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H7N9 influenza A virus (IAV) is an emerged contagious pathogen that may cause severe human infections, even death. Understanding the precise cross talk between virus and host is vital for the development of effective vaccines and therapeutics. In the present study, we identified the nucleoprotein (NP) of H7N9 IAV as a positive regulator of RIG-I like receptor (RLR)-mediated signaling. Based on a loss-of-function strategy, we replaced H1N1 (mouse-adapted PR8 strain) NP with H7N9 NP, by using reverse genetics, and found that the replication and pathogenicity of recombinant PR8-H7N9NP (rPR8-H7N9NP) were significantly attenuated in cells and mice. Biochemical and cellular analyses revealed that H7N9 NP specifically interacts with tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) after viral infection. Subsequently, we identified a PXXQXS motif in the H7N9 NP that may be a determinant for the NP and TRAF3 interaction. Furthermore, H7N9 NP stabilized TRAF3 expression via competitively binding to TRAF3 with cellular inhibitor of apoptosis 2 (cIAP2), leading to the inhibition of the Lys48-linked polyubiquitination and degradation of TRAF3. Taken together, these data uncover a novel mechanism by which the NP of H7N9 IAV positively regulates TRAF3-mediated type I interferon signaling. Our findings provide insights into virus and host survival strategies that involve a specific viral protein that modulates an appropriate immune response in hosts. IMPORTANCE The NS1, PB2, PA-X, and PB1-F2 proteins of influenza A virus (IAV) are known to employ various strategies to counteract and evade host defenses. However, the viral components responsible for the activation of innate immune signaling remain elusive. Here, we demonstrate for the first time that the NP of H7N9 IAV specifically associates with and stabilizes the important adaptor molecule TRAF3, which potentiates RLR-mediated type I interferon induction. Moreover, we reveal that this H7N9 NP protein prevents the interaction between TRAF3 and cIAP2 that mediates Lys48-linked polyubiquitination of TRAF3 for degradation. The current study revealed a novel mechanism by which H7N9 NP upregulates TRAF3-mediated type I interferon production, leading to attenuation of viral replication and pathogenicity in cells and mice. Our finding provides a possible explanation for virus and host commensalism via viral manipulation of the host immune system.

Dengue virus (DENV) is the most prevalent mosquito-transmitted viral pathogen in humans. The recently licensed dengue vaccine has major weaknesses. Therefore, there is an urgent need to develop improved dengue vaccines. Here, we report a virion assembly-defective DENV as a vaccine platform. DENV containing an amino acid deletion (K188) in nonstructural protein 2A (NS2A) is fully competent in viral RNA replication but is completely defective in virion assembly. When trans-complemented with wild-type NS2A protein, the virion assembly defect could be rescued, generating pseudoinfectious virus (PIV(NS2A)) that could initiate single-round infection. The trans-complementation efficiency could be significantly improved through selection for adaptive mutations, leading to high-yield PIV(NS2A) production, with titers of >10(7) infectious-focus units (IFU)/ml. Mice immunized with a single dose of PIV(NS2A) elicited strong T cell immune responses and neutralization antibodies and were protected from wild-type-virus challenge. Collectively, the results proved the concept of using assembly-defective virus as a vaccine approach. The study also solved the technical bottleneck in producing high yields of PIV(NS2A) vaccine. The technology could be applicable to vaccine development for other viral pathogens. IMPORTANCE Many flaviviruses are significant human pathogens that pose global threats to public health. Although licensed vaccines are available for yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue viruses, new approaches are needed to develop improved vaccines. Using dengue virus as a model, we developed a vaccine platform using a virion assembly-defective virus. We show that such an assembly-defective virus could be rescued to higher titers and infect cells for a single round. Mice immunized with the assembly-defective virus were protected from wild-type-virus infection. This vaccine approach could be applicable to other viral pathogens.

Superinfection exclusion is the ability of an established viral infection to interfere with a second viral infection. Using West Nile virus (WNV) as a model, we show that replicating replicons in BHK-21 cells suppress subsequent WNV infection. The WNV replicon also suppresses superinfections of other flaviviruses but not nonflaviviruses. Mode-of-action analysis indicates that the exclusion of WNV superinfection occurs at the step of RNA synthesis. The continuous culturing of WNV in the replicon-containing cells generated variants that could overcome the superinfection exclusion. The sequencing of the selected viruses revealed mutations in structural (prM S90R or envelope E138K) and nonstructural genes (NS4a K124R and peptide 2K V9M). Mutagenesis analysis showed that the mutations in structural genes nonselectively enhance viral infection in both naïve and replicon-containing BHK-21 cells; in contrast, the mutations in nonstructural genes more selectively enhance viral replication in the replicon-containing cells than in the naïve cells. Mechanistic analysis showed that the envelope mutation functions through the enhancement of virion attachment to BHK-21 cells, whereas the 2K mutation (and, to a lesser extent, the NS4a mutation) functions through the enhancement of viral RNA synthesis. Furthermore, we show that WNV superinfection exclusion is reversible by the treatment of the replicon cells with a flavivirus inhibitor. The preestablished replication of the replicon could be suppressed by infecting the cells with the 2K mutant WNV but not with the wild-type virus. These results suggest that WNV superinfection exclusion is a result of competition for intracellular host factors that are required for viral RNA synthesis.

VPg uridylylation is essential for picornavirus RNA replication. The VPg uridylylation reaction consists of the binding of VPg to 3D polymerase (3D pol ) and the transfer of UMP by 3D pol to the hydroxyl group of the third amino acid Tyr of VPg. Previous studies suggested that different picornaviruses employ distinct mechanisms during VPg binding and uridylylation. Here, we report a novel site (Site-311, located at the base of the palm domain of EV71 3D pol ) that is essential for EV71 VPg uridylylation as well as viral replication. Ala substitution of amino acids (T313, F314, and I317) at Site-311 reduced the VPg uridylylation activity of 3D pol by >90%. None of the Site-311 mutations affected the RNA elongation activity of 3D pol , which indicates that Site-311 does not directly participate in RNA polymerization. However, mutations that abrogated VPg uridylylation significantly reduced the VPg binding ability of 3D pol , which suggests that Site-311 is a potential VPg binding site on enterovirus 71 (EV71) 3D pol . Mutation of a polymerase active site in 3D pol and Site-311 in 3D pol remarkably enables trans complementation to restore VPg uridylylation. In contrast, two distinct Site-311 mutants do not cause trans complementation in vitro . These results indicate that Site-311 is a VPg binding site that stabilizes the VPg molecule during the VPg uridylylation process and suggest a two-molecule model for 3D pol during EV71 VPg uridylylation, such that one 3D pol presents the hydroxyl group of Tyr3 of VPg to the polymerase active site of another 3D pol , which in turn catalyzes VPg→VPg-pU conversion. For genome-length RNA, the Site-311 mutations that reduced VPg uridylylation were lethal for EV71 replication, which indicates that Site-311 is a potential antiviral target.

Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2′-OH methylations of the viral RNA cap (GpppA-RNA→m 7 GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5′-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5′-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5′ stem-loop of the genomic RNA.