Paul P R, Chattopadhyay D, Banerjee A K
Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
Virology. 1988 Oct;166(2):350-7. doi: 10.1016/0042-6822(88)90505-3.
A full-length cDNA clone of the mRNA encoding the phosphoprotein (NS) of the Indiana serotype of vesicular stomatitis virus was inserted into the SP6 transcription vector. By in vitro transcription of the inserted gene followed by translation of the mRNA in a rabbit reticulocyte lysate, NS protein was synthesized. The biological activity of the protein was demonstrated by RNA synthesis in vitro by reconstitution with L protein and N-RNA template purified from virions. Using oligonucleotide-directed RNase H cleavage of the full-length NS mRNA, a series of deleted RNAs were made which gave rise to corresponding size classes of truncated NS protein after translation in vitro. The N-RNA template binding site was located at the C-terminal domain (21 amino acids) of the NS protein and the L-protein binding site was present within 14 amino acids spanning the NH2-terminal side of the N-RNA binding site. These results are similar to that obtained with the NS protein of the New Jersey serotype of VSV, indicating conservation of the functional domains within the VSV serotypes.
将编码水疱性口炎病毒印第安纳血清型磷蛋白（NS）的mRNA的全长cDNA克隆插入到SP6转录载体中。通过对插入基因进行体外转录，然后在兔网织红细胞裂解物中对mRNA进行翻译，合成了NS蛋白。通过用从病毒粒子中纯化的L蛋白和N-RNA模板进行重组，在体外进行RNA合成，证明了该蛋白的生物学活性。利用寡核苷酸定向RNase H切割全长NS mRNA，制备了一系列缺失的RNA，这些RNA在体外翻译后产生了相应大小类别的截短NS蛋白。N-RNA模板结合位点位于NS蛋白的C末端结构域（21个氨基酸），L蛋白结合位点存在于跨越N-RNA结合位点NH2末端一侧的14个氨基酸内。这些结果与用VSV新泽西血清型的NS蛋白获得的结果相似，表明VSV血清型内功能结构域的保守性。
