Dynamics of the mammalian nuclear proteome during influenza viral infection using SILAC-based MS quantitative proteomics

Influenza has resided with the human race long before we have any written record of it. Its death toll is one of the highest among all other virus. In recent history, pandemic outbreaks of influenza have caused even more deaths. Therefore it is of great importance that we focus our resources on understanding its viral components and functions. In this study, chimeric mutagenesis was used to investigate the antigenic variance of antibodies I50C and I131B on H1N1 and H5N1 NP. It was revealed from previous study that antibodies I50C and I131B can detect H1N1 NP but not H5N1 NP. NP from influenza A strains A/Puerto Rico/8/1934 (PR8), A/Vietnam/3046/2004 (3046) and A/Indonesia/5/2005 (indo) were used to construct the NP chimeric mutants. Nucleotide sequence from the region spanning from bp 484-506 was chosen as template to design the primers for obtaining head and tail fragments which were components of the NP constructs. Results showed that antibodies I50C and I131B can only detect NP constructs with PR8 head fragments regardless of any tail fragments, and cannot detect NP constructs with 3046 or indo head fragments. Therefore the binding epitope on H1N1 NP tested by the antibodies I50C and I131B is deduced to be within bp 1-506. In order to understand the dynamics of host and viral nuclear proteome during the influenza A infection, the pulse SILAC (Stable Isotope Labeling of Amino acids on Cell lines) MS-proteomic approach was adopted. More and more research studies are MS-proteomic based as people recognize that proteins truly define the outcome of a cell, with fewer limitations by solely looking at the genome. The pulse SILAC technique involves incorporating “light” isotope-labeled amino acids such as arginine and lysine into cells’ proteins prior infection experiment. While the cells are under influenza infection, “heavy” isotope-labeled amino acids were used to label the cells 2 hour prior each harvesting time points. Since only proteins synthesized within the 2 hour windows are “heavy” isotope labeled, relative quantification of “heavy” isotope to “light” isotope by mass spectrometry (MS) can be calculated into heavy:light (H/L) ratios. Through this method we can know to what extents are the proteins affected and whether the effect is global or specific. Together with the temporal degree of the data, we can reveal the dynamics of host and viral nuclear proteome during the influenza A infection. MS results of the influenza viral proteins agree with the viral gene expression profile upon infections and corresponded well with time of viral protein expressions during influenza pathogenesis investigated by other research groups. A number of proteins were identified to increase in turnover rate at 8 hpi. This gives a partial view of up-regulated functions inside the nucleus during influenza A infection at that stage. The up-regulated proteins represent cellular functions that are related to: energy homeostasis, microtubule-dependent transport, DNA coiling regulation, transcription regulation, translation regulation and protein folding. The findings of this research present more information to understand influenza virus and provide a stepping stone for fellow influenza researchers.published_or_final_versionPathologyMasterMaster of Philosoph