Current Approaches on Viral Infection: Proteomics and Functional Validations

Zheng, Jie; Tan, Boon Huan; Sugrue, Richard; Tang, Kai

In the recent decade, this breakthrough technique has been playing an indispensable role in the realm of virology (Tan and Yin, 2004) . In addition, it could integrate and highlight proteomic data by shedding a light at the protein function level. And numerous researches have implemented gene silencing, especially siRNA knockdown, in validating the proteomic data in respect to virus infection. To explore the cellular interacting partners of NS5A of hepatitis C virus, a proteomic technique, Co-IP, was employed (Gonzalez et al., 2009) . Heat shock proteins, hsc40 and hsc70, were identified for interacting with NS5A and validated by western blot analysis. NS5A was known to be able to affect the internal ribosome entry site (IRES) mediated translation of HCV. To further determine the roles of these two proteins related to IRES mediated translation, sihsc70 and sihsc40 knockdown combined with a cell culture-based bicistronic luciferase reporter system were designed. The ratio of Firefly to Renilla luciferase expression could reflect the effectiveness of IRES mediated translation. And this ratio is significantly decreased by the knockdown of hsc70, suggesting the importance of hsc70's role involved in the NS5A alternation of IRES mediated translation. Similarly, Katoh Frontiers in Microbiology | Virology et al. (2011) also used Co-IP purification technique followed by MS to identify the binding partner of flavivirus core protein. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 was found to interact with flavivirus core protein. By applying siRNA knockdown of this protein, a 90% reduction of viral replication was discovered and vRNA synthesis was delayed, indicating its significant role in regulating virus replications. In another study, TAP based proteomic approach was utilized to explore the interacting complexes associated with viral polymerases of influenza A virus (Jorba et al., 2008) . KIAA0136 (NXP2), SFPQ/PSF protein, DEAD/H box polypeptide 3 (DDX3), HNRNP-M protein, coactivator activator, growth regulated nuclear 68 protein (DDX5), beta 5-tubulin, HNRP-H1 protein, ribosomal protein-small subunit S3, and similar to zinc finger protein 71 were specifically identified. Immunofluorescence imaging further confirmed the colocalizations of those host proteins with viral RNPs. And among those identified host factors, a series of functional studies was performed in vivo and in vitro for characterizing the nuclear associated protein SFPQ/PSF (Landeras-Bueno et al., 2011) . siRNA silencing of SFPQ/PSF was found to reduce virus propagations. In particular, the accumulations of vRNA and mRNA were interrupted and reduced; polyadenylation step in viral mRNA assembly was also disturbed when siRNA silencing of SFPQ/PSF was implemented.