Date of Graduation

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Du, Yuchun

Committee Member

Rhoads, Douglas D.

Second Committee Member

Henry, Ralph L.

Third Committee Member

Thallapuranam, Suresh

Keywords

Biological sciences; Health and environmental sciences; Host proteins; Influenza virus; Proteomics

Abstract

Influenza A viruses (IAVs) continue to be a threat to human health. Despite extensive studies, the mechanisms underlying the IAVs-host interactions during IAV infection remain elusive. We employed quantitative proteomic methods to systematically explore the host cell protein expression responses to IAV infection and examine the function of a critical IAV protein called NS1 by identifying its host binding partners. Specifically, we used a 2-dimentional gel electrophoresis (2-DE) based proteomic method to screen host proteins whose expression was substantially altered by IAV. One critical protein named IκB kinase-gamma (IKKγ) was found to be significantly down-regulated during IAV infection. Functional studies indicated that IKKγ and IAVs were mutually inhibitory and IKKγ might be the target for virus to inhibit IFN production.

IAV protein NS1 is known to play critical roles in viral pathogenesis and host immune responses. Through 2-DE proteomic approach and mass spectrometry, we identified several novel host cellular proteins that were associated with NS1. First, we found that heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) interacted with NS1, affected replication, transcription, expression and nucleo-cytoplasmic translocation of NS1 mRNA, and the eventual whole virus replication. Second, two ATPase proteins, RUVBL1 and RUVBL2, were identified to associate with NS1 for regulation of cell apoptosis in the absence of IFNs. Third, based on previous finding of the interaction between a DEAD family protein designated as DDX100 and NS1 through a more sensitive proteomic approach called SILAC (stable isotope labeling with amino acids in cell culture), we found this interaction promoted virus replication through enhancing viral NS1 gene replication, transcription, and dsRNA unwinding.

In summary, through quantitative proteomic, molecular and cell biology studies, we generated the global picture of host cell protein expression responses to IAV infection. For IAV NS1, several host cellular proteins were found to interact with NS1 to regulate the host cell action and virus proliferation.

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