Department of Virology II, National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo, Japan.
J Virol. 2024 Jul 23;98(7):e0052324. doi: 10.1128/jvi.00523-24. Epub 2024 Jun 5.
The picornavirus genome encodes a large, single polyprotein that is processed by viral proteases to form an active replication complex. The replication complex is formed with the viral genome, host proteins, and viral proteins that are produced/translated directly from each of the viral genomes (viral proteins provided in ). Efficient complementation of replication complex formation by viral proteins provided in , thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient complementation system for the replication of defective poliovirus (PV) mutants by a viral polyprotein precursor in HEK293 cells. Viral 3AB in the polyprotein, but not 2BC, was processed exclusively in . Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3C protease between 3C and 3D (3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in . Only a defect of 3D activity of the replicon could be rescued in ; inactivating mutations in 2C, 3B, and 3C of the replicon completely abrogated the -rescued replication. An intact N-terminus of the 3C domain of the 3CD provided in was essential for the -active function. By using this complementation system, a high-titer defective PV pseudovirus (PV) (>10 infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection.
Viral polyprotein processing is an elaborately controlled step by viral proteases encoded in the polyprotein; fully processed proteins and processing intermediates need to be correctly produced for replication, which can be detrimentally affected even by a small modification of the polyprotein. Purified/isolated viral proteins can retain their enzymatic activities required for viral replication, such as protease, helicase, polymerase, etc. However, when these proteins of picornavirus are exogenously provided (provided in ) to the viral replication complex with a defective viral genome, replication is generally not rescued/complemented, suggesting the importance of viral proteins endogenously provided (provided in ) to the replication complex. In this study, I discovered that only the viral polymerase activity of poliovirus (PV) (the typical member of picornavirus family) could be efficiently rescued by exogenously expressed viral proteins. The current study reveals potential roles for exogenous viral proteins in viral replication and offers insights into interactions during picornavirus infection.
微小核糖核酸病毒基因组编码一个大型的、单一的多蛋白，该多蛋白被病毒蛋白酶切割，形成一个活跃的复制复合物。复制复合物由病毒基因组、宿主蛋白和直接从每个病毒基因组中产生/翻译的病毒蛋白组成（在 中提供的病毒蛋白）。通过在 中提供的病毒蛋白有效地补充复制复合物的形成，从而补充外源性或异位表达的病毒蛋白，仍然有待证明。在这里，我们报告了一个有效的系统，用于在 HEK293 细胞中通过病毒多蛋白前体来补充有缺陷的脊髓灰质炎病毒（PV）突变体的复制。多蛋白中的病毒 3AB，但不是 2BC，仅在 中被专门加工。具有破坏的 3C 蛋白酶切割位点的缺陷型 PV 复制子突变体（3C/D[A/G]突变体）的复制可以通过在 中提供的病毒多蛋白来挽救。仅能挽救复制子的 3D 活性缺陷；复制子中的 2C、3B 和 3C 的失活突变完全阻断了 -挽救的复制。在 中提供的 3CD 的 3C 结构域的完整 N 端对于 -活性功能是必需的。通过使用这种 补充系统，可以用缺陷突变体生产高滴度的缺陷型 PV 假病毒（PV）（>每毫升 10 个感染单位），其复制完全依赖于 补充。这项工作揭示了外源性病毒蛋白在 PV 复制中的潜在作用，并为小核糖核酸病毒感染期间的蛋白/蛋白相互作用提供了见解。
病毒多蛋白的加工是由多蛋白中编码的病毒蛋白酶精心控制的步骤；为了复制，需要正确产生完全加工的蛋白质和加工中间体，即使多蛋白的微小修饰也会对其产生不利影响。纯化/分离的病毒蛋白可以保留其复制所需的酶活性，例如蛋白酶、解旋酶、聚合酶等。然而，当这些小核糖核酸病毒的蛋白被外源性地提供（在 中提供）给具有缺陷病毒基因组的病毒复制复合物时，复制通常不会被挽救/补充，这表明对于复制复合物内内源性提供（在 中提供）的病毒蛋白的重要性。在这项研究中，我发现只有脊髓灰质炎病毒（PV）（微小核糖核酸病毒科的典型成员）的病毒聚合酶活性可以通过外源性表达的病毒蛋白有效地挽救。目前的研究揭示了外源性病毒蛋白在病毒复制中的潜在作用，并为小核糖核酸病毒感染期间的相互作用提供了见解。
