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: The primary function of this protein is to encapsidate the viral RNA genome, and it is also thought to participate in the modulation of host cell biology and recruitment of cellular factors to facilitate virus infection.In order to the better understand these latter roles the cellular interactome of PRRSV N protein was defined using label free quantitative proteomics.This identified several cellular factors that could interact with the N protein including poly [ADP-ribose] polymerase 1 (PARP-1), a cellular protein, which can add adenosine diphosphate ribose to a protein.
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.