Insight Into the Interaction Between RNA Polymerase and VPg for Murine Norovirus Replication

Figures

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  FIGURE 1. Interactions in the RdRp-VPg(1-73) complex. (A) Cross-linking studies of RdRp-VPg-RNA complexes. The mixture containing 50 mM HEPES (pH 7.4), 5 mM MgCl2, 5 mM DTT, 1 μM oligo(A)15, 2 μM RdRp, and 6 μM VPg was incubated with or without 0.5% SDS. The reaction mixture was cross-linked by 0.001% glutaraldehyde treatment at room temperature for 6 min. Proteins were separated on 8% SDS-PAGE and analyzed by western blotting with anti-RdRp or anti-VPg antisera. The molecular masses of standard proteins are shown in kDa unit on the left. The RdRp, VPg monomer, and RdRp-VPg complex bands are indicated on the right of the gel. Monomer, dimer, trimer, and hexamer bands of RdRp were indicated with numbers 1, 2, 3, and 6, respectively. The experiments were repeated in triplicates. (B) SPR binding assay. RdRp or mutant proteins in sodium acetate buffer (pH 5.0) were immobilized onto a CM5 chip after being serially diluted in 20 mM Tris–HCl, pH 8.0, 100 mM NaCl, and 5 mM MgCl2. The binding affinity constant (KD) was determined from the association (kon) and dissociation (koff) rates by evaluating the 1:1 Langmuir binding model kinetics in the sensorgrams. Colored curves depict experimental data…

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  FIGURE 2. Structures of the MNV RdRp-VPg complex. (A) The RdRp-VPg(1-73) complex is shown in gray (RdRp) and red (VPg) (upper panel). Helical structure of VPg is shown below, where it was superimposed with the NMR structure (orange). (B) VPg is bound at the bottom of the palm domain of RdRp. Surface representations of VPg structures from MNV, EV71, FMDV, and CV are shown with red, pink, blue, and cyan color, respectively, highlighting diverse VPg binding modes (lower panel). (C) The interaction of RdRp with VPg in the RdRp-VPg(1-73) complex in which VPg binds to the base of the palm domain of RdRp. The critical amino acid residues (e.g., Asp331, Gln337, Leu354, and Glu355) of RdRp involved in the interaction are shown. (D) Surface representation of the hexameric RdRp-VPg complex is shown in the box, where a side view along the two-fold axis (upper) and a top view along the threefold axis (lower) are shown. Six monomers of RdRp are numbered from 1 to 6 and colored in rainbow colors, whereas VPg is in pink tint. The dimeric (upper panel) and trimeric (lower panel) arrangements found in the RdRp hexamer complex are shown in ribbon diagram on the right. (E) The crystal packing of the…

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  FIGURE 3. RdRp–VPg interaction for RNA synthesis and viral replication. (A) Representative autoradiograph of in vitro RNA synthesis assay. The RNA synthesis was monitored by the generation of double strand RNA (dsRNA) from poly(A) tailed sgRNA template in the presence of [α-32P]UTP. T7 indicates T7 RNA polymerase as a non-polymerizing control (upper panel). The experiments were repeated in duplicates. The RNA polymerase activity of recombinant RdRp (1 μg) in 50 mM HEPES, pH 7.4, 5 mM MgCl2, 10 mM dithiothreitol (DTT), 1 μg of in vitro transcribed (+)sgRNA, 250 μM of each NTP, 2.5 μCi [α-32P]UTP was determined in the presence of VPg native, Y26F or Y117F, analyzed by agarose gel electrophoresis in Tris–borate–EDTA buffer (lower panel). BSA was used as a control. (B) Representative western blot data showing the expression of RdRp at 24 hpt of plasmids producing the indicated recombinant MNVs into 293T-CD300LF cells. As a positive control, the cells were infected with MNV at the multiplicity of infection (MOI) of five for 24 h (MNV inf.). Actin as loading controls. N = 2 replicates. (C) Growth analysis of recombinant MNVs upon transfection of 293T-CD300LF cells with plasmids producing MNVs…

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  FIGURE 4. Essential role of RdRp–VPg interaction in viral replication. (A) Immunofluorescence assay for the localization of RdRp and VPg at 24 hpt of 293T-CD300LF cells with plasmids producing MNVs with the indicated mutations. Representative images (left) and quantitation (middle and right). Data as mean. N = 3 replicates. (B) Immunofluorescence assay for the localization of RdRp, dsRNA, and VP1 at 72 hpt as described in (A). Representative images (left) and quantitation (middle and right). Data as mean ± SEM. N = 2 replicates.