Formation of mixed hemagglutinin trimers in the course of double infection with influenza viruses belonging to different subtypes.

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.