INHIBITION OF VACCINIA VIRUS-REPLICATION BY NICOTINAMIDE - EVIDENCE FOR ADP-RIBOSYLATION OF VIRAL-PROTEINS
INHIBITION OF VACCINIA VIRUS-REPLICATION BY NICOTINAMIDE - EVIDENCE FOR ADP-RIBOSYLATION OF VIRAL-PROTEINS
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DOI：
10.1016/0168-1702(88)90027-5
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发表时间：
1988-02-01
期刊：
影响因子：
5
通讯作者：
HRUBY, DE
中科院分区：
文献类型：
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作者：
CHILD, SJ;FRANKE, CA;HRUBY, DE
在存在60 mM烟酰胺（NIC）（ADP-核糖基化反应的竞争性抑制剂）的情况下，BSC 40细胞单层中牛痘病毒（VV）的复制被抑制99.9%。利用VV DNA特异性探针的感染细胞提取物的斑点杂交分析表明，该药物对病毒DNA合成的影响很小。在感染后早期（2小时）或晚期（8小时）脉冲标记的新合成VV蛋白的SDS-聚丙烯酰胺凝胶电泳显示，尽管预期的病毒多肽的全谱是明显的，但在药物存在下观察到病毒蛋白的不同子集的表达水平的定量差异。病毒感染的细胞裂解物的速度沉降确定在药物处理的细胞中没有组装成熟颗粒。通过脉冲追踪标记实验获得了表明在NIC存在下VV形态发生失败的其他证据，该实验证明两个VV主要晚期核心多肽前体P94和P65在NIC存在下不被切割，所述前体P94和P65的蛋白水解加工为VP 62和VP 60与病毒组装密切相关。有趣的是，[3 H]腺苷存在下VV的生长导致与纯化的病毒体相关的8种蛋白质的代谢标记。这些蛋白质共迁移与[3 H]腺苷标记的蛋白质，存在于VV感染的提取物中，但未感染的细胞。这些分析还表明，VV感染使细胞蛋白质子集（MW 18-20 kDa，可能是组蛋白）的[3 H]腺苷标记增加了4倍。所观察到的这些18-20 kDa蛋白的合成增加或过度修饰的诱导被NIC抑制。这些结果进行了讨论是否一个或多个VV多肽进行强制性ADP-核糖基化修饰反应，以达到其活性的配置，如果是这样，是否催化thee反应的酶指定的病毒或宿主细胞。
Replication of vaccinia virus (VV) in monolayers of BSC40 cells was inhibited 99.9% in the presence of 60 mM nicotinamide (NIC), a competitive inhibitor of ADP-ribosylation reactions. Dot-blot hybridization analysis of infected cell extracts utilizing a VV DNA-specific probe indicated that the drug had only minimal effects on viral DNA synthesis. SDS-polyacrylamide gel electrophoresis of newly synthesized VV proteins pulse-labeled at early (2 h) or late (8 h) times post-infection revealed that although the full spectrum of expected viral polypeptides was evident, quantitative differences in the levels of expression of a distinct subset of viral proteins were observed in the presence of the drug. Velocity sedimentation of virus-infected cell lysates established that no mature particles were assembled in drug treated cells. Additional evidence suggesting that VV morphogenesis was abortive in the presence of NIC was obtained by pulse-chase labeling experiments that demonstrated that the two VV major late core polypeptide precursors P94 and P65, whose proteolytic processing to VP62 and VP60 is intimately assocaited with viral assembly, were not cleaved in the presence of NIC. Interestingly, growth of VV in the presence of [3H]adenosine resulted in the metabolic labeling of eight proteins that were associated with purified virions. These proteins co-migrated with proteins labeled with [3H]adenosine that were present in extracts of VV-infected, but not uninfected, cells. These analyses also revealed that the [3H]adenosine-labeling of a subset of cellular proteins (MW 18-20 kDa, possibly histones) was increased 4-fold by VV infection. The observed induction of either increased synthesis or hyper-modification of these 18-20 kDa proteins was inhibited by NIC. These results are discussed with respect to whether one or more VV polypeptides are subject to obligatory ADP-ribosylation modification reactions in order to attain their active configuration, and if so, whether the enzymes catalyzing thee reactions are specified by the virus or host cell.