초록
Ganciclovir(GCV)와 Vidarabine(ara-A)을 단독으로 또는 동시에 HSV-1에 작용시켰을때 HSV-1의 복제, DNA합성 및 단백질 합성에 미치는 영향을 관찰할 목적으로 본 연구를 시행하였다. 본 실험에서는 4가지의 다른 HSV-1(Wild type KOS, $VCV^r$, $IUdR^r$, 및 $PAA^r5$)를 사용하였다. Virus복제에 미치는 항 virus약의 효과는 Vero 세포단층 배양에서 Yield reduction assay에 의해서 관찰하였다. 항 virus약의 virus DNA 합성에 미치는 영향은 NaI 밀도 구배초원심침전후 $H^3-$표지 virus DNA의 방사능에 의해서 관찰하였다. Virus 단백질의 합성에 미치는 항 virus약의 효과는 $^{35}S$를 표지한 후 polyacrylamide 겔 전기영동법, 자가방사기록법 그러고 virus bands의 음영농도주사법을 이용, 관찰하여 다음과 같은 결과를 얻었다. 1. GCV는 wild trype HSV-1 KOS와 $PAA^r5$의 복제를 강력하게 억제하였으나 $ACV^r$와 $IUdR^r$는 GCV에 대해서 중등도의 저항을 보였다. ara-A는 실험대상의 모든 HSV-(KOS, $ACV^r$, $IUdR^r$ 및 $PAA^r5$)의 복제에 대해서 거의 비슷하게 억제효과를 보였다. GCV와 ara-A 동시첨가는 KOS와 $PAA^r5$의 복제에 대해서 상승적인 억제효과를 보였고 $ACV^r$와 $IUdR^r5$의 복제에 대해서는 상가작용이하의 억제 효과를 보였다. 2. GCV또는 ara-A는 HSV-1감염 Vero세포에서 virus DNA의 합성을 유의하게 억제하였다. GCV와 ara-A의 동시첨가는 KOS 또는 $PAA^r5$ 감염세포에서 virus DNA 합성을 GCV 또는 ara-A를 단독 첨가하였을때 보다 현저하게 억제하였다. $ACV^r$ 또는 $IUdR^r$ 감염세포에서는 이런 현상을 관찰할 수 없었다. 3. Wild type HSV-1 감염세포에서 virus-단백질의 합성은 GCV, ara-A의 단독 첨가 또는 GCV와 ara-A의 동시첨가에 의해서 변경되지 않았다. Wild type HSV-1 감염말기에 GCV 또는 ara-A 단독 혹은 동시첨가에 의해서 virus 단백질의 합성은 경미하나마 유의성있게 증가하였다. Wild type HSV-1과 $PAA^r5$에 의한 단백질의 합성은 GCV 또는 ara-A 단독 첨가에 의해서 유의성있게 억제되었다. GCV또는 ara-A의 동시첨가는 GCV또는 ara-A를 단독으로 첨가했을 경우보다 단백질의 합성을 더욱 억제하였다. 이상의 실험결과로 보아 GCV와 ara-A의 동시사용은 HSV-1 혹은 ACV저항 DNA polymerase변이주인 $PAA^r5$에 대해서 상승적인 억제작용을 나타냈으며 이 효과는 virus DNA 합성 억제에 의한 것으로 생각된다. ACV저항 thymidine kinase 변이주인 $ACV^r$ 및 $IUdR^r$에 대해서는 ara-A가 유효하였다. 항 virus 약물에 의한 virus 단백질합성의 변화는 virus DNA 합성에 대한 억제효과에 인한 것으로 사려된다.
