Arterivirus non-structural protein 1β co-operates with cellular poly (C) binding proteins to transactivate –2/–1 programmed ribosomal frameshifting

Porcine reproductive and respiratory syndrome virus (PRRSV) utilizes programmed ribosomal frameshifting (PRF) to direct efficient expression of a transframe protein (nsp2TF) from an alternative reading frame overlapping the viral replicase gene. This arterivirus frameshifting signal induces both –2 and –1 PRF and, unusually, lacks an obvious stimulatory RNA structure downstream of the shift site. The minimal RNA sequence required for frameshifting maps to a 34-nucleotide region that includes the slippery sequence (GG_GUU_UUU) and a downstream conserved C-rich motif (CCCAUCUCC). Unusually, efficient frameshifting is also dependent upon expression of a viral protein, non-structural protein (nsp) 1β, one of the 14 subunits produced from the PRRSV replicase polyproteins. Nsp1β is released by the combined action of two papain-like protease (PLP) domains, which reside in nsp1α and nsp1β and each cleave at their own C-terminus. Here we show that, in addition to this viral transactivator, frameshifting also requires the participation of cellular poly (C) binding proteins (PCBPs), which were previously identified as nsp1β interaction partners. In vitro translation assays demonstrated that both nsp1β and either PCBP1 or PCBP2 are required for efficient –2/–1 PRF. When cells were depleted for PCBP1 and PCBP2 by siRNA-mediated knockdown and subsequently transfected with a plasmid expressing nsp1β and nsp2, we observed a ~40% reduction of the expression of nsp2TF and nsp2N, the respective –2 and –1 PRF products. PCBP1 predominantly stimulates –2 PRF, while PCBP2 stimulates –1 PRF. We hypothesize that a complex of nsp1β and PCBP binds to the RNA signal downstream of the slippery sequence and here mimics the action of the more typical RNA pseudoknot stimulators of PRF. This unprecedented viral frameshift-stimulatory signal may provide new insights as to how the ribosomal elongation cycle can be modified by transacting protein factors. Furthermore, it broadens the repertoire of activities associated with poly (C) binding proteins and prototypes a new class of arterivirus-host interactions.