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Label-free quantitative proteomics reveals regulation of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) during respiratory syncytial virus infection.

ABSTRACT: 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.

SUBMITTER: Ternette N

PROVIDER: S-EPMC3190389 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Label-free quantitative proteomics reveals regulation of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) during respiratory syncytial virus infection.

Ternette Nicola N Wright Cynthia C Kramer Holger B HB Altun Mikael M Kessler Benedikt M BM

Virology journal 20110920 1

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 performanc ...[more]

PMID: 21933386

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Project description:Guanylate binding protein 5 (GBP5) belongs to the GTPase subfamily, which is mainly induced by interferon gamma (IFN-γ) and is involved in many important cellular processes, including inflammasome activation and innate immunity against a wide variety of microbial pathogens. However, it is unknown whether GBP5 inhibits respiratory syncytial virus (RSV) infection. In this study, we identified GBP5 as an effector of the anti-RSV activity of IFN-γ and found that in children, the weaker immune response, especially the weaker IFN-γ response and the decreased GBP5 expression, leads to RSV susceptibility. Furthermore, we revealed that GBP5 reduced the cell-associated levels of the RSV small hydrophobic (SH) protein, which was identified as a viroporin. In contrast, overexpression of the SH protein rescued RSV replication in the presence of GBP5. The GBP5-induced decrease in intracellular SH protein levels is because GBP5 promotes the release of the SH protein into the cell culture. Moreover, the GBP5 C583A mutants with changes at the C terminus or the GBP5 ΔC mutant lacking the C-terminal region, which impairs GBP5 localization in the Golgi, could not inhibit RSV infection, whereas the GTPase-defective GBP5 maintained RSV inhibition, suggesting that Golgi localization but not the GTPase activity of GBP5 is required for RSV inhibition. Interestingly, we found that RSV infection or RSV G protein downregulates GBP5 expression by upregulating DZIP3, an E3 ligase, which induces GBP5 degradation through the K48 ubiquitination and proteasomal pathways. Thus, this study reveals a complicated interplay between host restrictive factor GBP5 and RSV infection and provides important information for understanding the pathogenesis of RSV.IMPORTANCE RSV is a highly contagious virus that causes multiple infections in infants within their first year of life. It can also easily cause infection in elderly or immunocompromised individuals, suggesting that individual differences in immunity play an important role in RSV infection. Therefore, exploring the pathogenic mechanisms of RSV and identifying essential genes which inhibit RSV infection are necessary to develop an effective strategy to control RSV infection. Here, we report that the IFN-inducible gene GBP5 potently inhibits RSV replication by reducing the cell-associated levels of the RSV small hydrophobic (SH) protein, which is a viroporin. In contrast, the RSV G protein was shown to upregulate the expression of the DZIP3 protein, an E3 ligase that degrades GBP5 through the proteasomal pathway. Our study provides important information for the understanding of the pathogenic mechanisms of RSV and host immunity as well as the complicated interplay between the virus and host.

Dataset Information

Label-free quantitative proteomics reveals regulation of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) during respiratory syncytial virus infection.

Publications

Label-free quantitative proteomics reveals regulation of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) during respiratory syncytial virus infection.

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