Kanjanahaluethai A, Baker S C
Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153, USA.
J Virol. 2000 Sep;74(17):7911-21. doi: 10.1128/jvi.74.17.7911-7921.2000.
Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.
小鼠肝炎病毒（MHV）是一种31 kb的正链RNA病毒，通过一种称为复制酶的病毒RNA依赖性RNA聚合酶在受感染细胞的细胞质中进行复制。复制酶由基因组RNA 5'端最前面的22 kb编码，经翻译产生一个大于800 kDa的多聚蛋白。复制酶多聚蛋白由病毒蛋白酶以及可能还有细胞蛋白酶进行广泛加工，从而产生一个功能性复制酶复合体。到目前为止，已证实MHV复制酶编码的两种蛋白酶，即木瓜蛋白酶样蛋白酶1（PLP1）和脊髓灰质炎病毒3C样蛋白酶（3CLpro），可对复制酶多聚蛋白进行加工。在本报告中，我们描述了第三种MHV蛋白酶结构域PLP2的克隆、表达及活性。我们发现PLP2可切割一个编码复制酶多聚蛋白首个预测跨膜结构域（MP1）的底物。MP1的切割及150 kDa中间体p150的释放可能对于将复制酶复合体嵌入细胞膜至关重要。使用针对MP1结构域C末端的抗血清（抗D11），我们证实p150包含MP1结构域，并鉴定出一种44 kDa蛋白（p44）为p150的加工产物。脉冲追踪实验表明，p150在MHV感染的细胞中快速产生，且p44由p150前体加工而来。蛋白酶抑制剂研究显示，与3CLpro活性不同，PLP2活性对半胱氨酸蛋白酶抑制剂E64d不敏感。此外，使用PLP2结构域和一个编码MP1切割位点的底物进行的共表达研究表明，PLP2可有效地进行反式作用。定点诱变研究证实，半胱氨酸1715为PLP2的催化残基。本研究首次报道了PLP2结构域的酶活性，并证明三种不同的病毒蛋白酶活性可对MHV复制酶多聚蛋白进行加工。
