Sklyanskaya E I, Shie M, Komarov Y S, Yamnikova S S, Kaverin N V
D.I. Ivanovsky Institute of Virology, Academy of Medical Sciences, Moscow, U.S.S.R.
Virus Res. 1988 May;10(2-3):153-65. doi: 10.1016/0168-1702(88)90012-3.
Chick embryo primary cultured cells were infected with influenza viruses belonging to H1, H2, H3, H5 or H7 subtypes of hemagglutinin. The cells were subjected to a single or a double infection, labelled with 14C-amino acids from 2 to 6 hours postinfection, lysed with a mixture of ionic and non-ionic detergents, and the lysates were clarified by low-speed centrifugation. The clarified lysates contained 14C-labelled hemagglutinin mostly in the form of 9S trimers, as shown by velocity sedimentation in sucrose gradients with polyacrylamide gel electrophoresis (PAGE) analysis of the gradient fractions. The lysates were immunoprecipitated with antihemagglutinin antibodies specific for one of the co-infecting viruses. The immunoprecipitates were analysed by PAGE. Cells infected separately with each virus and mixed before lysis were used as a control sample in every experiment. In the lysates of cells doubly infected with H2 and H5 influenza viruses the analysis revealed the presence of structures containing HA monomers of both viruses, whereas no such structures were revealed in the lysate of a mixture of separately infected cells. Mixed structures (most likely HA trimers containing monomers of the two co-infecting viruses) were also found in the lysates of cells doubly infected with strains belonging to H1 and H2 subtypes. No such structures were revealed when the cells were co-infected with viruses belonging to H1 and H3 subtypes or H3 and H7 subtypes. The results suggest an extensive formation of mixed HA trimers in the course of double infection with viruses belonging to closely related subtypes, whereas the formation of mixed trimers by more distantly related HA monomers does not occur or is very scarce. The identity of the mixed structures as HA trimers was confirmed by immunoprecipitation experiments with 9S structures.
用属于血凝素H1、H2、H3、H5或H7亚型的流感病毒感染鸡胚原代培养细胞。细胞进行单次或双重感染，在感染后2至6小时用14C -氨基酸进行标记，用离子和非离子去污剂的混合物裂解，裂解物通过低速离心澄清。如在蔗糖梯度中进行速度沉降并用梯度级分的聚丙烯酰胺凝胶电泳（PAGE）分析所示，澄清的裂解物中14C标记的血凝素大多呈9S三聚体形式。用针对其中一种共同感染病毒的抗血凝素抗体对裂解物进行免疫沉淀。通过PAGE分析免疫沉淀物。在每个实验中，将分别用每种病毒感染并在裂解前混合的细胞用作对照样品。在同时感染H2和H5流感病毒的细胞裂解物分析中，发现存在含有两种病毒HA单体的结构，而在分别感染细胞的混合物裂解物中未发现此类结构。在同时感染属于H1和H2亚型毒株的细胞裂解物中也发现了混合结构（很可能是含有两种共同感染病毒单体的HA三聚体）。当细胞同时感染属于H1和H3亚型或H3和H7亚型的病毒时，未发现此类结构。结果表明，在密切相关亚型的病毒双重感染过程中广泛形成混合HA三聚体，而关系较远的HA单体形成混合三聚体的情况不发生或非常罕见。通过对9S结构的免疫沉淀实验证实了混合结构为HA三聚体。
