STRUCTURE OF POLIOVIRUS REPLICATIVE INTERMEDIATE RNA - ELECTRON-MICROSCOPE ANALYSIS OF RNA CROSS-LINKED INVIVO WITH PSORALEN DERIVATIVE
STRUCTURE OF POLIOVIRUS REPLICATIVE INTERMEDIATE RNA - ELECTRON-MICROSCOPE ANALYSIS OF RNA CROSS-LINKED INVIVO WITH PSORALEN DERIVATIVE
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DOI：
10.1016/0022-2836(84)90124-4
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发表时间：
1984-01-01
影响因子：
5.6
通讯作者：
EHRENFELD, E
中科院分区：
文献类型：
--
作者：
RICHARDS, OC;MARTIN, SC;EHRENFELD, E
用4‘’-氨基甲基-4，5‘’，8-三甲基补骨脂素(AMT)体内交联后，用电子显微镜观察脊髓灰质炎病毒复制中间体(RI)RNA的结构。从感染的[人宫颈癌HeLa]细胞中纯化后，未变性的RI呈现为基因组长度的双链骨架，平均有3条(有时多达8条)新生的单链尾巴。变性后，只看到不同长度的单链，表明细胞中的RI很少或没有双链结构，因此新生的链只在较短的区域与模板瞬时氢键。未变性RI的双链骨架是由于在脱蛋白和纯化过程中互补链的断裂。体内交联法的有效性通过处理细胞中病毒RNA合成的完全抑制和体内[~3H]AMT与RI分子的直接结合而得到证明。成熟的脊髓灰质炎病毒粒子对AMT是不渗透的；然而，在黑暗中与AMT一起孵育的细胞中的病毒生长导致含有RNA基因组的病毒颗粒的正常产量，其传染性随后可以被光灭活。通过对脊髓灰质炎病毒双链RNA(RF)的分析，确定AMT诱导的交联率。在体外交联，然后在变性条件下铺展EM，得到起泡的双链结构，至少有1个链间交联键/80个碱基对。体内RF交联物也表现出广泛的交联性，.apprx减少。5倍于体外交联值。因此，在这些条件下未能检测到交叉连接的RI表明在体内不存在广泛的碱基配对。
The structure of the poliovirus replicative intermediate (RI) RNA was examined by EM after cross-linking in vivo with 4''-aminomethyl-4,5'',8-trimethylpsoralen (AMT). After purification from infected [human cervical carcinoma HeLa] cells, undenatured RI appeared as a double-stranded backbone of genome length, with an averge of 3 (and ocasionally up to 8) nascent, single-stranded tails. After denaturation only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently H-bonded to their template over short regions. The double-stranded backbone of undenatured RI is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [3H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for EM under denaturing conditions yielded bubbled duplex structures with a minimum of 1 interstrand cross-link/80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased .apprx. 5-fold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.