小核糖核酸病毒基因组编码一个大的,由病毒蛋白酶加工形成活性复制复合物的单一多蛋白。复制复合物与病毒基因组形成,宿主蛋白,和直接从每个病毒基因组产生/翻译的病毒蛋白(以顺式提供的病毒蛋白)。通过反式提供的病毒蛋白在体内复制复合物形成的有效互补,因此,外源或异位表达的病毒蛋白,还有待证明。这里,我们报道了一种有效的反式互补系统,用于通过病毒多蛋白前体在HEK293细胞中复制缺陷型脊髓灰质炎病毒(PV)突变体。病毒3AB中的多蛋白,但不是2BC,完全在顺式中处理。有缺陷的PV复制子突变体的复制,在3Cpro和3Dpol(3C/D[A/G]突变体)之间的病毒3Cpro蛋白酶的裂解位点被破坏,可以通过反式提供的病毒多蛋白来挽救。只有复制子的3Dpol活性缺陷可以反式挽救;2CATPase/hel中的失活突变,3B,和3Cpro的复制子完全废除了反式拯救的复制。以反式提供的3CDpro的3Cpro结构域的完整N末端对于反式活性功能是必需的。通过使用这个反式互补系统,一个高滴度的有缺陷的PV假病毒(PVpv)(>107感染单位/毫升)可以产生与有缺陷的突变体,其复制完全依赖于反式互补。这项工作揭示了外源病毒蛋白在PV复制中的潜在作用,并提供了对微小核糖核酸病毒感染期间蛋白质/蛋白质相互作用的见解。
目的:病毒多蛋白加工是由多蛋白中编码的病毒蛋白酶精心控制的步骤;完全加工的蛋白质和加工中间体需要正确产生以进行复制,即使多蛋白的小修饰也可能受到不利影响。纯化/分离的病毒蛋白可以保留病毒复制所需的酶活性,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质被外源提供(反式提供)给具有缺陷病毒基因组的病毒复制复合物时,复制通常不被拯救/补充,提示内源性提供(以顺式提供)到复制复合物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)(小核糖核酸病毒家族的典型成员)的病毒聚合酶活性可以通过外源表达的病毒蛋白有效地挽救。目前的研究揭示了外源病毒蛋白在病毒复制中的潜在作用,并提供了对小核糖核酸病毒感染过程中相互作用的见解。
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 cis). Efficient complementation in vivo of replication complex formation by viral proteins provided in trans, thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient trans 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 cis. Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3Cpro protease between 3Cpro and 3Dpol (3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in trans. Only a defect of 3Dpol activity of the replicon could be rescued in trans; inactivating mutations in 2CATPase/hel, 3B, and 3Cpro of the replicon completely abrogated the trans-rescued replication. An intact N-terminus of the 3Cpro domain of the 3CDpro provided in trans was essential for the trans-active function. By using this trans complementation system, a high-titer defective PV pseudovirus (PVpv) (>107 infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on trans complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection.
OBJECTIVE: 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 trans) 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 cis) 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.