Wengler G, Wengler G, Boege U, Wahn K
Virology. 1984 Jan 30;132(2):401-12. doi: 10.1016/0042-6822(84)90045-x.
Core-like (CL) particles which closely resemble alphavirus cores in size, shape, and relative amount of nucleic acid and protein have been assembled in vitro from Sindbis (SIN) virus core (C) protein and single-stranded nucleic acids in buffer containing 1 M urea [G. Wengler, U. Boege, G. Wengler, H. Bischoff, and K. Wahn (1982) Virology 118, 401-410]. We have now analyzed the interaction of SIN virus C protein and nucleic acids in vitro under conditions designed to resemble those present in the cell during core assembly. In buffer containing 100 mM K-acetate, 1.7 mM Mg-acetate, pH 7.4, CL particles are efficiently assembled from all single-stranded nucleic acids analyzed, and even heparin and polyvinylsulfate are incorporated into such particles. A reticulocyte lysate translates SIN virus-specific mRNA into C protein under these ionic conditions. Interactions of C protein with nucleic acids and ribosomes in a reticulocyte lysate have also been analyzed. The following conclusions can be drawn from these analyses: (1) In accordance with earlier findings [N. Glanville and I. Ulmanen (1976) Biochem. Biophys. Res. Commun. 71, 393-399] the C protein translated in vitro efficiently binds to ribosomes. (2) Exogenously added C protein binds to the large subunit of the ribosomes in the lysate. (3) CL particles can be assembled in the lysate from exogenous added 42 S genome RNA and exogenous added C protein if both components are present at sufficiently high concentrations. (4) The C protein translated from viral mRNA in the lysate is transferred from the ribosomes into preassembled CL particles containing 42 S RNA in the lysate. (5) If only small amounts of CL particles are added into a lysate these particles disaggregate and core protein molecules are transferred from the particles to the large subunit of the ribosomes. The results on the assembly of CL particles in vitro allow the formulation of some hypotheses concerning the assembly and disassembly of core particles in vivo.
在含有1 M尿素的缓冲液中，已利用辛德毕斯（SIN）病毒核心（C）蛋白和单链核酸在体外组装出了核心样（CL）颗粒，其在大小、形状以及核酸与蛋白质的相对含量方面与甲病毒核心极为相似[G. 温格勒、U. 伯格、G. 温格勒、H. 比绍夫和K. 瓦恩（1982年），《病毒学》118卷，401 - 410页]。我们现在在旨在模拟核心组装过程中细胞内存在的条件下，对SIN病毒C蛋白与核酸在体外的相互作用进行了分析。在含有100 mM醋酸钾、1.7 mM醋酸镁、pH 7.4的缓冲液中，从所有分析的单链核酸都能高效组装出CL颗粒，甚至肝素和聚硫酸乙烯酯也能掺入此类颗粒中。在这些离子条件下，网织红细胞裂解物能将SIN病毒特异性mRNA翻译成C蛋白。还分析了C蛋白在网织红细胞裂解物中与核酸和核糖体的相互作用。从这些分析中可以得出以下结论：（1）与早期研究结果一致[N. 格兰维尔和I. 乌尔马宁（1976年），《生物化学与生物物理研究通讯》71卷，393 - 399页]，体外翻译的C蛋白能高效结合核糖体。（2）外源添加的C蛋白能与裂解物中的核糖体大亚基结合。（3）如果外源添加的42 S基因组RNA和外源添加的C蛋白都以足够高的浓度存在，CL颗粒就能在裂解物中组装而成。（4）在裂解物中从病毒mRNA翻译而来的C蛋白会从核糖体转移到裂解物中预先组装好的含有42 S RNA的CL颗粒中。（5）如果仅向裂解物中添加少量CL颗粒，这些颗粒会解聚，核心蛋白分子会从颗粒转移到核糖体大亚基上。体外CL颗粒组装的结果有助于提出一些关于体内核心颗粒组装和解聚的假说。
