Taddie J A, Traktman P
Department of Cell Biology, Cornell University Medical College, New York, New York 10021.
J Virol. 1993 Jul;67(7):4323-36. doi: 10.1128/JVI.67.7.4323-4336.1993.
In this report, we describe the isolation, molecular genetic mapping, and phenotypic characterization of vaccinia virus mutants resistant to cytosine arabinoside (araC) and phosphonoacetic acid (PAA). At 37 degrees C, 8 microM araC was found to prevent macroscopic plaque formation by wild-type virus and to cause a 10(4)-fold reduction in viral yield. Mutants resistant to 8 microM araC were selected by serial passage of a chemically mutagenized viral stock in the presence of drug. Because recovery of mutants required that initial passages be performed under less stringent selective conditions, and because plaque-purified isolates were found to be cross-resistant to 200 micrograms of PAA per ml, it seemed likely that resistance to araC required more than one genetic lesion. This hypothesis was confirmed by genetic and physical mapping of the responsible mutations. PAAr was accorded by the acquisition of one of three G-A transitions in the DNA polymerase gene which individually alter cysteine 356 to tyrosine, glycine 372 to aspartic acid, or glycine 380 to serine. AraCr was found to require one of these substitutions plus an additional T-C transition within codon 171 of the DNA polymerase gene, a change which replaces the wild-type phenylalanine with serine. Congenic viral stocks carrying one of the three PAAr lesions, either alone or in conjunction with the upstream araCr lesion, in an otherwise wild-type background were generated. The PAAr mutations conferred nearly complete resistance to PAA, a slight degree of resistance to araC, hypersensitivity to aphidicolin, and decreased spontaneous mutation frequency. Addition of the mutation at codon 171 significantly augmented araC resistance and aphidicolin hypersensitivity but caused no further change in mutation frequency. Several lines of evidence suggest that the PAAr mutations primarily affect the deoxynucleoside triphosphate-binding site, whereas the codon 171 mutation, lying within a conserved motif associated with 3'-5' exonuclease function, is postulated to affect the proofreading exonuclease of the DNA polymerase.
在本报告中，我们描述了对阿糖胞苷（araC）和膦甲酸（PAA）具有抗性的痘苗病毒突变体的分离、分子遗传定位及表型特征。在37℃时，发现8μM阿糖胞苷可阻止野生型病毒形成肉眼可见的噬斑，并使病毒产量降低10⁴倍。通过在药物存在下对化学诱变的病毒株进行连续传代，筛选出对8μM阿糖胞苷具有抗性的突变体。由于突变体的恢复需要在不太严格的选择条件下进行初始传代，并且发现噬斑纯化的分离株对每毫升200μg的PAA具有交叉抗性，因此对阿糖胞苷的抗性似乎需要不止一个遗传损伤。通过对相关突变进行遗传和物理定位，证实了这一假设。PAA抗性（PAAr）是由DNA聚合酶基因中三个G-A转换之一导致的，这三个转换分别将半胱氨酸356变为酪氨酸、甘氨酸372变为天冬氨酸或甘氨酸380变为丝氨酸。发现阿糖胞苷抗性（AraCr）需要这些替代之一，再加上DNA聚合酶基因第171密码子内的一个额外的T-C转换，该变化将野生型苯丙氨酸替换为丝氨酸。构建了在其他方面为野生型背景下携带三个PAAr损伤之一单独或与上游AraCr损伤一起的同基因病毒株。PAAr突变赋予了对PAA几乎完全的抗性、对阿糖胞苷的轻微抗性、对阿非科林的超敏感性以及降低的自发突变频率。在第171密码子处添加突变显著增强了对阿糖胞苷的抗性和对阿非科林的超敏感性，但未导致突变频率的进一步变化。几条证据表明，PAAr突变主要影响脱氧核苷三磷酸结合位点，而位于与3'-5'核酸外切酶功能相关的保守基序内的第171密码子突变，据推测会影响DNA聚合酶的校对核酸外切酶。
