CIS-ACTING REQUIREMENTS FOR THE REPLICATION OF FLOCK HOUSE VIRUS-RNA 2
CIS-ACTING REQUIREMENTS FOR THE REPLICATION OF FLOCK HOUSE VIRUS-RNA 2
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DOI：
10.1128/jvi.67.6.3544-3551.1993
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发表时间：
1993-06-01
影响因子：
5.4
通讯作者：
LI, Y
中科院分区：
文献类型：
--
作者：
BALL, LA;LI, Y
为了研究RNA复制的顺式作用要求，将鸡舍病毒（FHV）RNA 2的cDNA克隆转染到幼仓鼠肾细胞中并转录以产生具有不同长度的末端延伸或缺乏分子内部区域的RNA。测试这些RNA被FHV复制酶复制的能力，所述FHV复制酶由纯化的FHV RNA 1的共转染提供。结果表明，RNA复制受到末端延伸的抑制，特别是在RNA的5'末端的那些，尽管这些延伸在RNA复制期间被校正。具有12个核苷酸的3'延伸的负义转录物被复制以产生具有正确的5'末端的正义RNA，这表明复制酶可以从更长的模板中选择其正确的起始位点。正链第二位的尿苷残基是模板活性的重要决定因素。在RNA 2的1,400个核苷酸中，有近50%的大的内部缺失的RNA分子与全长分子一样有效地复制，但前提是它们包含位于核苷酸538和616之间的内部区域。在连续复制传代过程中出现的36个RNA 2的自发缺失都保留了分子的相同内部区域。这些结果表明，FHV RNA 2的末端和内部区域在使该分子成为复制的有效模板中起着重要作用。
To examine the cis-acting requirements for RNA replication, a cDNA clone of flock house virus (FHV) RNA 2 was transfected into baby hamster kidney cells and transcribed to yield RNAs that had terminal extensions of different lengths or that lacked internal regions of the molecule. These RNAs were tested for their ability to be replicated by FHV replicase that was provided by cotransfection of purified FHV RNA 1. The results showed that RNA replication was inhibited by terminal extensions, particularly those at the 5' end of the RNA, despite the fact that these extensions were corrected during RNA replication. A negative-sense transcript with a 12-nucleotide 3' extension was replicated to produce a positive-sense RNA that had the correct 5' end, showing that the replicase could select its correct initiation site from within a longer template. A uridine residue at the second position of the positive strand was an important determinant of template activity. RNA molecules with large internal deletions that amounted to almost 50% of the 1,400 nucleotides of RNA 2 replicated as efficiently as full-length molecules, but only if they contained an internal region that lay between nucleotides 538 and 616. Thirty-six spontaneous deletions of RNA 2 that arose during sequential replicative passages all conserved the same internal region of the molecule. These results establish that both terminal and internal regions of FHV RNA 2 play essential roles in making the molecule a competent template for replication.