Proliferating cell nuclear antigen impairs the nuclear import of influenza A virus PB2 and suppresses virus replication.

The genome of Influenza A virus (IAV) transcribes and replicates in the nucleus of cells and the viral ribonucleoprotein (vRNP) complex plays an important role in viral replication. As a major component of the vRNP complex, the polymerase basic protein 2 (PB2) is translocated to the nucleus via its nuclear localization signals mediated by the importins. Herein, it was identified proliferating cell nuclear antigen (PCNA) as an inhibitor of nuclear import of PB2 and subsequent viral replication. Mechanically, PCNA interacted with PB2 and inhibited the nuclear import of PB2. Furthermore, PCNA decreased the binding efficiency of PB2 with importin alpha (importin α) and the K738, K752, and R755 of PB2 were identified as the key sites binding with PCNA and importin α. Furthermore, PCNA was demonstrated to retrain the vRNP assembly and polymerase activity. Taken together, the results demonstrated that PCNA impaired the nuclear import of PB2, vRNP assembly and polymerase activity, which negatively regulated virus replication.

N6-methyladenosine reader protein YTHDC1 regulates influenza A virus NS segment splicing and replication.

N6-methyladenosine (m6A) modification on viral RNAs has a profound impact on infectivity. m6A is also a highly pervasive modification for influenza viral RNAs. However, its role in virus mRNA splicing is largely unknown. Here, we identify the m6A reader protein YTHDC1 as a host factor that associates with influenza A virus NS1 protein and modulates viral mRNA splicing. YTHDC1 levels are enhanced by IAV infection. We demonstrate that YTHDC1 inhibits NS splicing by binding to an NS 3' splicing site and promotes IAV replication and pathogenicity in vitro and in vivo. Our results provide a mechanistic understanding of IAV-host interactions, a potential therapeutic target for blocking influenza virus infection, and a new avenue for the development of attenuated vaccines.

Structural insights into DNA recognition by AimR of the arbitrium communication system in the SPbeta phage.

A newly identified arbitrium communication system regulates the lysis-to-lysogeny decision in a Bacillus bacteriophage. This system contains an arbitrium hexapeptide as a signal, the cellular receptor AimR, and the lysogenic negative regulator AimX. AimR specifically targets the downstream DNA to activate aimX gene expression. The arbitrium peptide binds to AimR, inhibiting its DNA-binding to promote phage lysogeny. Recently, we and other groups have elucidated how arbitrium peptide sensed by AimR. However, the molecular mechanisms of DNA recognition by AimR and the regulation of its DNA-binding activity by the peptide remain largely unknown. Here, we report the crystal structure of the AimR-DNA complex at 2.1 Å resolution. The N-terminal HTH motif recognizes the palindromic DNA sequence, buttressed by interactions between positively charged residues and the DNA phosphate groups. The DNA-bound AimR assembles a more closed dimer than the peptide-bound form. Single-molecule FRET and crosslinking assays revealed that the AimR protein samples both open and closed conformations in solution. Arbitrium peptide binding induces a closed-to-open conformational change of AimR, eliminating DNA targeting. Our structural and functional analysis provides new insights into the DNA recognition mechanism of AimR and its regulation by the arbitrium peptide in the context of phage lysis-lysogeny decisions.