Liu Baoming, Panda Debasis, Mendez-Rios Jorge D, Ganesan Sundar, Wyatt Linda S, Moss Bernard
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
J Virol. 2018 Mar 14;92(7). doi: 10.1128/JVI.02152-17. Print 2018 Apr 1.
Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5. Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess.
基因组脱壳对于大多数病毒的复制至关重要。对于痘病毒而言，该过程分为两个阶段：去除包膜以允许早期基因表达，以及突破核心壁以允许DNA释放、复制和晚期基因表达。随后的研究表明，宿主蛋白酶体和在DNA复制中起关键作用的病毒D5蛋白是痘苗病毒（VACV）基因组脱壳所必需的。在寻找其他VACV脱壳蛋白的过程中，我们注意到一份报告，该报告描述了从减毒的安卡拉痘苗病毒（MVA）中删除与细胞SCF（Skp1、cullin1、含F盒复合物）泛素连接酶复合物相关的编码68 kDa锚蛋白重复/F盒蛋白（68k-ank）的基因后，DNA复制和晚期表达出现缺陷。在这里，我们表明68k-ank缺失突变体的基因组脱壳、DNA预复制位点的形成以及病毒核心的降解均减少，并且在DNA合成中还存在另一个独立的缺陷。然而，删除VACV WR株的68k-ank同源物却没有影响，这表明存在补偿基因。通过将VACV基因插入MVA 68k-ank缺失突变体中，我们发现痘病毒免疫逃避（PIE）结构域超家族成员且是NF-κB调节剂的M2以及与基于cullin-3的连接酶复合物相关的BTB/Kelch超家族成员C5独立地挽救了68k-ank缺失表型。因此，痘病毒的脱壳和DNA复制是相互交织的过程，除了D5之外，还涉及至少三种具有相互冗余功能的病毒蛋白。痘病毒是一类感染脊椎动物和无脊椎动物并引起具有医学和动物学重要性疾病的大型DNA病毒家族。与大多数其他DNA病毒不同，痘病毒在细胞质中复制，其庞大的基因组通常编码200种或更多具有不同功能的蛋白质。基于其保守性或单个基因缺失研究，约90个基因可能是脊索痘病毒复制所必需的。然而，由于冗余性，这个数字可能低估了基本功能的真实数量。在这里，我们表明在尚未完全了解的基因组脱壳和DNA复制过程中，三种看似无关且单独非必需的蛋白质中的任何一种都是必需的，这是合成致死性的一个例子。因此，痘病毒似乎具有一个尚未被充分认识的复杂遗传相互作用网络，这需要多因素缺失筛选来评估。
