RNA REPLICATION BY RESPIRATORY SYNCYTIAL VIRUS (RSV) IS DIRECTED BY THE N-PROTEIN, P-PROTEIN, AND L PROTEIN - TRANSCRIPTION ALSO OCCURS UNDER THESE CONDITIONS BUT REQUIRES RSV SUPERINFECTION FOR EFFICIENT SYNTHESIS OF FULL-LENGTH MESSENGER-RNA
RNA REPLICATION BY RESPIRATORY SYNCYTIAL VIRUS (RSV) IS DIRECTED BY THE N-PROTEIN, P-PROTEIN, AND L PROTEIN - TRANSCRIPTION ALSO OCCURS UNDER THESE CONDITIONS BUT REQUIRES RSV SUPERINFECTION FOR EFFICIENT SYNTHESIS OF FULL-LENGTH MESSENGER-RNA
复制标题
DOI：
10.1128/jvi.69.9.5677-5686.1995
复制
发表时间：
1995-09-01
影响因子：
5.4
通讯作者：
COLLINS, PL
中科院分区：
文献类型：
--
作者：
GROSFELD, H;HILL, MG;COLLINS, PL
以前，构建了cDNA，使得通过T7 RNA聚合酶的转录产生类似于人呼吸道合胞病毒（RSV）基因组RNA的1-kb负义类似物，所述人呼吸道合胞病毒（RSV）基因组RNA含有在推定的RSV转录基序控制下的氯霉素乙酰转移酶（CAT）基因，并且侧翼为RSV基因组末端。当转染到RSV感染的细胞中时，该微型基因组被“拯救”，如通过高水平的CAT表达和在连续传代期间繁殖并表达高水平的CAT表达的可传播颗粒的产生所证明的（P. L.柯林斯，M. A. Mink和D. S. Stec，Proc. Natl. Acad. Sci. USA，88：9663-9667，1991）。在此，该cDNA与设计用于产生精确拷贝的正义RSV-CAT RNA反基因组的第二个cDNA一起，各自被修饰为含有自切割锤头状核酶，用于产生几乎精确的3'末端。将每种cDNA与编码RSV N、P和L蛋白的质粒一起转染到用表达T7 RNA聚合酶的牛痘病毒重组体感染的细胞中，所述质粒各自在T7启动子的控制下。当质粒提供的模板是微型反基因组时，产生微型基因组。当质粒提供的模板为小基因组时，产物为小反基因组、亚基因组多聚腺苷酸化mRNA和子代小基因组。从质粒提供的小基因组模板制备的子代小基因组的鉴定表明发生了完整的RSV RNA复制循环。RNA合成需要所有三种RSV蛋白，N、P和L，并且通过在L蛋白中的位置989处用Asn取代Asp而完全消除。因此，N、P和L蛋白足以合成正确的微型基因组和反基因组，但亚基因组mRNA的情况并非如此，这表明RNA复制和转录的要求并不相同。与N，P，和L单独互补产生了一个mRNA的模式，含有大部分的不完整的，异质性大小的分子。相反，与RSV互补（提供所有的RSV基因产物）产生一个单一的离散mRNA带。用RSV对基于RNA合成的分期为N/P/L的细胞进行超感染，产生单个离散的mRNA种类。一些额外的RSV重叠感染提供的因素似乎参与转录，最明显的可能性是一个或多个额外的RSV基因产物。
Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a similar to 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was ''rescued,'' as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P. L. collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3' end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L;based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.