Rao Ping, Yuan Weiming, Krug Robert M
Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712, USA.
EMBO J. 2003 Mar 3;22(5):1188-98. doi: 10.1093/emboj/cdg109.
In viral cap-snatching, the endonuclease intrinsic to the viral polymerase cleaves cellular capped RNAs to generate capped fragments that are primers for viral mRNA synthesis. Here we demonstrate that the influenza viral polymerase, which is assembled in human cells using recombinant proteins, effectively uses only CA-terminated capped fragments as primers for viral mRNA synthesis in vitro. Thus we provide the first in vitro system that mirrors the cap-snatching process occurring in vivo during virus infection. Further, we demonstrate that when a capped RNA substrate contains a CA cleavage site, the functions of virion RNA (vRNA) differ from those previously described: the 5' terminal sequence of vRNA alone is sufficient for endonuclease activation, and the 3' terminal sequence of vRNA functions solely as a template for mRNA synthesis. Consequently, we are able to identify the vRNA sequences that are required for each of these two separable functions. We present a new model for the influenza virus cap-snatching mechanism, which we postulate is a paradigm for the cap-snatching mechanisms of other segmented, negative-strand and ambisense RNA viruses.
在病毒的帽状结构抢夺过程中，病毒聚合酶固有的核酸内切酶切割细胞的带帽RNA，以产生带帽片段，这些片段作为病毒mRNA合成的引物。在此，我们证明，利用重组蛋白在人细胞中组装的流感病毒聚合酶，在体外仅有效地将以CA结尾的带帽片段用作病毒mRNA合成的引物。因此，我们提供了首个能反映病毒感染期间体内发生的帽状结构抢夺过程的体外系统。此外，我们证明，当带帽RNA底物含有CA切割位点时，病毒粒子RNA（vRNA）的功能与先前描述的不同：单独的vRNA 5'末端序列足以激活核酸内切酶，而vRNA的3'末端序列仅作为mRNA合成的模板。因此，我们能够确定这两种可分离功能各自所需的vRNA序列。我们提出了一种新的流感病毒帽状结构抢夺机制模型，我们推测该模型是其他分段的、负链和双义RNA病毒帽状结构抢夺机制的范例。
