Host Factors interacting with the Pestivirus N terminal protease, Npro are Components of the Ribonucleoprotein Complex.

The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defences through inhibition of IRF3. Here we use mass spectrometry to identify a new role for N(pro) through its interaction with 55 associated proteins, mainly ribosomal proteins and ribonucloproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumour protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3 and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoparticles and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a non-cytopathic BVDV strain Kyle. However, RNPs binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain, no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a pro-viral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins have roles in RISC function during RNA silencing. Using GAPDH silencing with siRNAs followed by Northern blot analysis of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex.