Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with β-arrestin1 and that β-arrestin1 interacted with the β2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either β-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the β-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells. IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2–β-arrestin1–AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of PIAS1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV.

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