Dingle K, Moraleda G, Bichko V, Taylor J
Fox Chase Cancer Center, Philadelphia, PA, USA.
J Virol Methods. 1998 Nov;75(2):199-204. doi: 10.1016/s0166-0934(98)00117-7.
Replication of hepatitis delta virus (HDV) is dependent on delta antigen (deltaAg), an HDV-encoded protein, which binds to HDV RNA and is capable of multimerization. To characterize HDV-specific ribonucleoprotein complexes (RNP) we used electrophoresis into non-denaturing agarose gels followed by northern analysis, to detect HDV RNA, and immunoblot, to detect deltaAg. We studied RNP from three sources: (i) vRNP, disrupted virions obtained from infected woodchuck serum; (ii) sRNP, disrupted particles secreted from transfected cultured cells; and (iii) cRNP, isolated from cells in which HDV genome replication was occurring. sRNP were approximately 28% smaller than vRNP. Treatment of vRNP with aurin tricarboxylic acid disrupted both deltaAg-deltaAg and deltaAg-RNA interactions while vanadyl ribonucleosides released the RNA without causing detectable disruption of the multimeric deltaAg complex. cRNP were smaller and more heterogeneous than vRNP and sRNP, and probably contained host components. The application of these electrophoretic procedures, and especially the use of prior treatments with vanadyl ribonucleoside complexes have provided valuable information on the RNP of HDV, and we expect they should find applicability in RNP studies of other RNA viruses.
丁型肝炎病毒（HDV）的复制依赖于丁型抗原（deltaAg），这是一种由HDV编码的蛋白质，它能与HDV RNA结合并具有多聚化能力。为了表征HDV特异性核糖核蛋白复合物（RNP），我们采用非变性琼脂糖凝胶电泳，随后进行Northern分析以检测HDV RNA，并采用免疫印迹法检测deltaAg。我们研究了来自三种来源的RNP：（i）vRNP，从感染的土拨鼠血清中获得的破坏病毒颗粒；（ii）sRNP，从转染的培养细胞中分泌的破坏颗粒；（iii）cRNP，从正在发生HDV基因组复制的细胞中分离得到。sRNP比vRNP小约28%。用金精三羧酸处理vRNP会破坏deltaAg-deltaAg和deltaAg-RNA相互作用，而钒核糖核苷会释放RNA，且不会导致多聚化deltaAg复合物出现可检测到的破坏。cRNP比vRNP和sRNP更小且更具异质性，可能含有宿主成分。这些电泳方法的应用，特别是钒核糖核苷复合物预处理的使用，为HDV的RNP提供了有价值的信息，我们预计它们在其他RNA病毒的RNP研究中也会有应用价值。
