Abstract
A large quantitative study was carried out to compare the proteome of respiratory syncytial virus (RSV) infected versus uninfected cells in order to determine novel pathways regulated during viral infection. RSV infected and mock-infected HEp2 cells were lysed and proteins separated by preparative isoelectric focussing using offgel fractionation. Following tryptic digestion, purified peptides were characterized using label-free quantitative expression profiling by nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry with collision energy ramping for all-ion fragmentation (UPLC-MSE). A total of 1352 unique cellular proteins were identified and their abundance compared between infected and non-infected cells. Ingenuity pathway analysis revealed regulation of several central cellular metabolic and signalling pathways during infection. Selected proteins that were found regulated in RSV infected cells were screened by quantitative real-time PCR for their regulation on the transcriptional level. Synthesis of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) mRNAs were found to be highly induced upon RSV infection in a time dependent manner. Accordingly, IFIT3 protein levels accumulated during the time course of infection. In contrast, little variation was observed in XRN2 protein levels, but different forms were present in infected versus non-infected cells. This suggests a role of these proteins in viral infection, and analysis of their function will shed further light on mechanisms of RNA virus replication and the host cell defence machinery.
Highlights
Human respiratory syncytial virus (RSV) is a pathogen of the family of Paramyxoviridae, causing severe infection of the lower respiratory tract predominantly in young children and the elderly
Viral titers were determined in a 96 flat well format by infection of 5000 cells/well using 10-fold serial dilutions of the virus preparations in 100 μl medium containing 0.5% fetal bovine serum (FBS). 48 hpi cells were washed, fixed in methanol and immunostained using a monoclonal antibody directed against RSV-P [27] followed by incubation with a secondary anti-mouse Fc antibody coupled to horseradish peroxidase (HRP) produced in goat (Dako)
A cytopathic effect (CPE) on the cells caused by viral infection should ideally be minimal
Summary
Human respiratory syncytial virus (RSV) is a pathogen of the family of Paramyxoviridae, causing severe infection of the lower respiratory tract predominantly in young children and the elderly. RSV is an enveloped virus encasing a single-strand negative RNA genome that encodes a total of 9 structural and 2 non-structural proteins that are present in the infected cells only. The nucleocapsid formed by the viral ss(-) RNA genome that is entirely complexed by the nucleocapsid (N) protein, is immediately released to the cell cytoplasm following viral and host cell membrane fusion. Whereas transcription and replication of the viral ss(-)RNA genome are catalyzed by the viral RNA dependent RNA polymerase, synthesis of viral proteins is conducted by the host cell translation machinery. The virus encodes for two non-structural (NS) proteins NS1 and NS2 that are only expressed in the infected cell but are not present in the mature viral particles. Viral proteins are encoded on the genome in the following manner: 3’NS1-NS2-N-P-M-SH-G-F-(M2-1/M2-2)-L-5’ [12,13,14] and transcription occurs in a sequential polar fashion from 3’ to 5’ which leads to a higher abundance of proteins encoded close to the 3’-end in the infected cell
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