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.

IMPORTANCE 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是必不可少的。对于痘病毒，该过程分为两个阶段：去除包膜，允许早期基因表达；破坏核心壁，允许DNA释放，复制和晚期基因表达。随后的研究表明，牛痘病毒（VACV）基因组脱壳需要宿主蛋白酶体和病毒D5蛋白（在DNA复制中具有重要作用）。在寻找其他VACV脱膜蛋白时，我们注意到了一份报告，该报告描述了当编码68 kDa锚蛋白重复序列​​/ F-box蛋白（68k-ank）的基因与细胞SCF相关时，DNA复制和后期表达存在缺陷。从减毒的改良牛痘病毒安卡拉（MVA）中删除了Skp1，cullin1，F-box复合物）泛素连接酶复合物。在这里，我们显示68k-ank缺失突变体表现出减少的基因组脱膜，DNA预复制位点的形成，病毒核心的降解以及DNA合成中的其他独立缺陷。但是，删除VACV株WR的68k-ank同源物无效，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M2（痘病毒免疫逃避（PIE）域超家族成员和NF-κB调节剂）和C5（与BTB / Kelch超家族相关的成员）基于cullin-3的连接酶复合物可独立拯救68k-ank缺失表型。因此，痘病毒脱壳和DNA复制是相互交织的过程，除了D5之外，还涉及至少三个具有相互冗余功能的病毒蛋白。DNA预复制位点的形成，病毒核心的降解以及DNA合成中的另一个独立缺陷。但是，删除VACV株WR的68k-ank同源物无效，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M2（痘病毒免疫逃避（PIE）域超家族成员和NF-κB调节剂）和C5（与BTB / Kelch超家族相关的成员）基于cullin-3的连接酶复合物可独立拯救68k-ank缺失表型。因此，痘病毒脱壳和DNA复制是相互交织的过程，除了D5之外，还涉及至少三个具有相互冗余功能的病毒蛋白。DNA预复制位点的形成，病毒核心的降解以及DNA合成中的另一个独立缺陷。但是，删除VACV株WR的68k-ank同源物无效，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M2（痘病毒免疫逃避（PIE）域超家族成员和NF-κB调节剂）和C5（与BTB / Kelch超家族相关的成员）基于cullin-3的连接酶复合物可独立拯救68k-ank缺失表型。因此，痘病毒脱壳和DNA复制是相互交织的过程，除了D5之外，还涉及至少三个具有相互冗余功能的病毒蛋白。但是，删除VACV株WR的68k-ank同源物无效，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M2（痘病毒免疫逃避（PIE）域超家族成员和NF-κB调节剂）和C5（与BTB / Kelch超家族相关的成员）基于cullin-3的连接酶复合物可独立拯救68k-ank缺失表型。因此，痘病毒脱壳和DNA复制是相互交织的过程，除了D5之外，还涉及至少三个具有相互冗余功能的病毒蛋白。但是，删除VACV株WR的68k-ank同源物无效，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M2（痘病毒免疫逃避（PIE）域超家族成员和NF-κB调节剂）和C5（与BTB / Kelch超家族相关的成员）基于cullin-3的连接酶复合物可独立拯救68k-ank缺失表型。因此，痘病毒脱壳和DNA复制是相互交织的过程，除了D5之外，还涉及至少三个具有相互冗余功能的病毒蛋白。痘病毒免疫逃避（PIE）域超家族的成员和NF-κB的调节剂，与基于cullin-3连接酶复合物相关的BTB / Kelch超家族的成员C5独立地拯救了68k-ank缺失表型。因此，痘病毒脱壳和DNA复制是相互交织的过程，除了D5之外，还涉及至少三个具有相互冗余功能的病毒蛋白。痘病毒免疫逃避（PIE）域超家族的成员和NF-κB的调节剂，与基于cullin-3连接酶复合物相关的BTB / Kelch超家族的成员C5独立地拯救了68k-ank缺失表型。因此，痘病毒脱壳和DNA复制是相互交织的过程，除了D5之外，还涉及至少三个具有相互冗余功能的病毒蛋白。

重要性痘病毒包括感染脊椎动物和无脊椎动物并引起医学和生态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，它们的大型基因组通常编码200种或200种以上具有多种功能的蛋白质。根据保守性或个别基因缺失研究，约有90个基因可能对脊索病毒复制至关重要。但是，由于存在冗余，此数字可能会低估基本功能的真实数目。在这里，我们显示了三个看似无关且各自非必需的蛋白质中的任何一种，对于基因组脱壳和DNA复制（合成杀伤力的一个例子）的不完全理解过程是必需的。因此，