Expression of the influenza protein M2 during viral infection inhibits CFTR activity

The purpose of these dissertation studies was to 1) determine whether influenza infection alters CFTR activity in polarized epithelium; 2) measure CFTR expression and activity in cells co-expressing influenza M2 protein; and 3) examine the role of M2 on the alteration of CFTR during viral infection. We determined that infection of polarized primary epithelial cells with influenza decreases CFTR expression and activity. In addition, individual cells infected with influenza had decreased CFTR conductance as measured by whole-cell patch clamp. We also found that the influenza ion channel, matrix protein 2 (M2), alone reduced CFTR expression and activity. M2 is expressed during viral infection and transported through the secretory pathway en route to the plasma membrane where it is incorporated into budding viral particles. During transit, activation of M2 by the low pH in secretory pathway results in proton leak from these organelles, thereby resulting in an increased pH. Inhibition of M2 ion transport with amantadine, or by introducing channel mutations that block ion transport, prevented the decreases in CFTR expression and activity. To determine the role of M2 in the inhibition of CFTR during viral infection, we knocked down M2 expression in virus infected cells with dicer-substrate RNA (DsiRNA) targeted against M2. M2 DsiRNA decreased expression of M2 protein without altering the expression of other viral proteins and prevented the influenza-mediated inhibition of CFTR activity. These results suggest that M2 protein is essential for the inhibition of CFTR activity during influenza infection.

Key words: CFTR, influenza, patch clamp, ion channel, M2