Paul Aniko V, Peters Julia, Mugavero JoAnn, Yin Jiang, van Boom Jacques H, Wimmer E
Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, 11794, USA.
J Virol. 2003 Jan;77(2):891-904. doi: 10.1128/jvi.77.2.891-904.2003.
The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.
脊髓灰质炎病毒（PV）RNA合成的第一步是UMP与末端蛋白VPg的共价连接。这个反应可以通过两种不同的体外试验来研究。较简单的试验基于聚（A）模板，需要合成的VPg、纯化的RNA聚合酶3D（pol）、UTP和一种二价阳离子。另一种试验使用特定的病毒序列[cre(2C)]作为VPg尿苷酸化的模板，并且需要添加蛋白酶3CD（pro）。使用这两种试验中的一种或两种，我们分析了尿苷酸化反应的VPg特异性和金属需求。我们确定了VPg中单个和双氨基酸取代对肽作为3D（pol）底物能力的影响。发现VPg中的突变在体外干扰尿苷酸化，会导致病毒生长停止。含有人类鼻病毒14（HRV14）VPg的嵌合PV是有活力的，但用HRV2和HRV89的VPg替换PV的VPg是致死性的。在所测试的三种鼻病毒VPg中，只有HRV14肽在体外被发现可作为PV1（M）3D（pol）的底物。我们还研究了聚（A）模板上VPg尿苷酸化反应的金属特异性。我们的结果表明，与Mg(2+)或其他二价阳离子相比，RNA聚合酶强烈偏好Mn(2+)作为辅因子。
