Mechanism of inhibition of vaccinia virus DNA polymerase by cidofovir diphosphate
Mechanism of inhibition of vaccinia virus DNA polymerase by cidofovir diphosphate
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DOI：
10.1128/aac.49.8.3153-3162.2005
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发表时间：
2005-08-01
影响因子：
4.9
通讯作者：
Evans, DH
中科院分区：
文献类型：
--
作者：
Magee, WC;Hostetler, KY;Evans, DH
西多福韦（CDV）是一种广谱抗病毒药物，已被批准用于治疗巨细胞病毒视网膜炎的临床使用。它也被用于标签外治疗各种其他病毒感染，包括由orf和传染性软疣痘病毒引起的感染。因为它是一种dCMP类似物，CDV被认为是通过抑制病毒DNA聚合酶发挥作用。然而，抑制机制的细节还没有很好地建立，并且对药物抑制痘病毒DNA聚合酶的机制一无所知。为了解决这个问题，我们研究了CDV的活性细胞内代谢产物CDV二磷酸（CDVpp）对牛痘病毒DNA聚合酶催化反应的影响。使用不同的引物-模板对和纯化的牛痘病毒聚合酶，我们观察到CDV被掺入与模板G相对的生长的DNA链中，但与dCTP相比，该酶表现出较低的催化效率。CDV终止的引物也是下一个脱氧核苷一磷酸加成步骤的良好底物，但这些CDV + 1反应产物是进一步合成的不良底物。我们还注意到，尽管CDV可以通过牛痘病毒聚合酶的3 '至5'校正核酸外切酶活性从引物3'末端切除，但携带CDV作为倒数第二个3'残基的DNA完全抵抗核酸外切酶攻击。这些结果表明，牛痘病毒DNA聚合酶可以使用CDVpp作为dCTP类似物，尽管其减慢了引物延伸的速率。通过抑制校正核酸外切酶的活性，CDV的错误掺入也可以促进痘病毒复制过程中的易错DNA合成。
Cidofovir (CDV) is a broad-spectrum antiviral agent that has been approved for clinical use in the treatment of cytomegalovirus retinitis. It has also been used off label to treat a variety of other viral infections, including those caused by orf and molluscum contagiosum poxviruses. Because it is a dCMP analog, CDV is thought to act by inhibiting viral DNA polymerases. However, the details of the inhibitory mechanism are not well established and nothing is known about the mechanism by which the drug inhibits poxvirus DNA polymerases. To address this concern, we have studied the effect of the active intracellular metabolite of CDV, CDV diphosphate (CDVpp), on reactions catalyzed by vaccinia virus DNA polymerase. Using different primer-template pairs and purified vaccinia virus polymerase, we observed that CDV is incorporated into the growing DNA strand opposite template G's but the enzyme exhibits a lower catalytic efficiency compared with dCTP. CDV-terminated primers are also good substrates for the next deoxynucleoside monophosphate addition step, but these CDV + 1 reaction products are poor substrates for further rounds of synthesis. We also noted that although CDV can be excised from the primer 3' terminus by the 3'-to-5' proofreading exonuclease activity of vaccinia virus polymerase, DNAs bearing CDV as the penultimate 3' residue are completely resistant to exonuclease attack. These results show that vaccinia virus DNA polymerase can use CDVpp as a dCTP analog, albeit one that slows the rate of primer extension. By inhibiting the activity of the proofreading exonuclease, the misincorporation of CDV could also promote error-prone DNA synthesis during poxvirus replication.