The nucleus represents a cellular compartment where the discrimination of self from non-self nucleic acids is vital. While emerging evidence establishes a nuclear non-self DNA sensing paradigm, the nuclear sensing of non-self RNA, such as that from nuclear-replicating RNA viruses, remains unexplored. Here, we report the identification of nuclear-resident RIG-I actively involved in nuclear viral RNA sensing. The nuclear RIG-I, along with its cytoplasmic counterpart, senses influenza A virus (IAV) nuclear replication leading to a cooperative induction of type I interferon response. Its activation signals through the canonical signaling axis and establishes an effective antiviral state restricting IAV replication. The exclusive signaling specificity conferred by nuclear RIG-I is reinforced by its inability to sense cytoplasmic-replicating Sendai virus and appreciable sensing of hepatitis B virus pregenomic RNA in the nucleus. These results refine the RNA sensing paradigm for nuclear-replicating viruses and reveal a previously unrecognized subcellular milieu for RIG-I-like receptor sensing.

The inflammasome represents a molecular platform for innate immune regulation and controls proinflammatory cytokine production. The NLRP3 inflammasome is comprised of NLRP3, ASC, and procaspase-1. When the NLRP3 inflammasome is activated, it causes ASC speck formation and caspase-1 activation, resulting in the maturation of interleukin-1β (IL-1β). The NLRP3 inflammasome is regulated at multiple levels, with one level being posttranslational modification. Interestingly, ubiquitination of ASC has been reported to be indispensable for the activation of the NLRP3 inflammasome. Influenza A virus (IAV) infection induces NLRP3 inflammasome-dependent IL-1β secretion, which contributes to the host antiviral defense. However, IAVs have evolved multiple antagonizing mechanisms, one of which is executed by viral NS1 protein to suppress the NLRP3 inflammasome. In this study, we compared IL-1β production in porcine alveolar macrophages in response to IAV infection and found that the 2009 pandemic H1N1 induced less IL-1β than swine influenza viruses (SIVs). Further study revealed that the NS1 C terminus of pandemic H1N1 but not that of SIV was able to significantly inhibit NLRP3 inflammasome-mediated IL-1β production. This inhibitory function was attributed to impaired ASC speck formation and suppression of ASC ubiquitination. Moreover, we identified two target lysine residues, K110 and K140, which are essential for both porcine ASC ubiquitination and NLRP3 inflammasome-mediated IL-1β production. These results revealed a novel mechanism by which the NS1 protein of the 2009 pandemic H1N1 suppresses NLRP3 inflammasome activation. Influenza A virus (IAV) infection activates the NLRP3 inflammasome, resulting in the production of IL-1β, which contributes to the host innate immune response. ASC, an adaptor protein of NLRP3, forms specks that are critical for inflammasome activation. Here, we report that the NS1 C terminus of the 2009 pandemic H1N1 has functions to suppress porcine IL-1β production by inhibiting ASC speck formation and ASC ubiquitination. Furthermore, the ubiquitination sites on porcine ASC were identified. The information gained here may contribute to an in-depth understanding of porcine inflammasome activation and regulation in response to different IAVs, helping to further enhance our knowledge of innate immune responses to influenza virus infection in pigs.

No 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.

Pigs are an important host of influenza A viruses due to their ability to generate reassortant viruses with pandemic potential. NS1 protein of influenza A viruses is a key virulence factor and a major antagonist of innate immune responses. It is also involved in enhancing viral mRNA translation and regulation of virus replication. Being a protein with pleiotropic functions, NS1 has a variety of cellular interaction partners. Hence, studies on swine influenza viruses (SIV) and identification of swine influenza NS1-interacting host proteins is of great interest. Here, we constructed a recombinant SIV carrying a Strep-tag in the NS1 protein and infected primary swine respiratory epithelial cells (SRECs) with this virus. The Strep-tag sequence in the NS1 protein enabled us to purify intact, the NS1 protein and its interacting protein complex specifically. We identified cellular proteins present in the purified complex by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and generated a dataset of these proteins. 445 proteins were identified by LC-MS/MS and among them 192 proteins were selected by setting up a threshold based on MS parameters. The selected proteins were analyzed by bioinformatics and were categorized as belonging to different functional groups including translation, RNA processing, cytoskeleton, innate immunity, and apoptosis. Protein interaction networks were derived using these data and the NS1 interactions with some of the specific host factors were verified by immunoprecipitation. The novel proteins and the networks revealed in our study will be the potential candidates for targeted study of the molecular interaction of NS1 with host proteins, which will provide insights into the identification of new therapeutic targets to control influenza infection and disease pathogenesis.

