| Project/Area Number |
02807052
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Virology
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| Research Institution | Fukushima Medical College |
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Principal Investigator |
SHIGETA Shiro Fukushima Medical College Department of Microbiology Professor, 医学部, 教授 (00045634)
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| Co-Investigator(Kenkyū-buntansha) |
BABA Masanori Fukushima Medical College Department of Microbiology Assistant (Lecturer), 医学部, 助手 (70181039)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Interferons / Herpes Simplex Virus / Varicella Zoster Virus / Thymidine Kinase / Acyclovir / Bromovinyl-Arauracil / Synergistic Effects / Antiviral Activity / ブロモビニ-ルアラウランル |
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| Research Abstract |
Combined use of several antiviral compounds against herpesvirus infections is important and sianificant because it may increase the potency of each drug selected for the combination or it may inhibit the appearance of drug resistant virus. Several workers have reported synergistic antiviral effects of nucleoside analogues(e. g., acyclovir)and interferons(IFN)on in vitro and in vivo herpesvirus infections. We also reported synergistic antiviral effect of acyclovir(ACV)and human IFN-beta on the plaque formation and virus yield of herpes simplex virus(HSV)-2. To express antiheroes activity, ACV has to be phosphorylated by virus encoded thymidine kinase(TK)of herpes simplex viruses. Whensoever ACV exhibits s-nergism with IFN to inhibit herpesvirus growth, the viral TK should be induced in virus infected cells which are pretreated by IFN. In this project, we examined the enzymatic characteristics of HSV-TK and VZV-TK which are induced in IFN pretreated cells comparing to that induced in IFN
… More
untreated cells.
HSV-TK and VZV-TK from IFN treated or mock treated human embryo fibroblast(HEF)were diluted to definite enzyme unit for dthd or dcyd phosphorylation and a phosphorylating efficiency for each dTK, ACV or BV-araU was compared. To determine the phospborylating efficiency of TK for ACV or BV-araU, each 100 mu1 of the TK preparation was added to equal volume of reaction mixture containing 100 muM ATP, 10 muC, of[gamma- ^<32>p]ATP and 1 muM each of ACV and BV-araU as substrates. The mixture was incubated at 37ﾟC for 15 min, and after the reaction 100 mu1 each of the sample was applied to HPLC. Total cpm in ACV-MP or BV-araU-MP fraction was counted by liquid scintillation counter. HSV-TK from IFN treated cells phosphorylated 1.3 to 1.7 times more efficiently ACV than that from IFN untreated cells. Km value of TK againstACV was also monitored with ACV plus[gamma- ^<32>p]ATP and from Lineweaver-Burk plot it was clear that the affinity of HSV-2 TK isolated from IFN pretreated HEF against ACV was markedly increased compared with that of TK isolated from IFN untreated cells(table 1). VZV TK scarecely phosphorylated ACV whether infected cells were pretreated with IFN or not. VZV-TK phosphorylated well BV-araU and the efficiency was much better in TK from IFN treated cells-than that from mock treated cells(data is not shown). Thus the mechanism of synergistic inhibitory effect of nucleoside analogues and IFN on herpesvirus replication proved to depend on the altered enzymatic affinity of viral TK to nucleoside analogues after the treatment of cells with IFN. Probably some cellular mediators are induced by IFN treatment and they may act directly or indirectly to viral TK and regulate the enzymatic activity by allosteric ways. Less
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