Morgan J R, Cohen L K, Roberts B E
J Virol. 1984 Oct;52(1):206-14. doi: 10.1128/JVI.52.1.206-214.1984.
The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5' and 3' termini were precisely located by S1 nuclease analysis.
编码痘苗病毒mRNA加帽酶大亚基的DNA序列位于病毒基因组上。用[α-32P]GTP标记形成的酶-鸟苷酸共价中间体，可用于鉴定加帽酶的大亚基，并用于监测感染周期中该酶的出现。该检测证实，痘苗病毒感染后，在病毒DNA复制之前，一种与大亚基相对应的新的84,000分子量多肽迅速合成。早期病毒mRNA的杂交选择无细胞翻译表明，痘苗病毒编码的一种多肽，其分子量与在痘苗病毒颗粒中发现的32P标记的84,000分子量多肽相同。与真正的加帽酶一样，这种病毒编码的无细胞翻译产物能特异性结合DNA纤维素。对V8蛋白酶、胰凝乳蛋白酶和胰蛋白酶产生的部分蛋白水解片段进行比较表明，32P标记的大亚基与[35S]甲硫氨酸标记的无细胞翻译产物相同。编码加帽酶大亚基的mRNA位于痘苗病毒基因组HindIII D限制片段左侧3.1千碱基对处。此外，该mRNA大小为3.0千碱基，通过S1核酸酶分析精确确定了其5'和3'末端的位置。
