Abstract
In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of beta-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy.
Highlights
In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation
To assist in the proteomics analysis of virus particles we first characterized RSV-infected cells using three-dimensional imaging, which allowed us to examine the distribution of the virus filaments and inclusion bodies in infected cells (Fig. 1) Cells were infected with RSV, and at 20 h postinfection, the cells were fixed and stained using MAb19 and anti-P, antibodies that recognize the F and P proteins respectively [16, 21, 41]
We observed similar levels of co-precipitated P protein in the absence or presence of drug, in drug-treated cells there was an approximate 7-fold increase in the co-precipitation of a band corresponding in size to actin (Fig. 8F (ii)), suggesting that inhibiting HSP90 may alter the Cellular Proteins Involved in RSV Assembly 1842 Molecular & Cellular Proteomics 9.9
Summary
The RSV A2 strain and the human respiratory airway cell line HEp2 were used throughout this study. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics, and infections were carried out in DMEM with 2% FCS. Infected cells were incubated at 33 °C in 5% CO2. The cells were infected with a multiplicity of infection (m.o.i.) of 2. Geldanamycin (GA) and 17AAG were purchased from Calbiochem and reconstituted in DMSO at 10 mM. This was diluted into tissue culture medium prior to use, giving a final concentration of 2 M (GA and 17AAG)
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