Author: Nisarg Patel, Scott Johnson, Jackelyn Crabtree, and Ralph A. Tripp
Date: March, 2013
Cleavage of influenza A virus hemagglutinin (HA) via host cell proteases is required for entry into the host cell. The serine protease, TMPRSS2, has been implicated as having an important role in HA cleavage and activation. To better understand the role of TMPRSS2 for avian, human and swine influenza virus infection, a lentivirus was constructed containing a doxycycline-inducible small hairpin ribonucleic acid (shRNA) to inhibit the TMPRSS2 gene via RNA interference (RNAi). This construct was used to transduce influenza-susceptible human cell lines with shRNA, and other necessary genes to facilitate development of a stable cell line. Transduced cells were isolated by cell sorting using flow cytometry, and puromycin selection was used to create cloned cell lines. TMPRSS2 gene silencing was validated by PCR. Despite blocking the production of TMPRSS2, human influenza virus infection was not substantially (p > 0.05) reduced, suggesting that other cellular proteases may compensate. Based on the insignificant reduction of infection, we can conclude that TMPRSS2 protease may not be the only protease used by influenza virus to cleave HA for infection. Other proteases may work alongside TMPRSS2 protease in the infection process.