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Virions and intracellular nucleocapsids produced during mixed heterotypic influenza infection of MDCK cells
Abstract:
Phenotypically mixed virus yields, obtained by coinfection of MDCK cells with influenza A/WSN/33 and B/Lee/40 viruses, contained both A/WSN/33 and B/Lee/40 NP proteins, as revealed by polyacrylamide gel electrophoresis of the purified 14C-amino acids-labeled virus. Virions were lysed with 0.6 M KCl-Triton X-100 buffer, and nucleocapsids were immunoprecipitated with antibodies against NP protein of influenza A virus. Polyacrylamide gel electrophoresis of the immunoprecipitate revealed NP protein of A/WSN/33 but not of B/Lee/40 virus. However, in similar experiments with the lysates of doubly infected cells, the band of B/Lee/40 NP protein was revealed in the polyacrylamide gel electrophoresis patterns of the immunoprecipitates. In an attempt to analyze the RNA content of the immune complexes, we absorbed the lysates of doubly infected [3H]uridine-labeled cells with protein A-containing Staphylococcus aureus covered with antibodies against the NP protein of influenza A virus. RNA extracted from the immune complexes contained genomic RNA segments of both A/WSN/33 and B/Lee/40 viruses. In control samples containing an artificial mixture of cell lysates separately infected with each virus, the analysis revealed homologous components (i.e., A/WSN/33 NP protein or RNA segments) in the immune complexes. The results suggest the presence of phenotypically mixed nucleocapsids in the cells doubly infected with influenza A and B viruses; in the course of the virion formation, the nucleocapsids lacking the heterologous NP protein are selected.
Insights
Coinfection with influenza A and B viruses creates phenotypically mixed virions containing both virus types. However, during virion formation, only nucleocapsids with homologous NP proteins are selected, indicating a selection process for influenza virus assembly.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Influenza viruses A and B are significant human respiratory pathogens.
- Coinfection with different influenza strains can lead to genetic reassortment and phenotypic mixing.
- Understanding the mechanisms of influenza virus assembly is crucial for antiviral strategies.
Purpose of the Study:
- To investigate the presence and characteristics of phenotypically mixed virions resulting from coinfection with influenza A and B viruses.
- To determine the composition of nucleocapsids within coinfected cells and during virion formation.
- To elucidate the selection mechanisms governing influenza virus assembly in coinfection scenarios.
Main Methods:
- Coinfection of Madin-Darby canine kidney (MDCK) cells with influenza A/WSN/33 and B/Lee/40 viruses.
- Metabolic labeling of viruses with 14C-amino acids and [3H]uridine.
- Immunoprecipitation using antibodies against influenza A virus nucleoprotein (NP).
- Polyacrylamide gel electrophoresis (PAGE) for protein analysis.
- RNA extraction and analysis from immune complexes.
Main Results:
- Phenotypically mixed virions contained NP proteins from both influenza A and B viruses.
- Immunoprecipitation with anti-influenza A NP antibodies isolated A/WSN/33 NP protein from purified virions.
- However, B/Lee/40 NP protein was detected in immunoprecipitates from doubly infected cell lysates.
- RNA extracted from immune complexes contained genomic segments from both A/WSN/33 and B/Lee/40 viruses.
- Control experiments confirmed the specificity of immunoprecipitation for homologous components.
Conclusions:
- The study provides evidence for the presence of phenotypically mixed nucleocapsids within cells coinfected with influenza A and B viruses.
- A selection process occurs during virion formation, favoring nucleocapsids that lack heterologous NP proteins.
- This selection mechanism influences the composition of progeny virions, impacting viral evolution and pathogenesis.