Genome-Wide siRNA Screening and Analysis of Tensin3 in Influenza Viral Entry

Despite advances to combat them, emerging infectious diseases remain a burden throughout the globe. Viruses are among the deadliest pathogens, and the development of innovative antiviral strategies is a continuous process as they emerge and evolve. The identification of required host factors can provide critical information about the route of viral infection which can aid in the design of novel antiviral drugs. In this study, a high throughput screen (HTS) was designed and executed to discover human genes involved in viral infection. Using RNAi technology, the mRNA of approximately 21,000 genes was reduced and the effect on both highly pathogenic avian influenza virus and Marburg virus entry was assessed in parallel. A pseudotyping system was utilized to allow safe study of these pathogenic viruses and focused this screen on the discovery of entry factors. The powerful dual screening strategy created a unique opportunity to identify both virus specific and shared entry factors, and also eliminated false positives caused by cytotoxic siRNAs. After the primary screen was completed, the data was mined for putative influenza and Marburg specific host factors, and two confirmation screens were performed. An initial list of 83 influenza host factors and 106 Marburg specific host factors was created. Among the influenza specific host factors was a previously unidentified viral entry factor, Tensin3 (TNS3). TNS3 has been previously identified as a cell migration factor, and it is speculated to be a cytoskeletal adaptor protein important for the maintenance of actin stress fibers. In the second part of this study, the involvement of TNS3 was further characterized to confirm its role in viral infection. Reduction of TNS3 protein consistently caused a reduction in influenza entry, both with H5N1 pseudovirus and with H1N1 infectious virus. Additionally, exogenous expression of TNS3 increased viral entry. Attempts to identify key domains of TNS3 were inconclusive, although we cautiously concluded that the actin binding domain (ABD) is involved. The mechanism by which TNS3 is involved in influenza entry must be further explored before its role in influenza infection can be fully elucidated.