 2000. UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells. Proc. Natl. Acad. Sci. U S A. 97: 14506-11.
Bresnahan WA and Shenk TE. 2000. A subset of viral transcripts packaged within human cytomegalovirus particles. Science 288: 2373-2376.
Bret AW, Robert AJ and Eng-shang Huang. Identification of positive and negative regulatory regions involved in regulating expression of the human cytomegalovirus UL94 late promoter : role of IE2-86 and cellular p53 in mediating negative regulatory function. J. Virol. 72: 1814-1825
Britt WJ and Alford CA. 1996. Cytomegalovirus. In: Fields, B.N., Knipe, D.M.,
Howley, P.M. (Eds), Fields Virology, Vol. 2, 3rd edition. Lippincott-Raven
publishers, Philadelphia, PA, p. 2493-2523.
Chalfie M., Tu Y., Euskirchen G., Ward WW., Prasher DC., 1994. Green fluorescent
protein as a marker for gene expression. Science 263: 802-805.
Chou S, Guentzel S, Micheal KR, Miner RC and Drew WL. 1995. Frequency of UL97 phosphotransferase mutations related to ganciclovir resistance in clinical cytomegalovirus isolates. J. Infect. Dis. 172: 239-242.
Compton T, Nepomuceno RR, Nowlin DM. 1992. Human cytomegalovirus penetrates host cells by pH-independent fusion at the cell surface. Virology 191: 387-395.
Compton T, Nowlin DM, Cooper NR. 1993. Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193: 834-841.
Cormack BP, Valdivia RH, Falkow S. 1996. FACS-optimized mutants of the green
fluorescent protein (GFP). Gene 173: 33-38.
Crumpacker CS. 1996. Ganciclovir. N. Engl. J. Med. 335: 721-729.
Drew WL, Miner R and Saleh E. 1993. Antiviral susceptibility testing of cytomegalovirus: criteria for detecting resistance to antivirals. Clin. Diagn. Virol. 1: 179-185.
Duclos H, Elfassi E, Michelson S, Arenzana-Seisdedos F, Hazan U, Munier A, Virelizier JL. 1989. Cytomegalovirus infection and trans- activation of HIV-1 and HIV-2 LTRs in human astrocytoma cells. AIDS Res. Hum. Retroviruses. 5: 217-24.
Erice A. 1999. Resistance of human cytomegalovirus to antiviral drugs. Clin. Microbiol. Rew.12 : 286-297.
Fox JC, Griffiths PD and Emery VC. 1992. Quantification of human cytomegalovirus DNA using the polymerase chain reaction. J. Gen. Virol. 73: 2405-2408.
Grefte JM, BT van der G.un, Schmolke S, M van der Giessen, van Son WJ, Plachter B, Jahn G, The TH. 1992 The lower matrix protein pp65 is the principal viral antigen present in peripheral blood leukocyte during an active cytomegalovirus infection. J. Gen. Virol. 73: 2923-2932.
Griffiths PD and Emery VC. 2002. Cytomegalovirus. In: Richman,D.D., Whitley,
R.J., Hayden, F.G. (Eds), Clinical Virology. Churchill Livingstone, New York
Hahn G, Jores R, Mocarski ES. 1998. Cytomegalovirus remains latent in a common
precursor of dendritic and myeloid cells. Proc. Natl . Acad. Sci .U S A; 95: 3937-3942.
Heim R, Prasher DC and Tsien RY. (1994). Wavelength mutations and
posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci.
U S A 91: 12501-4.
Hensel GM, Meyer HH, Buchmann I, Pommerehne D, Schmolke S, Plachter B, Radsak K, and Kern HF.1996. Intracellular localization and expression of the human cytomegalovirus matrix phosphoprotein pp71 (ppUL82): evidence for its translocation into the nucleus. J. Gen. Virol. 77: 3087-97.
Hiyoshi M TS, Takubo T, Tanakn K, Nakao T, Higeno Y, Tamura K, Shimaoka M, Fujii A, Higashihata M, Yasui Y, Kim T, Hiraoka A, Tatsumi N. (1997). Evaluation of the AMPLICOR CMV test for direct detection of cytomegalovirus in plasma specimens. J. Clin. Microbiol. 35: 2692-2694.
Inouye RT, Panther LA, Hay CM and Hammer SM. 2002. Antiviral agents. In:
Richman DD., Whitley RJ., Hayden FG. (Eds), Clinical Virology. Churchill
Livingstone, New York, p. 171-242.
Jault FM, Spector SA, Spector DH. 1994. The effects of Cytomegalovirus on
human immunodeficiency virus replication in brain-derived cells correlate with
permissiveness of the cells for each virus. J. Virol. 68: 959-73.