Zika virus (ZIKV) infection is a serious public threat with cases reported in about 70 countries and territories. One of the most serious consequences of ZIKV infection is congenital microcephaly in babies. Congenital microcephaly has been suggested to result from infection of neural progenitor cells (NPCs) in the developing fetal brain. However, the molecular and cellular mechanisms underlying microcephaly development remains to be fully elucidated. In this study, we employed quantitative proteomics to determine protein expression profile that occur during viral replication in NPCs. Bioinformatics analysis of the protein expression changes resulted in the identification of a wide range of cell signaling pathways. Specifically, pathways involved in neurogenesis and embryonic development were markedly altered, along with those associated with cell cycle, apoptosis, lipid metabolism and oxidative stress. Notably, the differential regulation of Ephrin Receptor and PPAR signaling pathways, as revealed by quantitative proteomics and validated by qPCR array, underscores the need to explore these pathways in disease development. Collectively, these results indicate that ZIKV-induced pathogenesis involves complex virus-host reactions; the findings reported here could help shed light on the mechanisms underlying ZIKV-induced microcephaly and ZIKV replication in NPCs.

Respiratory syncytial virus (RSV) is a leading cause of respiratory infections worldwide and disease management measures are hampered by the lack of a safe and effective vaccine against the infection. We constructed a novel recombinant RSV vaccine candidate based on a deletion mutant vaccinia virus platform, in that the host range genes E3L and K3L were deleted (designated as VACVΔE3LΔK3L) and a poxvirus K3L ortholog gene was used as a marker for the rapid and efficient selection of recombinant viruses. The safety of the modified vaccinia virus was investigated by intranasal administration of BALB/c mice with the modified vaccinia vector using a dose known to be lethal in the wild-type Western Reserve. Only a minor loss of body weight by less than 5% and mild pulmonary inflammation were observed, both of which were transient in nature following nasal administration of the high-dose modified vaccinia virus. In addition, the viruses were cleared from the lung in 2 days with no viral invasions of the brain and other vital organs. These results suggest that the virulence of the virus has been essentially abolished. We then investigated the efficiency of the vector for the delivery of vaccines against RSV through comparison with another RSV vaccine delivered by the widely used Modified Vaccinia virus Ankara (MVA) backbone. In the cotton rats, we found a single intramuscular administration of VACVΔE3LΔK3L-vectored vaccine elicited immune responses and protection at a level comparable to the MVA-vectored vaccine against RSV infection. The distinct features of this novel VACV vector, such as an E3L deletion for attenuation and a K3L ortholog for positive selection and high efficiency for vaccine delivery, could provide unique advantages to the application of VACV as a platform for vaccine development.

SARS-CoV-2 infections present a tremendous threat to public health. Safe and efficacious vaccines are the most effective means in preventing the infections. A variety of vaccines have demonstrated excellent efficacy and safety around the globe. Yet, development of alternative forms of vaccines remains beneficial, particularly those with simpler production processes, less stringent storage conditions, and the capability of being used in heterologous prime/boost regimens which have shown improved efficacy against many diseases. Here we reported a novel DNA vaccine comprised of the SARS-CoV-2 spike protein fused with CD40 ligand (CD40L) serving as both a targeting ligand and molecular adjuvant. A single intramuscular injection in Syrian hamsters induced significant neutralizing antibodies 3-weeks after vaccination, with a boost substantially improving immune responses. Moreover, the vaccine also reduced weight loss and suppressed viral replication in the lungs and nasal turbinates of challenged animals. Finally, the incorporation of CD40L into the DNA vaccine was shown to reduce lung pathology more effectively than the DNA vaccine devoid of CD40L. These results collectively indicate that this DNA vaccine candidate could be further explored because of its efficacy and known safety profile.