Molecular Mechanism By Which P300 Regulates Cholesterol Biosynthesis And Promotes Influenza Virus Replication

Influenza is an acute respiratory infectious disease caused by influenza A virus(IAV).The symptoms of virus infection include high fever,cough,fatigue,muscle pain etc.Immunodeficient or elderly patients often develop pneumonia.Severe infections even cause heart,kidney and other multi-organ failure,and death.The past century has witnessed several influenza pandemics,which cause losses of millions of lives.The main approach to preventing influenza is vaccination.However,the virus is prone to mutation due to high infidelity of viral RNA polymerase during virus replication.Once the prediction of dominant epidemic strains fails,it often leads to poor immune protection and a significant increase in morbidity.In addition,traditional anti-influenza drugs have unknown efficacy and limited effect on emerging subtypes.There is an urgent need to develop broad-spectrum antiviral drugs that target cellular factors.Cholesterol is the main lipid component of cell membranes,accounting for about 20%of total lipid content.Cholesterol-based lipid rafts play an important role in virus entry,replication,and the release of viral particles.Virus endocytosis often takes place in lipid rafts,cholesterol depletion from the cell membrane impairs the formation of lipid rafts and inhibits endocytosis and replication.The HA and NA proteins of IAV are transported to the cell membrane where they bind to sphingophospholipids,gather in lipid rafts,and form a transmembrane structure for virus budding and release.Most importantly,the viral envelop is derived from the infected host cell membrane during virus budding.Cholesterol in the envelop of IAV accounts for 42%of total lipids and 12%of the mass of the virus.Influenza virus replication fully relies on host cells and often enforces cells to undergo metabolic reprogramming.Inhibitors that target host cell metabolism are expected to have antiviral activity.The cholesterol biosynthesis pathway involves more than a dozen of enzymes whose gene expression is largely regulated by two transcription factors,RORγ and SREBP2.When the intracellular cholesterol level is low,the SCAP-SREBP2 complex is translocated to the membrane of the Golgi where SREBP2 is cleaved by two proteases,S1P and S2P.p300/CBP is a histone acetyltransferase that acetylates histones and some transcription factors such as p53,NF-kB,and SREBP2,and plays important roles in regulating cell cycle,differentiation,proliferation,and apoptosis.p300 is also a co-activator of SREBP2 that can acetylate SREBP2 and enhance its transcriptional activity.A recent study revealed that the p300 inhibitor C646 can inhibit influenza virus replication.A-485 is a stronger and more specific competitive p300 inhibitor of acetyl coenzyme A,which can inhibit H3K27ac(acetylation of the 27th lysine residue on histone H3).Whether A-485 can suppress influenza replication has not been reported.The aim of this study is to explore whether p300 regulates cholesterol biosynthesis through SREBP2,thereby promoting viral replication.We first investigated the anti-influenza virus activity of A-485.NL20 and 293T cells infected with H5N1 and H1N1 viruses were treated with different concentrations of A-485.The results showed that A-485 dose-dependent downregulated the expression levels of viral proteins PB2,NP and NS1.The TCID50 value in the cultured supernatant was measured,and it was found that A-485 inhibited the EC50 of H5N1 and H1N1 viruses on NL20 cells at 0.52 and 1.89 μM,respectively,and the EC50 of H5N1 and H1N1 viruses on 293T cells at 1.23 and 0.49 μM,respectively.The results of the CellTiter-Glo kit for measuring cell viability showed that the CC50 of A-485 on NL20 and 293T cells was 19.45 and 15.5μM,respectively.The selectivity index(SI)of A485 to inhibit viral replication is greater than 10,indicating that A-485 does not inhibit IAV replication by reducing cell viability.A-485 has synergistic antiviral activity with the RORγ inhibitor XY018 and the SREBP2 inhibitor Fatostatin.The antiviral activity of XY018 or Fatostatin alone is relatively low.We next investigated the molecular mechanism by which A-485 inhibits influenza virus replication.Without affecting viral replication,A-485 dose-dependent inhibited the expression of genes related to cholesterol synthesis.Overexpression of p300 in 293T cells promotes IAV replication and the expression of SREBP2,RORy and HMGCR,andincreased the titer of influenza virus in the conditioned media.The opposite results were obtained with the use of p300 knockout cells.We further showed that the antiviral activity of A-485 in HMGCR-deficient NL20 cells was significantly reduced.Filipin staining revealed that A485 can significantly reduce intracellular cholesterol levels.Exogenous cholesterol blocked the antiviral activity of A-485.These results collectively suggest that A-485 inhibits influenza virus replication by inhibiting cholesterol biosynthesis.We finally investigated the antiviral effect of A-485 in mice.A485 was adNasal administration of A485 at 12 hours before the attack and three consecutive days after the attack(10 mg/kg,twice a day),A-485 significantly reduced the level of viral protein and virus titers in the lung tissue of the attack group;H&E staining results showed that A-485 significantly reduced the infiltration of inflammatory cells in the lung tissue of IAV-infected mice.In summary,our study demonstrates that p300 promotes the synthesis of cholesterol in host cells by upregulating enhancing the expression of the cholesterol biosynthesis-related genes SREBP2 and HMGCR,thereby leading to increased viral replication.This study provides a foundation for developing broad-spectrum antiviral agents for treating influenza virus infection.