Fig. 4: Mechanism of TSWV inhibition by ribavirin.
a, Effects of ribavirin treatment on TSWV infection. N. benthamiana plants inoculated with TSWV were subjected to treatment with either buffer or ribavirin (100 μg ml−1). Left: disease development was photographed at 7, 14 and 21 dpi. Right: the accumulation level of nucleocapsid protein (NP) in the systemic leaves of treated plants was determined via immunoblot analysis using an NP-specific antibody. CK+, TSWV-infected sample. b, Left: cut-away global view of the L-ribavirin complex (PDB ID: 8KI6). The upper right panel provides a close-up and sliced-surface view of ribavirin located in the middle of the 5′-hook-binding pocket and the template-binding channel. The lower right panel offers a magnified view of ribavirin positioned in the active site. Ribavirin is shown as a blue shape. c, vRNA structures within the 5′-hook-binding pocket and template-binding channel of the L–ribavirin complex, achieved by superimposing the vRNA-bound L structure onto the ribavirin-bound L structure described here. The two surface views display the location of ribavirin in vRNA-binding position. d, Illustration of the entrance and exit tunnels and active site of L depicting how ribavirin inhibits activity of the polymerase. The L structure (grey) with vRNA (coloured) is displayed with tunnels (cyan) labelled with arrows as template entry, NTP entry, product and template exit tunnel, as calculated using Caver v.3.0.3 (left). Ribavirin is superimposed on the same tunnels as in the left panel and is depicted in blue (right). e, 5′ hook RNA binding analysis of the L protein in the absence or presence of ribavirin by EMSA. Increasing concentrations of L protein were incubated with FAM-labelled 5′ hook RNA(1–10 nts) in the absence of ribavirin (lanes 1–4), and the L and FAM-labelled 5′ hook RNA mixtures were incubated with decreasing amounts of ribavirin (lanes 5–8). f, Effect of ribavirin on in vitro RNA synthesis directed by TSWV RNP. Digoxin-labelled RNA synthesized in vitro by heat-inactivated virus (lane 2) and untreated virus in the absence (lane 1) and presence (lanes 3–5) of increasing concentrations of ribavirin (25, 50 and 100 μg ml−1, respectively).
