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Through bioinformatics analysis of the NS1-interacting complex, we grouped the identified cellular partners into diverse functional categories. This shows that the large protein complex associating with NS1 form part of smaller complexes with distinct cellular functions. Even though our data cannot characterize the cellular partners as direct or indirect NS1

interacting partners, our analysis provides an insight into the potential NS1 regulated cellular pathways important for influenza infection. Validation and characterization of these associations will provide a deeper understanding of the virus-host interplay, the co-evolution mechanisms that moulded the host-pathogen relationship and will help in the identification of new therapeutic

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CHAPTER 4: DDX3 INTERACTS WITH INFLUENZA A NS1 AND NP PROTEINS AND EXERTS ANTIVIRAL FUNCTION THROUGH THE REGULATION OF STRESS

GRANULE FORMATION

Relationship of this study to the dissertation.

In the previous study, DDX3 was identified to be associated with NS1 as part of a larger NS1 interacting protein complex. Therefore, in this study we tested and characterized the

interaction of DDX3 with NS1 and other viral proteins. Existing literature about DDX3 function in SGs as an essential nucleating factor (486), prompted us to explore the function of DDX3 in influenza virus-induced SGs.

4.1 Abstract

DDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. DDX3 was identified as a NS1 associating partner in chapter 3. However, its role in influenza virus infection is unknown. In this study, the potential role of DDX3 in influenza A virus life cycle was explored. The interaction of DDX3 and NS1 was first validated. In the search of other potential viral proteins that interact with DDX3, NP was also observed to interact with DDX3 in virus-infected cells. The contribution of the different domains in DDX3 to its NS1- and NP- interaction was examined. Stress granules (SGs) are known to be antiviral and do form in influenza A virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. Thus, the role of DDX3 in affecting influenza A virus infection through regulation of SGs was further explored. Results from this work showed that SGs were formed in infected cells upon infection with a mutant influenza A virus lacking a functional NS1 (del NS1) protein and DDX3

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co-localized with NP in SGs. Furthermore, the DDX3 helicase domain was identified not to interact with NS1 and NP; however, it was essential for DDX3 localization in virus induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers.

DDX3-NP interaction may be important for NP recruitment into SGs and DDX3-NS1 interaction may play a role in NS1 mediated SG inhibition in an eIF2α-independent manner. Taken together, these results identified DDX3 as an antiviral protein with a role in virus-induced SG formation.

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4.2 Introduction

DDX3 belongs to the DEAD box RNA helicase family and harbors ATPase and RNA helicase activities (145). Like most other DEAD box helicases, DDX3 is a multifunctional protein with functions related to RNA metabolism, RNA export, ribosome biogenesis, cellular signalling and apoptosis (19, 480). DDX3 is known to enhance antiviral innate immunity by interacting with specific proteins of the type I IFN pathway (547). However, many viruses employ viral proteins such as Vaccinia virus (VACV) K7, Hepatitis B virus (HBV) pol and Hepatitis C virus (HCV) core protein to counteract DDX3 function and in turn use DDX3 to enhance their own replication (20, 76, 459, 472, 563). Contrary to its antiviral function, DDX3 is required for the replication of several viruses such as Human Immunodeficiency virus (HIV), West Nile virus (WNV), Japanese encephalitis virus (JEV), HCV and Norovirus (547).

Therefore, existing literature portrays DDX3 both as a host factor required for viral replication as well as a component of the antiviral innate immune response.

Stress granules (SGs) are discrete cytoplasmic foci containing untranslated mRNA in nucleoprotein aggregates. They form in eukaryotic cells in response to a variety of environmental stress conditions including viral infections (409). The first step in the signalling cascade leading to SG assembly is the phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which can be regulated by any of the four serine/threonine kinases, namely double stranded RNA-dependent protein kinase R (PKR), Heme-regulated translation inhibitor kinase (HRI), PKR-like endoplasmic reticulum kinase (PERK) and General Control Nonderepressible 2 (GCN2) (17, 402, 434). PKR is activated by heat, UV irradiation and viral infections (580), HRI is activated in erythroid cells subject to oxidative stress and when levels of free heme are limiting during hemoglobin assembly (167, 191), PERK is activated in response to unfolded protein

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accumulation in endoplasmic reticulum (195, 422) and GCN2 is activated during amino acid deprivation (281). The phosphorylation of eIF2α reduces the availability of ternary complex eIF2-GTP-tRNAiMet, which is required to load the initiator tRNAiMet onto the small ribosomal subunit to initiate translation (267). This results in the accumulation of stalled translation

preinitiation complexes containing the translationally inactive messenger ribonucleoproteins, which recruit the RNA-binding proteins such as T-cell intracellular antigen-1 (TIA-1) and TIA-1 related protein (TIAR). These RNA-binding proteins in turn mediate the formation of SGs (266).

Several RNA helicases including DDX3 have been shown to localize in SGs (69). A recent study reported that DDX3 localized in the SGs, induced by a variety of cellular stresses including sorbitol, arsenite, DTT, heat shock treatment and UV irradiation. Further, DDX3 was found to be a SG-nucleating factor and DDX3-eIF4E interaction is essential for SG formation (486). Many viruses induce SGs through the activation of PKR kinase and in some cases GCN2 by the detection of viral RNA in the cytoplasm (46, 350). Most viruses including influenza A virus have mechanisms to inhibit SG formation, implicating the antiviral role of SGs in the virus life cycle (409). In case of influenza A viruses, NS1 protein is known to inactivate PKR, thereby preventing eIF2α phosphorylation and SG formation (272). Besides NS1, the nucleoprotein (NP) and polymerase-acidic protein-X (PA-X) have also been shown to aid influenza virus in

overcoming stress induced translation arrest (271). Besides inducing translation arrest, SGs have also been shown to play a role in interferon (IFN) synthesis by sequestering retinoic acid

inducible gene I (RIG-I) and influenza viral RNA (vRNA), thereby serving as a platform for the sensing of viral RNA by RIG-I (408). In addition, other antiviral proteins such as MDA5, LGP2, RNaseL, OAS and PKR also localize in influenza virus-induced SGs (408). These studies

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of DDX3 in virus life cycle and SG formation. These studies prompted the exploration of the role of DDX3 in influenza A virus induced SG formation.

Several host factors involved in RNA metabolism including DDX3 have been shown to associate with the viral polymerase complex and co-localize with NP (244). However, detailed studies on the viral interaction partners and the function of DDX3 during influenza A virus replication were not conducted. Another study, attempting to assess the effect of DDX3 downregulation on influenza A virus polymerase activity, could not determine conclusively whether DDX3 regulates this function (58).Thus, in this study the role of DDX3 during influenza virus infection and the mechanism of DDX3-mediated regulation on influenza virus replication was investigated. DDX3 was established as an interaction partner with the viral NS1 and NP proteins and it was observed to localize in virus induced SGs. NS1 was able to counteract virus- induced SG formation and DDX3 localization into these SGs. The domains in DDX3 that are critical for interaction with NS1, NP and SG formation was also identified. Moreover,

knockdown of DDX3 impaired SG formation and increased virus titers upon infection with PR8 NS1 deletion virus. Thus, DDX3 was established as an antiviral protein for influenza A virus infection with a prominent role in regulating SG formation, which warrants further study and understanding.

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