Abstract
Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell.
Highlights
We showed that M can interact with all three members of the Caveolin family (Fig. 2 and Table I)
Together with cellular colocalization of Cav1 and Cav2 with M at the viral filaments (Figs. 3B and 4A), this implies that interaction with Caveolin proteins occurs in lipid rafts at the plasma membrane where M oligomerizes and virion budding takes place, and presumably it is the mechanism by which Cav1 can be incorporated into mature virions
Our results showed that depletion of Cav2 rather than Cav1 by Small Interfering RNA (siRNA) has a significant effect on infectivity of both cell-associated and released virus (Fig. 5D)
Summary
Microfluidics—The microfluidic devices were fabricated on silicone molds performed as described previously [29, 30]. Small Interfering RNA (siRNA) Depletion—For knockdown of Caveolin, Caveolin, and Cofilin, overnight cultures of HEp-2 cells were seeded at 4 ϫ 105 cells/well in six-well plates or 2 ϫ 105 cells/well in 12-well plates on 16 mm glass coverslips and siRNA depletion experiments were performed 24 h later with siRNA (20 nM Qiagen FlexiTube siRNA using Lipofectamine RNAiMax, Invitrogen), the medium changed 6 h later, and the cells infected at 48 h after siRNA treatment with WT A2 RSV at an MOI of three. Fortyeight hours post transfection, cells were infected with (r) A2 RSV strain at an MOI of one, and cell associated and supernatant released virus was harvested for analysis by plaque assay 24 h postinfection. Western blots were developed using freshly prepared chemiluminescent substrate (100 mM Tris-HCl, pH 8.8, 1.25 mM luminol, 0.2 mM p-coumaric acid, and 0.05% H2O2) and exposed to FUJI autoradiography films
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