Weiss B, Nitschko H, Ghattas I, Wright R, Schlesinger S
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
J Virol. 1989 Dec;63(12):5310-8. doi: 10.1128/JVI.63.12.5310-5318.1989.
We investigated the interaction of the capsid protein of Sindbis virus with Sindbis viral RNAs and defined a region of the genome that is required for binding in vitro and for packaging in vivo. The binding studies were performed with purified capsid protein immobilized on nitrocellulose and 32P-labeled RNAs transcribed in vitro from viral and nonspecific cDNAs. Genomic and defective interfering (DI) RNAs bound capsid protein significantly better than either the subgenomic (26S) RNA or nonspecific RNAs. Transcripts prepared from either truncated or deleted cDNAs were used to define the segment required for binding. This segment, which is represented twice in DI RNA, lies between nucleotides 746 and 1226 of the genomic RNA and is within the coding region of the nonstructural protein nsP1. Insertion of a domain covering these sequences into a nonviral RNA was able to convert it from a background level of binding to an activity that was 80% that of the Sindbis virus DI RNA. We analyzed DI RNA transcripts in detail because they could be studied not only for the ability to bind capsid protein in vitro but also for the ability to be replicated and packaged in vivo in the presence of helper virion RNA. The results obtained with three DI RNAs are reported. One (CTS14), which has one copy of the binding domain, bound efficiently to capsid protein in vitro and was packaged in vivo as measured by amplification on passaging. In contrast, a DI RNA (CTS1) which lacked this region did not bind to capsid protein and was not detected on passaging. By using lipofectin (P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J.P. Northrop, G. M. Ringold, and M. Danielson, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987) to enhance RNA uptake, we were able to demonstrate that CTS1 RNA was replicated in the transfected cells. It was replicated to the same level as another DI RNA (CTS253) which has only the 3' 279 nucleotides of the binding domain and these are located near the 3' terminus of the RNA. CTS253 bound capsid protein to an intermediate level but was amplified on passaging. The binding studies and the in vivo packaging data, taken together, provide strong support for the conclusion that there is a specific capsid recognition domain in Sindbis virus RNA that plays a role in nucleocapsid assembly.
我们研究了辛德毕斯病毒衣壳蛋白与辛德毕斯病毒RNA的相互作用，并确定了基因组中一个在体外结合及体内包装所必需的区域。结合研究使用固定在硝酸纤维素上的纯化衣壳蛋白以及从病毒和非特异性cDNA体外转录的32P标记RNA进行。基因组RNA和缺陷干扰（DI）RNA与衣壳蛋白的结合明显优于亚基因组（26S）RNA或非特异性RNA。从截短或缺失的cDNA制备的转录本用于确定结合所需的片段。该片段在DI RNA中出现两次，位于基因组RNA的746至1226核苷酸之间，且在非结构蛋白nsP1的编码区内。将覆盖这些序列的结构域插入非病毒RNA能够使其从背景结合水平转变为具有辛德毕斯病毒DI RNA 80%活性的结合能力。我们详细分析了DI RNA转录本，因为它们不仅可用于研究体外结合衣壳蛋白的能力，还可用于研究在辅助病毒粒子RNA存在下在体内复制和包装的能力。报告了用三种DI RNA获得的结果。其中一种（CTS14）有一个结合结构域拷贝，在体外能有效结合衣壳蛋白，并且通过传代扩增测定在体内被包装。相比之下，一种缺乏该区域的DI RNA（CTS1）不与衣壳蛋白结合，传代时未被检测到。通过使用脂质体转染试剂（P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J.P. Northrop, G. M. Ringold, and M. Danielson, Proc. Natl. Acad. Sci. USA 84:7413 - 7417, 1987）增强RNA摄取，我们能够证明CTS1 RNA在转染细胞中复制。其复制水平与另一种DI RNA（CTS253）相同，CTS253仅具有结合结构域的3'端279个核苷酸，且这些核苷酸位于RNA的3'端附近。CTS253与衣壳蛋白的结合处于中等水平，但传代时会扩增。结合研究和体内包装数据共同为以下结论提供了有力支持：辛德毕斯病毒RNA中存在一个特定的衣壳识别结构域，它在核衣壳组装中起作用。
