The influenza A virus matrix protein as a marker to monitor initial virus internalisation

The uptake of influenza A viruses (IAV) into cells represents an attractive antiviral drug target, e.g., by interfering with essential cellular or viral entry factors. So far, this process could only be studied by time-consuming microscopical methods. Thus, there is a lack of rapid and easy assay systems to monitor viral entry. Here, we describe a rapid procedure to analyse internalisation of AV via Western blot detection of virion-associated matrix protein (M1), the most abundant protein within the viral particle. The assay is broadly applicable and detects different virus strains of various subtypes. As a proof of principle, treatment of cells with various known or presumed entry inhibitors resulted in reduced M1 levels. Removal of sialic acids, the receptors for IAV, led to a complete loss of the M1 signal, indicating that virus internalisation can be monitored already at the stage of attachment. Prevention of endosomal acidification resulted in a delayed degradation of M1 indicative of IAV particles trapped in endosomes. Thus, early detection of the virus-associated M1 protein is a rapid method to monitor different steps of influenza virus internalisation and has potential for application as a screening method for drugs that interfere with the uptake of IAV.