Antiviral activity of baicalin against influenza virus H1N1-pdm09 is due to modulation of NS1-mediated cellular innate immune responses.
OBJECTIVE
Baicalin, a flavonoid, has been shown to have antiviral and anti-inflammatory activities, although the mechanism of action has been unknown. Therefore, attempts were made to analyse the mechanism behind the antiviral effects of baicalin using an influenza A virus (IAV) model in vitro and in vivo.


METHODS
Baicalin's anti-influenza activity was elucidated (in vitro and in vivo) utilizing pandemic influenza strain A/H1N1/Eastern India/66/pdm09 (H1N1-pdm09). Anti-influenza activity was measured by plaque inhibition, fluorescent focus-forming units (ffu) and quantifying viral transcripts using quantitative real-time PCR following treatment with baicalin in a dose- and time-dependent manner. The role of the IAV non-structural protein 1 (NS1) gene in modulating host responses was measured by immunoblotting, co-immunoprecipitation and molecular docking.


RESULTS
Baicalin treatment following IAV infection revealed up-regulation of interferon (IFN)-induced antiviral signalling and decreased phosphoinositide 3-kinase/Akt (PI3K/Akt) activation compared with infected, untreated controls. Baicalin exerts its antiviral effects by modulating the function of the IAV-encoded NS1 protein. NS1 has been shown to counteract cellular antiviral responses by down-regulating IFN induction and up-regulating PI3K/Akt signalling. Baicalin disrupted NS1-p85β binding. Molecular docking predicted the binding site of baicalin in the RNA binding domain (RBD) of NS1. Site-directed mutagenesis within the RBD region of NS1 and the difference in the fluorescence quenching pattern of full-length NS1 and mutant NS1 proteins in the presence of baicalin confirmed the interaction of baicalin with the NS1 RBD. Amino acid residues 39-43 of the NS1 RBD were found to be crucial for the baicalin-NS1 interaction.


CONCLUSIONS
Overall, this study highlights that baicalin exerts its anti-influenza virus activity by modulating viral protein NS1, resulting in up-regulation of IFN-induced antiviral signalling and a decrease in PI3K/Akt signalling in cells.
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata, West Bengal 700010, India.
Mukti Kant
MK
Nayak
Anurodh S
AS
Agrawal
Sudeshna
S
Bose
Shaon
S
Naskar
Rahul
R
Bhowmick
Saikat
S
Chakrabarti
Sagartirtha
S
Sarkar
Mamta
M
Chawla-Sarkar
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D005419:Flavonoids / Q000378:metabolism / Q000494:pharmacology*; D007113:Immunity, Innate; D007155:Immunologic Factors / Q000378:metabolism / Q000494:pharmacology*; D053118:Influenza A Virus, H1N1 Subtype / Q000276:immunology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction; D019562:Viral Load; D017361:Viral Nonstructural Proteins / Q000276:immunology*; D010948:Viral Plaque Assay
D000998:Antiviral Agents; D005419:Flavonoids; C085820:INS1 protein, influenza virus; D007155:Immunologic Factors; D017361:Viral Nonstructural Proteins; C038044:baicalin
PI3K/Akt; anti-influenza virus; flavonoids; interferons; non-structural protein 1