Influenza virus NS1 protein upregulates epithelial sodium channel expression and function by inhibiting ubiquitin ligase Nedd4‐2

Abstract

Influenza is a contagious respiratory virus that infects epithelial cells throughout the respiratory tract. The viral infection is facilitated by the attachment of the viral hemagglutinin to the sialic acid residues on the host cell membrane. Once the virus is inside the host cell, the acidic endosomal environment facilitates the release of the viral ribonucleoproteins, which are, subsequently, transported to the host cell nucleus for replication. The mechanism by which replicating influenza viruses affect the function of ion channels in the lung epithelia is not well understood. We have studied the effects of the matrix protein 2 (M2) on the Epithelial Sodium Channel (ENaC) and the Cystic Fibrosis Transmembrane Conductance regulator1,2. However, the effects of other viral proteins is still unknown. In this study we explored the mechanisms by which the Non‐Structural protein 1 (NS1) regulates the expression and the function of ENaC heterologously expressed in Xenopus Laevis oocytes. in‐vivo, NS1 acts by, mainly, inhibiting the host immune response. Herein, we found NS1 to act by a dual mechanism when heterologously expressed with ENaC in Xenopus Laevis oocytes. 1) We observe an inhibition of ENaC expression and function (−5.64±2.7 μA to −1.14±0.71 μA; p<0.001) when both cRNAs, ENaC's and NS1's, were consecutively injected the same day, NS1 acted by inhibiting the replication and proteins synthesis, a known effect of NS1 on host proteins and RNAs. 2) NS1 acted by enhancing ENaC expression and function when its cRNA was injected 24 hours after ENaC cRNA was injected, a situation simulating the infection of an epithelail cell by a flu virus. NS1 increased the expression of ENaC proteins at the surface of the oocytes, at least, three times the control values, measured by western blot. The increased ENaC proteins at the membrane of the oocyte resulted in a 3 fold increase of recorded amiloride sensitive sodium currents (from −6.62±0.67 μA to −17.15±0.62 μA; p<0.001) measured by the double voltage clamp technique. In conclusion, NS1 acted by increasing the number of active ENaC at the surface of oocytes. NS1 may have acted by inhibiting Nedd4‐2, the ubiquitin ligase, responsible th regulation of ENaC expression at the cell surface. Nedd4‐2 acts by ubiquitinilating and marking ENaC proteins for removal by endocytosis, internalization, and degradation in the proteasome or lysosome.Support or Funding Information5U01ES026458 and 1U01ES02769701This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.