The herpes simplex virus (HSV) type I alkaline nuclease, UL12, has 5′-to-3′ exonuclease activity and shares homology with nucleases from other members of the Herpesviridae family. We previously reported that a UL12-null virus exhibits a severe defect in viral growth. To determine whether the growth defect was a result of loss of nuclease activity or another function of UL12, we introduced an exonuclease-inactivating mutation into the viral genome. The recombinant virus, UL12 D340E (the D340E mutant), behaved identically to the null virus (AN-1) in virus yield experiments, exhibiting a 4-log decrease in the production of infectious virus. Furthermore, both viruses were severely defective in cell-to-cell spread and produced fewer DNA-containing capsids and more empty capsids than wild-type virus. In addition, DNA packaged by the viral mutants was aberrant, as determined by infectivity assays and pulsed-field gel electrophoresis. We conclude that UL12 exonuclease activity is essential for the production of viral DNA that can be packaged to produce infectious virus. This conclusion was bolstered by experiments showing that a series of natural and synthetic α-hydroxytropolones recently reported to inhibit HSV replication also inhibit the nuclease activity of UL12. Taken together, our results demonstrate that the exonuclease activity of UL12 is essential for the production of infectious virus and may be considered a target for development of antiviral agents.

IMPORTANCE Herpes simplex virus is a major pathogen, and although nucleoside analogs such as acyclovir are highly effective in controlling HSV-1 or -2 infections in immunocompetent individuals, their use in immunocompromised patients is complicated by the development of resistance. Identification of additional proteins essential for viral replication is necessary to develop improved therapies. In this communication, we confirm that the exonuclease activity of UL12 is essential for viral replication through the analysis of a nuclease-deficient viral mutant. We demonstrate that the exonuclease activity of UL12 is essential for the production of viral progeny and thus provides an attractive, druggable enzymatic target.

单纯疱疹病毒（HSV）I型碱性核酸酶UL12具有5'至3'核酸外切酶活性，并且与疱疹病毒科其他成员的核酸酶具有同源性家庭。我们之前曾报道过，无UL12的病毒在病毒生长中表现出严重的缺陷。为了确定生长缺陷是核酸酶活性丧失还是UL12另一功能的结果，我们将核酸外切酶失活突变引入了病毒基因组。重组病毒UL12 D340E（D340E突变体）在病毒产量实验中的行为与无效病毒（AN-1）相同，传染性病毒的产量减少了4个对数。此外，与野生型病毒相比，这两种病毒在细胞间传播方面都存在严重缺陷，产生的含DNA衣壳和空衣壳更多。另外，由病毒突变体包装的DNA是异常的，如通过传染性测定和脉冲场凝胶电泳所确定的。我们得出的结论是，UL12核酸外切酶活性对于可包装产生传染性病毒的病毒DNA的生产至关重要。实验表明，最近报道的一系列抑制HSV复制的天然和合成α-羟基对苯二酚也抑制UL12的核酸酶活性，这一结论为实验提供了支持。两者合计，我们的结果表明，UL12的核酸外切酶活性对于生产传染性病毒至关重要，并可能被视为开发抗病毒剂的目标。

重要事项单纯疱疹病毒是主要病原体，尽管核苷类似物（例如无环鸟苷）在控制免疫能力强的个体中对控制HSV-1或-2感染非常有效，但由于抗药性的提高，它们在免疫受损患者中的使用变得复杂。鉴定对于病毒复制必不可少的其他蛋白质对于开发改进的疗法是必要的。在此通讯中，我们通过分析核酸酶缺陷型病毒突变体，确认UL12的核酸外切酶活性对于病毒复制至关重要。我们证明UL12的核酸外切酶活性对于病毒后代的产生至关重要，因此提供了一个有吸引力的，可药物化的酶促靶标。