Bárcena J, Ochoa M, de la Luna S, Melero J A, Nieto A, Ortín J, Portela A
Centro Nacional de Microbiología Virología e Inmunología Sanitarias, Instituto de Salud Carlos III, Madrid, Spain.
J Virol. 1994 Nov;68(11):6900-9. doi: 10.1128/JVI.68.11.6900-6909.1994.
Two panels of monoclonal antibodies (MAbs) specific for the influenza A virus PA and PB2 polypeptides have been obtained from mice immunized with denatured proteins produced in Escherichia coli. All MAbs (13 specific for the PA polypeptide and 8 specific for the PB2 protein) reacted to the corresponding influenza virus protein in Western blotting (immunoblotting), immunoprecipitation, and immunofluorescence assays. To gain information about the roles of the nucleoprotein (NP) and PB2 and PA proteins during viral mRNA synthesis, the 21 anti-P antibodies and 3 anti-NP antibodies (J. A. López, M. Guillen, A. Sánchez-Fauquier, and J. A. Melero, J. Virol. Methods 13:255-264, 1986) were purified and tested for their ability to inhibit the transcriptase activity associated with viral cores purified from virions. Four of the antibodies (one anti-PB2 and the three anti-NP MAbs) inhibited transcription by more than 50% compared with unrelated control antibodies. The inhibitory effect was not due to a nonspecific effect of the antibody preparations, because these MAbs did not inhibit transcription when tested on influenza B virus nucleocapsids, which are not recognized by the antibodies. To determine whether the antibodies were acting on an early transcription step, transcription reactions were carried out in the presence of globin mRNA (a mixture of alpha- and beta-globin chains) and only one labeled nucleoside triphosphate (either GTP or CTP). The results obtained showed that MAbs to the PB2 and NP polypeptides interfered with the initiation step of mRNA-primed transcription. The implications of these results regarding initiation of viral mRNA synthesis are discussed.
从用大肠杆菌中产生的变性蛋白免疫的小鼠体内获得了两组针对甲型流感病毒PA和PB2多肽的单克隆抗体（MAb）。所有单克隆抗体（13种针对PA多肽，8种针对PB2蛋白）在蛋白质免疫印迹法（免疫印迹）、免疫沉淀和免疫荧光试验中均与相应的流感病毒蛋白发生反应。为了了解核蛋白（NP）以及PB2和PA蛋白在病毒mRNA合成过程中的作用，对21种抗P抗体和3种抗NP抗体（J. A. López、M. Guillen、A. Sánchez - Fauquier和J. A. Melero，《病毒学方法杂志》13:255 - 264，1986年）进行了纯化，并测试它们抑制从病毒粒子中纯化的病毒核心相关转录酶活性的能力。与不相关的对照抗体相比，其中四种抗体（一种抗PB2单克隆抗体和三种抗NP单克隆抗体）抑制转录的程度超过50%。这种抑制作用并非由于抗体制剂的非特异性作用，因为当在乙型流感病毒核衣壳上进行测试时，这些单克隆抗体并不抑制转录，而乙型流感病毒核衣壳不能被这些抗体识别。为了确定这些抗体是否作用于转录的早期步骤，在存在珠蛋白mRNA（α - 和β - 珠蛋白链的混合物）和仅一种标记的核苷三磷酸（GTP或CTP）的情况下进行转录反应。所获得的结果表明，针对PB2和NP多肽的单克隆抗体干扰了mRNA引发转录的起始步骤。讨论了这些结果对于病毒mRNA合成起始的意义。
