Expression of hepatitis A virus precursor protein P3 in vivo and in vitro: polyprotein processing of the 3CD cleavage site.
Expression of hepatitis A virus precursor protein P3 in vivo and in vitro: polyprotein processing of the 3CD cleavage site.
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甲型肝炎病毒前体蛋白 P3 体内外表达：3CD 切割位点的多蛋白加工。
DOI：
10.1006/viro.1994.1063
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发表时间：
1994
期刊：
影响因子：
3.7
通讯作者：
Ehrenfeld,E
中科院分区：
文献类型：
--
作者：
Tesar,M;Pak,I;Jia,XY;Richards,OC;Summers,DF;Ehrenfeld,E
为研究甲型肝炎病毒（HAV）3D蛋白的特性和30 - 3D之间的切割位点，在体内和体外表达了编码HAV P3区蛋白的cDNA。将蛋白质编码序列置于T7启动子和EMCV翻译起始信号的控制下。通过用重组牛痘病毒vTF 7 -3（T. R. Fuerstet等人，Proc. Natl. Acad. Sci. USA83，8122-8126，1986）。P3蛋白的有效合成和加工产生3CD（78 kDa）、3D（54 kDa）、3ABC（33 kDa）、3BC（25 kDa）和3C（23 kDa）。通过比较3D蛋白在SDS-PAGE中的迁移率和3D蛋白在两个建议切割位点中的每一个处开始的3D蛋白，对3C/3D切割位点进行定位;此外，对来自体外培养的放射性标记的3D蛋白进行直接N-末端测序。结果表明，HAV多聚蛋白的1738和1739位的谷氨酰胺-精氨酸（Q/R）对通过切割形成3D。在BS-C-1细胞中通过P3前体蛋白的自催化切割产生的HAV 3D蛋白实际上是完全不溶的，并且在低速离心后沉淀。这与脊髓灰质炎病毒3D蛋白形成对比，脊髓灰质炎病毒3D蛋白由类似的构建体产生，其中很大一部分保持可溶性。含有脊髓灰质炎病毒3D蛋白质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蛋白质的提取物通过相同的测定显示出不可检测的活性。
Hepatitis A virus (HAV) cDNAs encoding the P3 region proteins were expressedin vivoandin vitroto characterize the HAV 3D protein and to identify the cleavage site between 30 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. Fuerstet al., Proc. Natl. Acad. Sci. USA83, 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 lysatein vitro.The 3C/3D 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 translationin vitrowas 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.