Abstract
Non-structural protein 1 (NS1) of influenza virus is a multifunctional protein that plays an important role in virus replication and virulence. In this study, an acetylation modification was identified at the K108 residue of the NS1 protein of H1N1 influenza virus. To further explore the function of the K108 acetylation modification of the NS1 protein, a deacetylation-mimic mutation (K108R) and a constant acetylation-mimic mutation (K108Q) were introduced into the NS1 protein in the background of A/WSN/1933 H1N1 (WSN), resulting in two mutant viruses (WSN-NS1-108R and WSN-NS1-108Q). In vitro and mouse studies showed that the deacetylation-mimic mutation K108R in the NS1 protein attenuated the replication and virulence of WSN-NS1-108R, while the constant acetylation-mimic mutant virus WSN-NS1-108Q showed similar replication and pathogenicity as the wild-type WSN virus (WSN-wt). The results indicated that acetylation at K108 of the NS1 protein has an important role in the replication and virulence of influenza virus. To further explore the potential mechanism, the type I interferon (IFN-I) antagonistic activity of the three NS1 proteins (NS1-108Q, NS1-108R, and NS1-wt) was compared in cells, which showed that the K108R mutation significantly attenuated the IFN-β antagonistic activity of the NS1 protein compared with NS1-wt and NS1-108Q. Both NS1-wt and NS1-108Q inhibited the IFN-β response activated by RIG-I CARD domain, MAVS, TBK1, and IRF3 more efficiently than the NS1-108R protein in cells. Taken together, the results indicated that acetylation at NS1 K108 is important for the IFN antagonistic activity of the NS1 protein and virulence of the influenza virus.
Highlights
Influenza virus non-structural protein 1 (NS1) is a multifunctional protein that is responsible for interacting with cellular factors to antagonize the host antiviral response during viral infection [1]
A K108R substitution was introduced into the NS1 protein, since an R substitution prevents acetylation but preserves the positive charge, and a mutant virus containing the NS1-K108R substitution was generated in the background of WSN/1933 H1N1 (WSN) virus (WSN-NS1-108R)
To mimic constantly acetylated lysine at K108 of the NS1 protein, a K108Q substitution that is a known acetylation mimic was introduced into NS1, resulting in a mutant virus containing NS1K108Q (WSN-NS1-108Q)
Summary
Influenza virus non-structural protein 1 (NS1) is a multifunctional protein that is responsible for interacting with cellular factors to antagonize the host antiviral response during viral infection [1]. IFN signalling response by regulating other host factors, such as phosphoinositide 3-kinase (PI3K) activity, Crklike protein (CRKL), and the JAK-STAT signalling pathway [7–11]. Multiple basic amino acids (e.g., 35R, 38R, 41K, and 46R) in the RBD are important for RNA binding activity and suppressing the activation of PKR [12, 13]. The ED plays an important role by targeting multiple host factors, such as PKR, CPSF30, and p85β (PI3K), to inhibit antiviral responses and enhance viral replication [1]. Residues 186E, 189D, and 194V play important roles in the binding of NS1 to cleavage and polyadenylation specificity factor 30 (CPSF30), and mutations in those residues weaken the binding of NS1 to CPSF30 and impair the ability of the NS1 protein to shut off host gene expression [15, 16]
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