Hill K R, Hajjou M, Hu J Y, Raju R
Department of Microbiology, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208, USA.
J Virol. 1997 Apr;71(4):2693-704. doi: 10.1128/JVI.71.4.2693-2704.1997.
Sindbis virus (SIN), a mosquito-transmitted animal RNA virus, carries a 11.7-kb positive-sense RNA genome which is capped and polyadenylated. We recently reported that the SIN RNA-dependent RNA polymerase (RdRp) could initiate negative-strand RNA synthesis from a 0.3-kb 3'-coterminal SIN RNA fragment and undergo template switching in vivo (M. Hajjou, K. R. Hill, S. V. Subramaniam, J. Y. Hu, and R. Raju, J. Virol. 70:5153-5164, 1996). To identify and characterize the viral and nonviral sequences which regulate SIN RNA synthesis and recombination, a series of SIN RNAs carrying altered 3' ends were tested for the ability to produce infectious virus or to support recombination in BHK cells. The major findings of this report are as follows: (i) the 3'-terminal 20-nucleotides (nt) sequence along with the abutting poly(A) tail of the SIN genome fully supports negative-strand synthesis, genome replication, and template switching; (ii) a full-length SIN RNA carrying the 3'-terminal 24 nt but lacking the poly(A) tail is noninfectious; (iii) SIN RNAs which carry 3' 64 nt or more without the poly(A) tail are infectious and regain their poly(A) tail in vivo; (iv) donor templates lacking the poly(A) tail do not support template switching; (v) full-length SIN RNAs lacking the poly(A) tail but carrying 3' nonviral extensions, although debilitated to begin with, evolve into rapidly growing poly(A)-carrying mutants; (vi) poly(A) or poly(U) motifs positioned internally within the acceptor templates, in the absence of other promoter elements within the vicinity, do not induce the jumping polymerase to reinitiate at these sites; and (vii) the junction site selection on donor templates occurs independently of the sequences around the acceptor sites. In addition to furthering our understanding of RNA recombination, these studies give interesting clues as to how the alphavirus polymerase interacts with its 3' promoter elements of genomic RNA and nonreplicative RNAs. This is the first report that an in vitro-synthesized alphavirus RNA lacking a poly(A) tail can initiate infection and produce 3' polyadenylated viral genome in vivo.
辛德毕斯病毒（SIN）是一种由蚊子传播的动物RNA病毒，其携带一个11.7 kb的正链RNA基因组，该基因组有帽状结构并进行了多聚腺苷酸化修饰。我们最近报道，SIN RNA依赖性RNA聚合酶（RdRp）能够从一个0.3 kb的3' 共末端SIN RNA片段起始负链RNA合成，并在体内发生模板转换（M. Hajjou、K. R. Hill、S. V. Subramaniam、J. Y. Hu和R. Raju，《病毒学杂志》70:5153 - 5164，1996年）。为了鉴定和表征调控SIN RNA合成与重组的病毒和非病毒序列，对一系列携带改变的3' 末端的SIN RNA进行了测试，以检测它们在BHK细胞中产生感染性病毒或支持重组的能力。本报告的主要发现如下：（i）SIN基因组的3' 末端20个核苷酸（nt）序列以及相邻的多聚（A）尾完全支持负链合成、基因组复制和模板转换；（ii）携带3' 末端24 nt但缺少多聚（A）尾的全长SIN RNA无感染性；（iii）携带3' 端64 nt或更多且无多聚（A）尾的SIN RNA具有感染性，并在体内重新获得其多聚（A）尾；（iv）缺少多聚（A）尾的供体模板不支持模板转换；（v）缺少多聚（A）尾但携带3' 非病毒延伸序列的全长SIN RNA，尽管一开始功能减弱，但会演变成快速生长的携带多聚（A）的突变体；（vi）在受体模板内部位置的多聚（A）或多聚（U）基序，在附近没有其他启动子元件的情况下，不会诱导跳跃聚合酶在这些位点重新起始；（vii）供体模板上的连接位点选择独立于受体位点周围的序列。除了加深我们对RNA重组的理解外，这些研究还为甲病毒聚合酶如何与其基因组RNA和非复制性RNA的3' 启动子元件相互作用提供了有趣的线索。这是第一份关于体外合成的缺少多聚（A）尾的甲病毒RNA能够在体内起始感染并产生3' 多聚腺苷酸化病毒基因组的报告。
