Replication of murine hepatitis virus is regulated by papain-like proteinase 1 processing of nonstructural proteins 1, 2, and 3
Replication of murine hepatitis virus is regulated by papain-like proteinase 1 processing of nonstructural proteins 1, 2, and 3
复制标题
DOI：
10.1128/jvi.01428-06
复制
发表时间：
2006-12-01
影响因子：
5.4
通讯作者：
Denison, Mark R.
中科院分区：
文献类型：
--
作者：
Graham, Rachel L.;Denison, Mark R.
冠状病毒是正链RNA病毒，可将其基因组RNA翻译成多蛋白，多蛋白经共翻译和翻译后加工成中间和成熟复制酶非结构蛋白（nsps）。在小鼠肝炎病毒（MHV）中，nsp1、2和3通过nsp3 （PLP1和PLP2）内的两种木瓜蛋白酶活性加工产生nsp1、nsp2-3中间体、成熟的nsp2和nsp3。为了确定nsp2和nsp3在切割位点2 （CS2）和PLP1蛋白酶活性上的复制过程中的作用，在MHV基因组的CS2、CS1和CS2以及PLP1催化位点上单独和联合设计了突变。在CS2上切断的突变病毒的生长和RNA合成延迟，但生长到野生型滴度为bbb10 (7) PFU/ml。缺失CS1和CS2的突变病毒表现出生长延迟和峰值病毒滴度下降到类似于10(4)PFU/ml。PLP1催化残基的失活导致突变病毒在CS1或CS2中都没有加工，并且生长严重减弱，仅达到10(2)PFU/ml。然而，当在PLP1灭活的情况下同时删除CS1和CS2时，产生的突变病毒的生长得到了部分补偿，与CS1和CS2缺失突变体的生长相当。这些结果表明，PLP1与CS1和CS2的相互作用对蛋白质加工至关重要，并表明这种相互作用在调节nsp1、2和3在病毒RNA合成中的功能中发挥特定作用。
Coronaviruses are positive-strand RNA viruses that translate their genome RNA into polyproteins that are co- and posttranslationally processed into intermediate and mature replicase nonstructural proteins (nsps). In murine hepatitis virus (MHV), nsps 1, 2, and 3 are processed by two papain-like proteinase activities within nsp3 (PLP1 and PLP2) to yield nspl, an nsp2-3 intermediate, and mature nsp2 and nsp3. To determine the role in replication of processing between nsp2 and nsp3 at cleavage site 2 (CS2) and PLP1 proteinase activity, mutations were engineered into the MHV genome at CS2, at CS1 and CS2, and at the PLP1 catalytic site, alone and in combination. Mutant viruses with abolished cleavage at CS2 were delayed in growth and RNA synthesis but grew to wild-type titers of > 10(7) PFU/ml. Mutant viruses with deletion of both CS1 and CS2 exhibited both a delay in growth and a decrease in peak viral titer to similar to 10(4) PFU/ml. Inactivation of PLP1 catalytic residues resulted in a mutant virus that did not process at either CS1 or CS2 and was severely debilitated in growth, achieving only 10(2) PFU/ml. However, when both CS1 and CS2 were deleted in the presence of inactivated PLP1, the growth of the resulting mutant virus was partially compensated, comparable to that of the CS1 and CS2 deletion mutant. These results demonstrate that interactions of PLP1 with CS1 and CS2 are critical for protein processing and suggest that the interactions play specific roles in regulation of the functions of nspl, 2, and 3 in viral RNA synthesis.