Department of Medical Microbiology & Immunology.
Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada.
PLoS Pathog. 2022 Mar 15;18(3):e1010392. doi: 10.1371/journal.ppat.1010392. eCollection 2022 Mar.
Poxvirus genomes consist of a linear duplex DNA that ends in short inverted and complementary hairpin structures. These elements also encode loops and mismatches that likely serve a role in genome packaging and perhaps replication. We constructed mutant vaccinia viruses (VACV) where the native hairpins were replaced by altered forms and tested effects on replication, assembly, and virulence. Our studies showed that structure, not sequence, likely determines function as one can replace an Orthopoxvirus (VACV) hairpin with one copied from a Leporipoxvirus with no effect on growth. Some loops can be deleted from VACV hairpins with little effect, but VACV bearing too few mismatches grew poorly and we couldn't recover viruses lacking all mismatches. Further studies were conducted using a mutant bearing only one of six mismatches found in wild-type hairpins (SΔ1Δ3-6). This virus grew to ~20-fold lower titers, but neither DNA synthesis nor telomere resolution was affected. However, the mutant exhibited a particle-to-PFU ratio 10-20-fold higher than wild-type viruses and p4b/4b core protein processing was compromised, indicating an assembly defect. Electron microscopy showed that SΔ1Δ3-6 mutant development was blocked at the immature virus (IV) stage, which phenocopies known effects of I1L mutants. Competitive DNA binding assays showed that recombinant I1 protein had less affinity for the SΔ1Δ3-6 hairpin than the wild-type hairpin. The SΔ1Δ3-6 mutant was also attenuated when administered to SCID-NCR mice by tail scarification. Mice inoculated with viruses bearing wild-type hairpins exhibited a median survival of 30-37 days, while mice infected with SΔ1Δ3-6 virus survived >70 days. Persistent infections favor genetic reversion and genome sequencing detected one example where a small duplication near the hairpin tip likely created a new loop. These observations show that mismatches serve a critical role in genome packaging and provide new insights into how VACV "flip and flop" telomeres are arranged.
痘病毒基因组由线性双链 DNA 组成，其末端为短的反向互补发夹结构。这些元件还编码环和错配，可能在基因组包装中发挥作用，也许在复制中也发挥作用。我们构建了突变痘苗病毒（VACV），其中天然发夹被改变形式取代，并测试了对复制、组装和毒力的影响。我们的研究表明，结构而不是序列可能决定功能，因为可以用来自兔痘病毒的发夹取代正痘病毒（VACV）发夹，而对生长没有影响。可以从 VACV 发夹中删除一些环，影响很小，但 VACV 携带的错配太少，生长不良，我们无法恢复缺乏所有错配的病毒。进一步的研究使用仅携带野生型发夹中发现的六个错配之一的突变体（SΔ1Δ3-6）进行。该病毒的生长滴度降低了约 20 倍，但 DNA 合成和端粒分辨率均不受影响。然而，该突变体的粒子-效价比（PFU）比野生型病毒高 10-20 倍，并且 p4b/4b 核心蛋白加工受损，表明存在组装缺陷。电子显微镜显示，SΔ1Δ3-6 突变体的发育在不成熟病毒（IV）阶段受阻，这模拟了已知的 I1L 突变体的影响。竞争性 DNA 结合测定表明，重组 I1 蛋白与 SΔ1Δ3-6 发夹的亲和力低于野生型发夹。通过尾巴划伤将 SΔ1Δ3-6 突变体施用于 SCID-NCR 小鼠时，其也会减弱。接种野生型发夹病毒的小鼠的中位存活期为 30-37 天，而感染 SΔ1Δ3-6 病毒的小鼠的存活期超过 70 天。持续性感染有利于遗传回复，基因组测序检测到一个例子，即在发夹尖端附近的一个小重复可能创建了一个新的环。这些观察结果表明，错配在基因组包装中起关键作用，并为痘苗病毒“翻转”端粒的排列方式提供了新的见解。