Combined effects of ganciclovir (GCV) and vidarabine (ara-A) on the replication, DNA synthesis, and gene expression of wild type-1 herpes simplex virus (HSV-1) and three acyclovir (ACV)-resistant HSV-1 mutants were studied. These mutants include a virus expressing no thymidine kinase $(ACV^r)$, a virus expressing thymidine kinase with altered substrate specificity $(IUdR^r)$, and a mutant expressing altered DNA polymerase $(PAA^r5)$. GCV, an agent activated by herpesvirus specific thymidine kinase, showed potent antiviral activity against the wild type HSV-1(KOS) and DNA polymerase mutant $(PAA^r5)$. The ACV-resistant mutants with thymidine kinase gene $(ACV^r\;and\;IUdR^r)$ were resistant to GCV. All tested wild type HSV-1 or ACV-resistant HSV-1 mutants did not display resistance to vidarabine (are-A). Combined GCV and ara-A showed potentiating synergistic antiviral activity against wild type KOS and $PAA^r5$, and showed subadditive combnined ativiral activity against thymidine kinase mutants. Combined GCV and ara-A more significantly inhibited the viral DNA synthesis in wild type KOS and $PAA^r5-infected$ cells to a greater extent than either agent alone, but the synergism was not determined in $ACV^r$ or $IUdR^r-infected$ cells. These data clearly indicate that combined GCV and ara-A therapy might be useful for the treatment of infections caused by wild type HSV-1 or ACV-resistant HSV-1 with DNA polymerase mutation. ACV-resistant viruses with the mutation in thymidine kinase gene are also, resistant to GCV, but susecptible to ara-A, indicating that ara-A would the drug of choice for the treatment of ACV-resistant HSV-1 which does not express thymidine kinase or expresses thymidine kinase with altered substrate specificity. While the synthesis of viral ${\alpha}-proteins$ of wild type HSV-1 was not affected by ACV, GCV, ara-A, or combined GCV and ara-A, the synthesis of ${\beta}-proteins$ was slightly but significantly increased at the later stage of viral infection by the antiviral agents. The synthesis of ${\gamma}-proteins$ of wild type HSV- 1 was significantly inhibited by ACV, GCV, ara-A, and combined GCV and ara-A. Combined GCV $(5-{\mu}M)$ and ara-A $(100-{\mu}M)$ also significantly altered the expression of viral ${\beta}-and$ ${\gamma}-proteins$, of which efffct was similar to that of GCV $(10-{\mu}M)$ alone. Although ACV at the concentration of $10-{\mu}M$ did not alter the expression of ${\alpha}-$, ${\beta}-$, and ${\gamma}-proteins$ of ACV-resistant $PAA^r5$, GCV and ara-A significantly alter the epression of ${\beta}-and$ ${\gamma}-proteins$, not ${\alpha}-protein$, as same manner as they altered the expression of those proteins in cells inffcted with wild type HSV-1. Combined GCV $(5-{\mu}M)$ and ara-A $(100-{\mu}M)$ altered the expression ${\beta}-and$ ${\gamma}-proteins$ in $PAA^r5$ infected cells, and the effect of combined regimen was comparable of that of GCV $(10-{\mu}M)$. These data indicate that the alteration in the expression of ${\beta}-and$ ${\gamma}-proteins$ in wild type HSV-1 or $PAA^r5$ infected cells could be more significantly affected by combined GCV and are-A than individual GCV or ara-A. In view of the fact that (a) viral ${\alpha}-$, ${\beta}-$, and ${\gamma}-proteins$ are synthesized in a cascade manner; (b) ${\beta}-proteins$ are essential for the synthesis of viral DNA; (c) the synthesis of ${\beta}-proteins$ are inhibited by ${\gamma}-proteins$; and (d) most ${\gamma}-proteins$ are made from the newly synthesized progeny virus, it is suggested that GCV and ara-A, alone or in combination, primarily inhibit the synthesis of viral DNA, and by doing so might exhibit their antiherpetic activity. The alteration in viral protein synthesis in the presence of tested antiviral agents could result from the alteration in viral DNA synthesis. From the present study, it can be concluded that (a) combined GCV and ara-A therapy would be beneficial for the control of inffctions caused by wild type HSV-1 or ACV-resistant DNA polymerase mutants; (b) the combined synergistic activity of GCV and ara-A is due to further decrease in the viral DNA by the combined regimen; (c) ara-A is the drug of choice for the infection caused by ACV-resistant HSV-1 with thymidine kinase mutation; and (d) the alteration in viral protein synthesis by GCV and ars-A, alone or in combination, is mostly due to the decreased synthesis of viral DAN.