单纯疱疹病毒1(HSV-1)编码能够进行核酸外切校对的家族BDNA聚合酶(Pol),其功能在过去已得到广泛研究。对纯化的Pol蛋白的体外活性的早期研究发现,全酶的酶功能在很大程度上是分开的。因此,外切核酸酶活性可以通过催化重要区域内的某些点突变来降低或消除,对聚合酶活性没有或只有很小的影响。尽管聚合酶活性没有受损,用无催化活性的核酸外切酶回收HSV-1突变体迄今尚未成功。因此,突变,如D368A,消除外切核酸酶活性,被认为是致命的.这里,我们表明,HSV-1可以在缺乏Pol内在核酸外切酶活性的情况下恢复,并证明缺乏校对会导致可能的有害突变的快速积累。尽管消除外切核酸酶活性的突变似乎并不致命,缺乏校对会产生具有自杀表型的病毒,该病毒在重建后的几段内停止复制。因此,我们得出的结论是,通过校对获得的高复制保真度对于保持HSV-1基因组完整性至关重要,并且缺乏外切核酸酶活性会产生最初可行但快速自杀的表型.然而,通过突变位于保守的核酸外切酶结构域内的催化不太重要的位点,可以产生具有降低的核酸外切酶活性并因此提高突变率的稳定复制的病毒。重要性完全外切核酸酶缺陷型单纯疱疹病毒1(HSV-1)DNA聚合酶突变体的回收迄今尚未成功。然而,核酸外切酶活性不是病毒复制直接必需的,因此,某些HSV-1聚合酶突变体的致死表型归因于外切核酸酶活性以外的因素。这里,我们表明,可以回收各种核酸外切酶缺陷的HSV-1聚合酶突变体,并且这些突变体最初具有复制能力。我们,然而,观察到在细胞培养传代后,突变体生存力逐渐丧失,这与核酸外切酶缺陷病毒中突变的快速积累相吻合。因此,我们得出结论,缺乏核酸外切酶缺陷病毒的DNA校对会导致最初可行但快速自杀的超突变子表型,因此,突变病毒在恢复后几代内灭绝。这将使缺乏外切核酸酶活性成为长期以来报道的培养外切核酸酶缺陷型HSV-1突变体的困难的主要原因。
Herpes simplex virus 1 (HSV-1) encodes a family B DNA polymerase (Pol) capable of exonucleolytic proofreading whose functions have been extensively studied in the past. Early studies on the in vitro activity of purified Pol protein found that the enzymatic functions of the holoenzyme are largely separate. Consequently, exonuclease activity can be reduced or abolished by certain point mutations within catalytically important regions, with no or only minor effects on polymerase activity. Despite unimpaired polymerase activity, the recovery of HSV-1 mutants with a catalytically inactive exonuclease has been so far unsuccessful. Hence, mutations such as D368A, which abolish exonuclease activity, are believed to be lethal. Here, we show that HSV-1 can be recovered in the absence of Pol intrinsic exonuclease activity and demonstrate that a lack of proofreading causes the rapid accumulation of likely detrimental mutations. Although mutations that abolish exonuclease activity do not appear to be lethal, the lack of proofreading yields viruses with a suicidal phenotype that cease to replicate within few passages following reconstitution. Hence, we conclude that high replication fidelity conferred by proofreading is essential to maintain HSV-1 genome integrity and that a lack of exonuclease activity produces an initially viable but rapidly suicidal phenotype. However, stably replicating viruses with reduced exonuclease activity and therefore elevated mutation rates can be generated by mutating a catalytically less important site located within a conserved exonuclease domain. IMPORTANCE Recovery of fully exonuclease-deficient herpes simplex virus 1 (HSV-1) DNA polymerase mutants has been so far unsuccessful. However, exonuclease activity is not known to be directly essential for virus replication, and the lethal phenotype of certain HSV-1 polymerase mutants is thus attributed to factors other than exonuclease activity. Here, we showed that the recovery of a variety of exonuclease-deficient HSV-1 polymerase mutants is possible and that these mutants are initially replication competent. We, however, observed a progressive loss of mutant viability upon cell culture passaging, which coincided with the rapid accumulation of mutations in exonuclease-deficient viruses. We thus concluded that a lack of DNA proofreading in exonuclease-deficient viruses causes an initially viable but rapidly suicidal hypermutator phenotype and, consequently, the extinction of mutant viruses within few generations following recovery. This would make the absence of exonuclease activity the primary reason for the long-reported difficulties in culturing exonuclease-deficient HSV-1 mutants.