Kalejta RF & Shenk T. 2002. Manipulation of the cell cycle by human cytomegalovirus. Front. Biosci. 7: 295–306.
Kalejta RF & Shenk T. 2003. The human cytomegalovirus UL82 gene product (pp71) accelerates progression through the G1 phase of the cell cycle. J. Viol. 77: 3451-3459.
Kari B, Radeke R and Gehrz R. 1992. Processing of human cytomegalovirus envelope glycoproteins in and egress of cytomegalovirus from human astrocytoma cell. J. Gen. Virol. 73: 253-260.
Landry ML, Stanat S, Biron K, Brambilla D, Britt W, Jokela J, Chou S, Drew WL, Erice A, Gilliam B, Lurain N, Manischewitz J, Miner R, Nokta M, Rechelderfer P, Spector S, Weinberg A, Yen-Lieberman Band Crumpacker C. 2000. A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates. Antimicrob. Agents Chemother. 44: 688-692.
Liu WT, Sun JR, Lin CH, Kuo RL and Kung SH. 2001. An indicator cell assay for detection of human cytomegalovirus based on enhanced green fluorescent protein. J. Virol. Mthods. 96: 85-92.
Liu B and Stinski MF. 1992. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements.J. Virol. 66: 4434-44.
Lurain NS, Thompson KD, Holmes EW, and Read GS. 1992. Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents. J. Viol. 66: 7146-7152.
McSharry JJ. 1999. Antiviral drug susceptibility assays: going with the flow. Antiviral Res. 43: 1-21. Review.
Mocarski ES and Courcelle CT. 2001. Cytomegaloviruses and their replication. In: Fields, B.N.,Knipe, D.M., Howley, P.M. (Eds), Fields Virology, Vol.2, 4th edition. Lippincott-Raven publicashers, Philadelphia, PA, p.2629-2674.
Mocarski ES, Liu AC, Spaete RR. 1987. Structure and variability of the a sequence in the genome of human cytomegalovirus (Townw strain). J. Gen. Viol. 68: 2223-2230.
Mc Voy MA and Adler SP. 1994. Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within the concatemer. J. Viol. 68: 1040-1051.
Ogilvie M. 2001. Molecular techniques should not now replace cell culture in diagnostic virology laboratories. Rev. Med. Virol. 11: 351-354.
Pass RF., 2001. Cytomegalovirus. In: Fields, B.N., Knipe, D.M., Howley, P.M.
(Eds), Fields Virology, Vol. 2, 4th edition. Lippincott-Raven publishers,
Philadelphia, PA, p. 2493-2523.
Pietropaolo R and Compton T. 1999. Interference with annexin II has no effect on
entry of human cytomegalovirus into fibroblast cells. J. Gen. Virol. 80: 1807-1816.
Plachter B, Sinzger C and Jahn G. 1996. Cell types involved in replication and distribution of human cytomegalovirus. Advances in Virus Research 46: 195-261.
Rowe WP, Hartley JW, Waterman S, et al. 1956. Cytopathogenic agent resembling salivary gland virus recovered from tissue cultures of human adenoids. Proc. Soc. Exp. Biol. Med. 92: 418-424
Sinzger C and Jahn G. 1996. Human cytomegalovirus cell tropism and pathogenesis. Interviology 39: 302-319.
Sinzger C, Grefte A, Plachter B, et al. 1995. Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. J. Gen. Virol. 76: 741-750.
Sinclair J and Sissons P. 1996. Latent and persistent infections of monocytes and
Macrophages. Interviology 39: 283-301.
Smith IL, Cherrington JM, Jiles RE, Fuller MD , Freeman WR and Spector SA. 1997. High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J. Infect. Dis. 176: 69-77.
Soderberg-Naucler C and Nelson JY. 1999. Human cytomegalovirus latency and
reactivation-A delicate balance between the virus and its host’s immune system.
Interviology 42: 314-321.
Tatarowicz WA, Lurain NS, and Thompson KD.1991. In situ ELISA For the evaluation of antiviral compounds effective against human cytomegalovirus. J. Virol. Methods 35: 207-215.
Weller TH, Hanshaw JB and Scott DE. 1960. Serological differentiation of viruses responsible for cytomegalic inclusion disease. Virology 12: 130-132.
Weller TH, Macauley JC, Craig JM and Wirth P. 1957. Isolation of intranuclear inclusion producing agents from infants with illnesses resembling cytomegalic inclusion disease. Proc. Soc. Exp. Biol. Med. 94: 4-12.
Yang TT, Cheng L and Kain S.R. 1996. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res. 24: 4592-4593.
第一頁 上一頁 下一頁 最後一頁 top