Figure 3.

Structure-guided mutational analysis of PCBP2 and nsp1β binding to PRRSV RNA.A, probing nucleic acid interaction sites of PCBP2. A schematic of full-length PCBP2 showing KH1 (gray), KH2 (green), and KH3 (teal) domains with accompanying three-dimensional structures (PDB entries 2P2R (26), 2JZX (24), and 2PQU (25) for DNA-bound KH1, KH1-KH2 fusion, and DNA-bound KH3, respectively). The KH1 guanidino groups of Arg⁴⁰ and Arg⁵⁷ appear to hydrogen-bond with the keto group of a cytosine nucleobase, whereas the side chain of Asn³²⁵ in KH3 is within hydrogen-bonding distance of an adenine nucleobase. Whereas the published structure has the amino group of Asn³²⁵ interacting with adenine, it is more likely that the carboxamide is rotated 180° to allow the carbonyl group to interact with the base instead. B, probing PRRSV nsp1β interactions with nucleic acid. Shown is a schematic of nsp1β from PRRSV strain XH-GD (PDB entry 3MTV (22)) with the putative RNA-binding motif (14) and residues Tyr¹³¹ and Arg¹³⁵ shown in purple. Figures were generated using PyMOL (27). C and D, EMSAs performed with a 20 μm concentration of the 34-nt ssRNA (Fig. 1B). In C, WT nsp1β and two mutants (Y131A and R135A) were combined with PCBP2 and the ssRNA probe. In D, WT PCBP2 and three mutants (single mutant (N325D), double mutant (N325D/R40A), and triple mutant (N325D/R40A/R57A)) were combined with nsp1β and the ssRNA probe. Molar excess of each protein is listed below each well compared with the probe.