Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus
Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus
复制标题
痘苗病毒编码的 DNA 聚合酶的遗传证据：从感染突变病毒的细胞质中分离出抗膦酰乙酸酶
作者：
B. Moss;N. Cooper
浓度为200微克/ml或更高的膦酰乙酸酯（PAA）可阻止HeLa和BSC-1细胞中牛痘病毒的生长。在高PAA水平下选择的自发性牛痘病毒突变体对药物的抗病毒作用具有抗性。PAA的作用针对早期病毒功能，因为药物仅在约15小时生长周期的前4小时内具有抑制作用。相反，只有在感染的第四小时或之前移除药物时，才能获得抗病毒作用的显著逆转。[3 H]胸腺嘧啶核苷掺入细胞质病毒DNA被严重抑制，在感染野生型病毒的细胞，但不是在感染突变病毒的细胞。从感染野生型或突变型病毒的细胞的细胞质中分离的病毒诱导的DNA聚合酶在DEAE-纤维素和磷酸纤维素柱上具有不可区分的色谱特性。然而，野生型酶被相对低浓度的PAA抑制，而突变酶的等效抑制需要10倍的浓度。动力学分析表明，PAA的抑制是非竞争性的脱氧核苷三磷酸，Ki值为野生型和突变型DNA聚合酶分别约为25和300 μ M。野生型DNA聚合酶的抑制是立即和完整的，甚至当PAA在体外DNA合成开始后加入，这表明链延伸受到影响。这些结果证实了DNA聚合酶是PAA抗病毒作用的靶点，并提供了这种酶是病毒编码的遗传证据。
Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.