32 JYI | March 2013 | Vol. 25 Issue 3  2013 Journal of Young Investigators RESEARCH ARTICLE Journal of Young Investigators 1* College of Veterinary Medicine, Department of Infectious Diseases,University of Georgia, Athens, GA 30602 *To whom correspondence should be addressed: patel1804@gmail.com Reducing Influenza Virus Infection through shRNA- Targeting TMPRSS2 Protease Nisarg Patel 1* , Ralph A. Tripp 1 ,Scott Johnson 1 , and Jackelyn Crabtree 1 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. INTRODUCTION Influenza A virus causes seasonal outbreaks of mild to severe respiratory tract infection that can cause 3,000 to 49,000 deaths in the US alone, and between 250,000 to 500,000 deaths a year throughout the world (Mueller et al., 2010). Influenza virus is in the Orthomyxoviridae family, which contains three genera: influenza A, B, and C (Gangurde et al., 2011). Typically, humans are infected with influenza A and B viruses, while group C rarely causes illness (Potter, 2002). The most recent notable incidence of an influenza virus pandemic is the swine-origin H1N1 pandemic that emerged in Mexico in 2009 and took the lives of 7,000-13,000 individuals throughout the world (Secencan et al., 2011; Korteweg & Gu, 2010). Influenza viruses are enveloped and contain a segmented, single-stranded, negative-sense ribonucleic acid (RNA) genome (Liu & Zhang, 2010). The virus acquires its envelope from the host cell membrane during budding, and has a morphology that can be pleomorphic or spherical with a diameter of 800-1200 angstroms (Potter, 2002). The genome of the virus consists of seven or eight segments of linear negative-sense RNA that code for 11 proteins (Chiu et al., 2003). The outer envelope consists of two major glycoproteins, which are hemagglutinin (HA) and neuraminidase (NA), and the less prominent matrix protein. The HA and NA are major antigenic determinants of the virus and their variation is responsible for the appearance of new epidemic and pandemic strains of influenza (Potter, 2002). The influenza virus HA is the viral attachment protein. The HA also has an important role in the release of the viral RNA into the cell by causing fusion of viral and cellular membranes. HA must be cleaved by cellular proteases to be active as a fusion protein. The interaction between the virion and the sialic acid receptors on host cells leads to virus endocytosis, and the acidic pH in the endosome initiates a structural change in HA causing viral and endosomal membrane bonding (Garten & Klenk, 2008). During the fusion process, the precursor HA glycoprotein (HA0) is cleaved in two subunits: HA1 and HA2 (Potter, 2002; Garten & Klenk, 2008). These two subunits remain together at the surface of the virus particle after the cleavage process (Garten et al., 2004). The new N-terminal end of HA2 display a sequence of hydrophobic amino acids called fusion peptide, which is inserted into the endosomal membrane and causes fusion of the viral and cell membranes (Bosch et al., 1981). Consequently, the influenza viral RNAs can enter the human cell cytoplasm (Skehel & Wiley, 2000; Steinhuer, 1999; Garten et al., 2011). The cleavage sites present in HA vary between viral strains – a feature that contributes to virus spread, pathogenicity, and tissue tropism (Garten et al., 2011). Viral spread is restricted by tissue type because proteases that can cleavage HA are tissue specific (Bottcher-Friebertshauser et al., 2010). Most influenza viruses, including the H1, H2, and H3 subtypes typically infecting humans, require activation by trypsin-like proteases (Bosch et al., 1981). Trypsin-like proteases such as TMPRSS2 (transmembrane proteases serine S1 member 2) and HAT (Human airway trypsin-like protease) are known to be present in human airway epithelial cells and their full-length coding sequences are also known (Bottcher et al., 2006; Shulla et al., 2011). TMPRSS2 protease was evaluated in this study because TMPRSS2 has been shown to play an important role in