title: Host Factors Modulating RSV Infection: Use of Small Interfering RNAs to Probe Functional Importance.
abstract: Although respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants and the elderly worldwide [1], the protein-protein interactions between the host cell and virus remain poorly understood. We have used a focused small interfering RNA (siRNA) approach to knock-down and examine the role(s) of various host cell proteins. Here, we describe approaches for casein kinase 2α (CK2α) as a key example. We show how to study the effect of host gene (CK2α) knockdown using siRNA on cell-associated and released virus titers, using both quantitative RT-PCR, which measures the level of viral RNA, and plaque assay, which measures infectious virus directly. Both assays identified reduced viral titers with CK2α gene knock-down, indicating that it is likely required for efficient viral assembly and/or release. Effects were confirmed in RSV infected cells using the specific CK2α inhibitor 4,5,6,7-tetrabromobenzotriazole, revealing a similar reduction in viral titers as CK2α specific siRNA. This demonstrates that siRNA can be used to characterize critical host cell-RSV protein-protein interactions, and establishes CK2α as a future druggable target.
authors.affiliation: Biochemistry and Molecular Biology, Monash University, Bldg. 77, Wellington Road, Clayton, VIC, 3800, Australia. leon.caly@monash.edu.
authors.forename: Leon
authors.initials: L
authors.lastname: Caly
authors.affiliation: Biochemistry and Molecular Biology, Monash University, Bldg. 77, Wellington Road, Clayton, VIC, 3800, Australia.
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Li
authors.affiliation: Biochemistry and Molecular Biology, Monash University, Bldg. 77, Wellington Road, Clayton, VIC, 3800, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Jans
mesh_terms: D000072283:A549 Cells; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D054884:Host-Pathogen Interactions; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D018357:Respiratory Syncytial Virus Infections / Q000235:genetics* / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000502:physiology*; D019562:Viral Load; D010948:Viral Plaque Assay; D019065:Virus Assembly
chemical_list: D034741:RNA, Small Interfering; C570325:CSNK2A1 protein, human; D047390:Casein Kinase II
keywords: Casein kinase 2α; Focussed screen; Plaque assay; RSV; Respiratory syncytial virus; qRT-PCR; siRNA