Tesar M, Pak I, Jia X Y, Richards O C, Summers D F, Ehrenfeld E
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Virology. 1994 Feb;198(2):524-33. doi: 10.1006/viro.1994.1063.
Hepatitis A virus (HAV) cDNAs encoding the P3 region proteins were expressed in vivo and in vitro to characterize the HAV 3D protein and to identify the cleavage site between 3C and 3D. Protein coding sequences were placed under control of a T7 promoter and an EMCV translational initiation signal. T7 RNA polymerase was provided by simultaneous infection of transfected BS-C-1 cells with a recombinant vaccinia virus vTF7-3 (T. R. Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126, 1986). Efficient synthesis and processing of P3 proteins occurred to yield 3CD (78 kDa), 3D (54 kDa), 3ABC (33 kDa), 3BC (25 kDa), and 3C (23 kDa). Similar products were produced by translation of T7 transcripts in a rabbit reticulocyte lysate in vitro. The 3C/D cleavage site was mapped by comparing the mobility of 3D in SDS-PAGE with 3D proteins engineered to begin at each of the two proposed cleavage sites; in addition, direct N-terminal sequencing of radiolabeled 3D protein from translation in vitro was performed. The results showed that 3D was formed by cleavage at the glutamine-arginine (Q/R) pair at position 1738 and 1739 of the HAV polyprotein. HAV 3D protein produced by autocatalytic cleavage of P3 precursor proteins in BS-C-1 cells is virtually completely insoluble and sediments after low-speed centrifugation. This is in contrast to the poliovirus 3D protein, produced from a similar construct, a significant portion of which remains soluble. Extracts containing the poliovirus 3D protein manifested high levels of RNA-dependent RNA polymerase activity, whereas those containing the HAV 3D protein showed no detectable activity by the same assay.
编码P3区域蛋白的甲型肝炎病毒（HAV）cDNA在体内和体外进行表达，以表征HAV 3D蛋白并确定3C和3D之间的切割位点。蛋白质编码序列置于T7启动子和EMCV翻译起始信号的控制之下。通过用重组痘苗病毒vTF7-3同时感染转染的BS-C-1细胞来提供T7 RNA聚合酶（T.R. Fuerst等人，《美国国家科学院院刊》83，8122-8126，1986）。P3蛋白发生高效合成和加工，产生3CD（78 kDa）、3D（54 kDa）、3ABC（33 kDa）、3BC（25 kDa）和3C（23 kDa）。通过在兔网织红细胞裂解物中体外翻译T7转录本也产生了类似的产物。通过比较SDS-PAGE中3D的迁移率与设计从两个提议的切割位点中的每一个开始的3D蛋白，来确定3C/D切割位点；此外，对体外翻译的放射性标记3D蛋白进行了直接N端测序。结果表明，3D是由HAV多蛋白1738和1739位的谷氨酰胺-精氨酸（Q/R）对处的切割形成的。在BS-C-1细胞中由P3前体蛋白自催化切割产生的HAV 3D蛋白实际上几乎完全不溶，在低速离心后沉淀。这与由类似构建体产生的脊髓灰质炎病毒3D蛋白形成对比，其中很大一部分仍然可溶。含有脊髓灰质炎病毒3D蛋白的提取物表现出高水平的RNA依赖性RNA聚合酶活性，而含有HAV 3D蛋白的提取物通过相同测定未显示可检测到的活性。
