Inhibition of Influenza A Replication Using Cell Penetrating Protein Mimetics

Abstract

The Influenza virus is a major human respiratory pathogen responsible for seasonal ‘flu’ outbreaks and sporadic global pandemics. The Influenza polymerase complex is necessary for viral RNA synthesis and full virulence and requires the assembly of three conserved subunits: PA, PB1 and PB2. A recombinant chimeric protein mimetic consisting of the N-terminus (20 amino acids) of PB1 fused to Maltose Binding Protein (MBP) and Tat Nuclear Localization Signal (NLS) was designed and purified with the aim of inhibiting the assembly of the polymerase by mimicking PB1. The cell-penetrating protein mimetic was shown to efficiently enter the cell nucleus and prevent assembly of the Influenza polymerase, thus inhibiting viral replication. When MDCK cells were incubated with the mimetic and subsequently challenged with Influenza A virus, viral replication decreased up to 98% at 50 µM. Using a nuclear extraction assay, the mimetic was shown to efficiently penetrate the plasma membrane and enter the host nucleus. GST pull-down assays showed that the mimetic interacts with PA. Molecular modeling was then employed to predict the improved hypothetical free energy of binding between PB1 and PA and determined two significant substitutions for PB1 threonine at position six: glutamic acid (T6E) and arginine (T6R). These mutations increased potency of the mimetic at 25 µM (71% for T6E and 77% for T6R compared to 36% for the native construct) and 12.5 µM (27% for T6E and 70% for T6R compared to 16% for the native construct), suggesting a more stable interaction with PA consistent with molecular modeling. Using various in vitro assays, the mimetic was shown to be non-toxic to host cells. Targeting critical protein-protein interactions using a peptide fused to a cell-penetrating carrier protein presents a novel and intriguing approach in designing anti-viral therapeutics.