In vitro, the partially purified recombinant ANDV GST-N protein does not stimulate translation of an Acapped-RNA, nor does it substitute for eIF4G.

(A)In vitro translation of the cap-N-RNA-3’UTR and Acap-N-RNA-3’UTR (0.01 pmol) under similar conditions as described in (Fig 1). The Acap-N-RNA-3’UTR RNA was also translated in the presence of increasing concentrations of the ANDV GST-N protein (protein/RNA molar ratio from 0.25:1 to 250:1). GST alone at a protein/RNA molar ratio of 1:1 was used as a control. Data are shown as ratios, the number of ANDV GST-N protein molecules added to each reaction, relative to the estimated number of Acap-N-RNA-3’UTR molecules added to the reaction (see legend to Fig 1). (B) RRL (35% v/v) was supplemented with L-protease-RRL (4% v/v), RRL programmed with FMDV L protease RNA template, or not (lane 1), from 1 to 30 minutes (lanes 2 to 6). Western analysis was performed using polyclonal antibodies against eIF4GI [74]. Positions of molecular mass standards (in kDa) are shown. The cleavage product (CP) has been previously characterized [34]. (C) In vitro translation (90 min at 30°C) of RRL (35% v/v) treated for 30 min with L-protease-RRL (4% v/v) before the addition of the cap-N-RNA-3’UTR (0.01 pmol) RNA, in the presence of increasing concentrations of recombinant ANDV GST-N protein (0.5:1 to 250:1 protein to RNA ratio). Values are the mean (+/- SEM) of at least three independent experiments, each conducted in duplicate. Statistical analysis was performed by a one-way ANOVA with Dunnett’s multiple comparisons test (P<0.05).