TREX (transcription/export)-NP complex exerts a dual effect on regulating polymerase activity and replication of influenza A virus.

Influenza A viruses effectively hijack the intracellular "resources" to complete transcription and replication, which involve extensive interactions between the viral and host proteins. Herein, we screened the host factors, which belong to DExD/H-box protein family members, RNA-binding proteins or mitochondrial anchoring proteins, to investigate their effects on polymerase activity. We observed DDX39B and DDX39A, DEAD-box RNA-Helicases, exert a dual effect on regulating polymerase activity and replication of influenza A viruses. We further revealed that DDX39B and DDX39A interact with viral NP and NS1 proteins. Interestingly, the viral NP proteins could reverse the inhibitory effect of excess DDX39B or DDX39A on polymerase activity. Mechanistically, the TREX complex subunits, THOC1, THOC4 and CIP29, were recruited to DDX39B-DDX39A-NP complex in an ATP-dependent manner, via the interaction with DDX39B or DDX39A, followed by excess TREX-NP complexes interfere with the normal oligomerization state of NP depending on the ratio between the viral and host proteins. On the other hand, the TREX complex, an evolutionarily conserved protein complex, is responsible for the integration of several mRNA processing steps to export viral mRNA. Knockdown of TREX complex subunits significantly down-regulated viral titers and protein levels, accompanied by retention of viral mRNA in the nucleus. Taken together, screening the host factors that regulate the replication of influenza virus advances our understanding of viral pathogenesis and our findings point out a previously unclear mechanism of TREX complex function.

DDX5/METTL3-METTL14/YTHDF2 Axis Regulates Replication of Influenza A Virus.

DEAD-box helicase 5 (DDX5), a member of the DEAD/H-box helicases, is known to participate in all aspects of RNA metabolism. However, its regulatory effect in antiviral innate immunity during replication of influenza virus remains unclear. Herein, we found that human DDX5 promotes replication of influenza virus in A549 cells. Moreover, our results further revealed that DDX5 relies on its N terminus to interact with the nucleoprotein (NP) of influenza virus, which is independent of RNA. Of course, we also observed colocalization of DDX5 with NP in the context of transfection or infection. However, influenza virus infection had no significant effect on the protein expression and nucleocytoplasmic distribution of DDX5. Importantly, we found that DDX5 suppresses antiviral innate immunity induced by influenza virus infection. Mechanistically, DDX5 downregulated the mRNA levels of interferon beta (IFN-ß), interleukin 6 (IL-6), and DHX58 via the METTL3-METTL14/YTHDF2 axis. We revealed that DDX5 bound antiviral transcripts and regulated immune responses through YTHDF2-dependent mRNA decay. Taken together, our data demonstrate that the DDX5/METTL3-METTL14/YTHDF2 axis regulates the replication of influenza A virus. IMPORTANCE The replication and transcription of influenza virus depends on the participation of many host factors in cells. Exploring the relationship between viruses and host factors will help us fully understand the characteristics and pathogenic mechanisms of influenza viruses. In this study, we showed that DDX5 interacted with the NP of influenza virus. We demonstrated that DDX5 downregulated the expression of IFN-ß and IL-6 and the transcription of antiviral genes downstream from IFN-ß in influenza virus-infected A549 cells. Additionally, DDX5 downregulated the mRNA levels of antiviral transcripts via the METTL3-METTL14/YTHDF2 axis. Our findings provide a novel perspective to understand the mechanism by which DDX5 regulates antiviral immunity.

Enhanced immunogenicity of foot and mouth disease DNA vaccine delivered by PLGA nanoparticles combined with cytokine adjuvants.

Although the immunogenicity of DNA vaccines is nonideal, they are still considered as potential alternative vaccine candidates to conventional vaccines. Various DNA delivery systems, including nanoparticles, have been extensively explored and validated to further enhance the immunogenicity of DNA vaccines. DNA vaccines are considered as alternative vaccine candidates. Various DNA delivery systems, including nanoparticles, have been extensively explored to enhance the immunogenicity of DNA vaccines. In this study, positively charged Poly (D, l-lactide-co-glycolic acid) (PLGA) nanoparticles were generated and characterized as a delivery system for O-serotype foot-and-mouth DNA vaccine. A recombinant plasmid encoding swine interleukin (IL)-18, IL-2, or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was introduced into the DNA vaccine to further improve its immunogenicity, which was evaluated in a guinea pig model. PLGA-pVAX-VP013/IL-18 elicited significantly (P = 0.0149) higher FMDV-specific antibody levels than naked DNA before the challenge. The level of neutralizing antibodies induced by PLGA-pVAX-VP013/IL-18, PLGA-pVAX-VP013/IL-2, and PLGA-pVAX-VP013/GM-CSF significantly increased compared with that induced by naked DNA (P < 0.0001). The lymphocyte proliferation assay showed that cellular immunity induced by PLGA-pVAX-VP013/IL-18 and PLGA-pVAX-VP013/GM-CSF was dramatically enhanced compared with that induced by the inactivated vaccine. The protection by PLGA-pVAX-VP013/IL-18 was consistent with that by the inactivated vaccine post-challenge and was followed by PLGA-pVAX-VP013/GM-CSF. Therefore, cationic PLGA nanoparticles can deliver DNA vaccines and induce humoral and cellular immune responses. The co-administration of FMD DNA vaccine with IL-18 formulated with PLGA nanoparticles was the optimal strategy to improve the immunogenicity of FMD DNA vaccines.