洋地黃毒苷抑制第一型單純皰疹病毒立即早期基因之蛋白質表現

Abstract

第一型單純皰疹病毒（herpes simplex virus type 1；HSV-1）的基因，依據其調控順序及出現的時期，可分為立即早期基因（Immediate early gene；IE gene）、早期基因（Early gene；E gene）及晚期基因（Late gene；L gene）。在之前的研究中，我們發現原本用於治療心臟疾病的洋地黃毒苷（digitoxin）可抑制HSV-1在細胞中的複製。在本研究中，我們發現digitoxin亦可抑制第二型HSV（HSV-2）及對傳統藥物－無環鳥苷（acyclovir）具抗藥性的HSV-1病毒株；而digoxin、ouabain octahydrate 及 G-strophanthin等與digitoxin結構相似之化合物亦具有抑制HSV的活性。從作用時間點及抑制病毒基因的情形推斷，digitoxin應該是作用在HSV-1的早期基因表現之前。以digitoxin處理後，受HSV-1感染的Vero細胞內立即早期基因ICP4及ICP27蛋白質的表現量及表現ICP4的細胞比例皆明顯下降。由於digitoxin在病毒感染後3-4小時加入時對ICP4的抑制情形最為顯著，顯示digitoxin應該不是作用在病毒複製最早的階段，如病毒進入、脫殼（uncoating）或入核。此外，digitoxin並未改變ICP4及ICP27蛋白質的穩定性，且其mRNA表現量亦不受影響，暗示其可能藉由抑制基因轉譯來減少蛋白質表現量。目前我們已確定digitoxin並非藉由調控HSV-1小片段RNA－miR-H6或細胞的轉譯因子eIF3m、eIF4G及eIF4E的表現量來抑制HSV-1基因轉譯；但是此特異性的抑制作用需要細胞的Na+/K+-ATPase (NKA)受器的存在。藉由特異性地抑制HSV-1立即早期基因的蛋白質轉譯，digitoxin有機會成為抗病毒候選化合物。

Human herpes virus type 1 (HSV-1) is known to have a tightly-regulated cascade expression of its immediate early (IE), early (E), and late (L) genes. Previously, digitoxin, which was widely-used as cardiac drug, was shown to inhibit in vitro replication of HSV-1. In this study, we found that digitoxin can repress HSV-2 and acyclovir-resistant HSV-1, and several structural analogues of digitoxin such as digoxin, ouabain octahydrate and G-strophanthin also exhibited anti-HSV activity. The inhibitory effects of digitoxin are likely to be introduced at the early stage of HSV-1 replication. In HSV-infected Vero cells, the protein levels of IE gene ICP4 and ICP27 as well as the ratio of ICP4-positive cells decreased in the presence of digitoxin. The decrease of ICP4 protein level by digitoxin was most effective when digitoxin was added at 3-4 h post-infection, indicating that the inhibitory effect was less likely attributed to blocking of virus entry or post-entry events, such as uncoating and nuclear localization. Moreover, this reduction was not due to degradation of ICP4 and ICP27 protein, and the steady mRNA level suggests that the ICP4 and ICP27 protein translation was interrupted by digitoxin. At present, the involvement of HSV-encoded small RNA, miR-H6, and some eukaryotic translation initiation factors, including eIF3m, eIF4G and eIF4E in digitoxin-induced interruption of viral protein translation was excluded, and the specific interaction between digitoxin and Na+/K+-ATPase (NKA) pump was proved to be essential for such inhibition. Our results demonstrated that digitoxin is a specific inhibitor of HSV IE protein translation and could be a candidate of anti-HSV agents.