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Molecular studies on IAV nucleoprotein: interaction with host factors and its role in virus life cycle
thesisposted on 24.02.2017, 01:57 by Batra, Jyoti
Influenza A viruses (IAV) are obligate intracellular pathogens, causing substantial health and economic impacts worldwide. Like other RNA viruses, IAV greatly rely on the exploitation and subversion of host cellular proteins and pathways to facilitate virus replication. Insight into the molecular biology of these relationships could lead to novel antiviral strategies and has the potential to identify host specific interactions that would act as a barrier to pandemic emergence. IAV genome consists of eight negative sense RNA strands that encode for at least twelve proteins. IAV nucleoprotein (NP) is a most abundantly expressed viral protein that plays pivotal role in virus transcription and replication. One key function of NP is nuclear trafficking of viral ribonucleoproteins (vRNPs) by interacting with importins through its nuclear localization signals. Few cellular factors involved in nuclear import of influenza viral polymerase complex have been well characterized but many remain to be defined. In the present study, it was demonstrated that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of IAV vRNPs and is essential for efficient IAV replication. Hsp40 interacts with NP component of influenza virus ribonucleoprotein complex during early stages of infection. Attenuation of Hsp40 through chemical inhibitor or specific siRNAs, impedes the nuclear accumulation of viral NP. Consecutively, Hsp40 knockdown results in reduced viral transcription, translation and overall viral titers. Interestingly, Hsp40 was also found to promote the interaction between NP and importin-α. These studies demonstrate an important role for cellular chaperone Hsp40 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins. The IAV NP is a most profusely expressed multifunctional protein, indicating that its function is affected by interactions with various cellular factors. Therefore, co-immunoprecipitation in combination with mass spectrometry (MS) based high-throughput proteomic approach was used to identify the cellular interactors of NP in context of infected cells. Using this approach, we identified a total of 195 cellular factors as putative interactors of nucleoprotein in different subcellular compartments during course of infection. The analysis revealed that IAV NP interacts/interferes with cellular proteins involved in immune response, cellular metabolism, cell cycle, unfolded protein response, transcription/translation machinery. This method complements previous studies that have aimed to identify cellular factors that are involved in the influenza A virus life cycle. Further, the results of MS screening were validated for a select number of proteins by co-immunoprecipitation and immunofluorescence microscopy. Functional validation of these cellular targets will provide better insights of viral pathogenesis. Hence, the data presented in the study contribute to the understanding of the role of cellular proteins in influenza virus replication and underpin further studies into the molecular mechanisms that govern role of nucleoprotein at different stages of virus life cycle. An improved understanding of how virus use host cell pathways and cellular factors is essential to identify mechanisms of virus pathogenicity, host adaptation and, ultimately, new targets for antiviral intervention.