iTRAQ‐based quantitative subcellular proteomic analysis of Avibirnavirus‐infected cells

Abstract

Infectious bursal disease virus (IBDV) enters the host cells via endocytic pathway to achieve viral replication in the cytoplasm. Here, we performed LC-MS/MS coupled with isobaric tags for relative and absolute quantification labeling of differentially abundant proteins of IBDV-infected cells using a subcellular fractionation strategy. We show that the viral infection regulates the abundance and/or subcellular localization of 3211 proteins during early infection. In total, 23 cellular proteins in the cytoplasmic proteome and 34 in the nuclear proteome were significantly altered after virus infection. These differentially abundant proteins are involved in such biological processes as immune response, signal transduction, RNA processing, macromolecular biosynthesis, energy metabolism, virus binding, and cellular apoptosis. Moreover, transcriptional profiles of the 25 genes corresponding to the identified proteins were analyzed by quantitative real-time RT-PCR. Ingenuity Pathway Analysis clustered the differentially abundant proteins primarily into the mTOR pathway, PI3K/Akt pathway, and interferon-β signaling cascades. Confocal microscopy showed colocalization of the viral protein VP3 with host proteins heterogeneous nuclear ribonucleoprotein H1, nuclear factor 45, apoptosis inhibitor 5, nuclear protein localization protein 4 and DEAD-box RNA helicase 42 during the virus infection. Together, these identified subcellular constituents provide important information for understanding host-IBDV interactions and underlying mechanisms of IBDV infection and pathogenesis.

Transcriptomic Analysis and Functional Characterization Reveal the Duck Interferon Regulatory Factor 1 as an Important Restriction Factor in the Replication of Tembusu Virus

“…Whereas in birds, only one member, IFIT5, has been identified. Human IFIT5 may participate in the innate immunity, and in cells infected with infectious bursa disease virus (IBDV), IFIT5 was significantly up-regulated ( Sun et al, 2015 ).…”

Transcriptome and Proteomic Analysis Reveals Up-Regulation of Innate Immunity-Related Genes Expression in Caprine Herpesvirus 1 Infected Madin Darby Bovine Kidney Cells

“…IFIT genes encode a family of proteins that is induced after IFN treatment, viral infection, or pathogen-activated molecular pattern (PAMPS) recognition. IFIT proteins are poised to confer inhibitory effects after infection, and they have been found to be increased in abundance in target cells infected with various viruses, such as infectious bursal disease virus (IBDV) [ 47 ], Japanese encephalitis virus (JEV) [ 48 ], and porcine reproductive and respiratory syndrome virus (PRRSV) [ 49 ]. Recently, progress was made in identifying how IFIT proteins inhibit through distinct mechanisms of action and the replication of multiple families of viruses [ 50 ], such as vesicular stomatitis virus [ 51 ] and hepatitis C virus [ 52 ].…”

Cellular proteomic analysis of porcine circovirus type 2 and classical swine fever virus coinfection in porcine kidney‐15 cells using isobaric tags for relative and absolute quantitation‐coupled LC‐MS/MS

“…Using the 4‐plex iTRAQ reagents according to the manufacturer's instructions (Applied Biosystems, Foster city, CA), peptide mixtures were respectively labeled as mock‐infected (NE)‐114/117/116, PCV2‐infected (SP)‐115/116/117, CSFV‐infected (SC)‐116/115/114 and PCV2‐CSFV coinfection (PC)‐117/114/115. Protein samples from independent experiments were mixed, vacuum dried and fractionated by strong cation exchange chromatography using an AKTA Purifier 100 system (GE Healthcare) and analyzed on a Q Exactive mass spectrometer coupled with an EASY nLC system (ProxeonBiosystems, now Thermo Fisher Scientific) as described previously .…”