DDX3 Interacts with Influenza A Virus NS1 and NP Proteins and Exerts Antiviral Function through Regulation of Stress Granule Formation

Abstract

DDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein, and DDX3 colocalized with NP in SGs. We further determined that the DDX3 helicase domain did not 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. Taken together, our results identified DDX3 as an antiviral protein with a role in virus-induced SG formation. IMPORTANCEDDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza virus NS1 and NP proteins; DDX3 and NP colocalize in the del NS1 virus-induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection and its antiviral activity is through regulation of SG formation. Our findings provide knowledge about the function of DDX3 in the influenza virus life cycle and information for future work on manipulating the SG pathway and its components to fight influenza virus infection.

Venezuelan equine encephalitis virus non-structural protein 3 (nsP3) interacts with RNA helicases DDX1 and DDX3 in infected cells

“…demonstrated that DDX3 interacted with influenza virus NS1 and NP proteins, and localized to virus induced SGs (Thulasi Raman et al, 2016). Furthermore, that study indicated that DDX3 is capable of nucleating stress granule formation, and that the core helicase domain of DDX3 was sufficient for its localization to SGs (Thulasi Raman et al, 2016). This offers further support to the loss of interactions we observed between VEEV-nsP3 and the host translational machinery with treatment of RK-33.…”

“…ND ¼ not detectable. demonstrated that DDX3 interacted with influenza virus NS1 and NP proteins, and localized to virus induced SGs (Thulasi Raman et al, 2016). Furthermore, that study indicated that DDX3 is capable of nucleating stress granule formation, and that the core helicase domain of DDX3 was sufficient for its localization to SGs (Thulasi Raman et al, 2016).…”

“…Alternatively, VEEV-nsP3 could usurp these host factors to disassemble the SGs to create an environment suitable for efficient viral multiplication. A similar subversion of SGs is necessary for establishment of a successful influenza infection, as knockdown of DDX3 impaired stress granule formation and increased viral titers (Thulasi Raman et al, 2016). The effect of VEEV-nsP3 interactions with host translational machinery on viral translation are future avenues being explored by our laboratory.…”

Schlafen 14 (SLFN14) is a novel antiviral factor involved in the control of viral replication

“…Recent studies have highlighted an important function of other viral RNA helicases such as DDX3, DDX21, and DDX60 in virus sensing (Fullam and Schroder, 2013; Miyashita et al, 2011; Thulasi Raman et al, 2016). DDX3 was shown to interact with influenza NS1 and NP prote ins and to act as an antiviral protein by regulating stress granule formation, whereas DDX21 was shown to inhibit viral RNA and protein synthesis during infection through sequential interactions with PB1 and NS1 (Chen et al, 2014).…”

Diverse Roles of DEAD/DEAH-Box Helicases in Innate Immunity and Diseases

“…On the other hand, stress granules are assembled during cellular stress conditions to store mRNA and ribonucleoproteins, and to stall translation initiation. Both of these structures participate in cellular response against viruses and restriction of viral replication [51,133]. Importantly, the fact that RLRs can also be recruited to and activated in stress granules further strengthens and interconnects the multiple roles of DDX3 in innate immunity [134].…”