Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology.
Bottom Line: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry and food security worldwide.The nucleocapsid (N) protein is a major structural protein of PRRSV.Use of the PARP-1 small molecule inhibitor, 3-AB, in PRRSV infected cells demonstrated that PARP-1 was required and acted as an enhancer factor for virus biology.
Affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.Show MeSH
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Mentions: Label free proteomics was used to compare the proteome isolated from the N interaction studies to the proteome isolated from the UBC9 pulldown. Peptide frequency was then used as a measure of the abundance of a cellular protein binding to the N protein bait compared to the UBC9 control protein. Confidence in protein identification is routinely achieved with the use of 2 or more peptides. Examination of this ratio for the N interactome dataset (Fig. 2A) indicated that the modal ratio was approximately Log2-1, and work by ourselves and others has previously suggested that the Log2-2 cutoff or more represents a working threshold for assigning specific from non-specific interactions that arise due to binding to either the matrix or the single chain EGFP antibody component of the GFP-trap (Trinkle-Mulcahy et al., 2008). Cellular interacting proteins exhibiting ratios of a conservative value of 4-fold or greater were therefore considered to potentially represent specific interacting partners of the N protein. This resulted in the identification of 108 proteins that could potentially interact with N protein either individually or as part of larger protein complexes (supplementary data). These proteins were used in downstream analysis.
Affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.