The ANDV-N protein enhances translation of the ANDV-like SmRNA in cells.

HEK-293T cells were transfected with increasing amounts (50–500 ng) of a plasmid encoding for the recombinant ANDV His-N protein. Total transfected DNA was kept constant at 500 ng using pSP64 Poly(A) DNA. In vitro transcribed cap-N-RNA-3’UTR or cap-N-RNA-poly(A) RNAs, were transfected in cells together with a capped and polyadenylated mRNA encoding for RLuc (RLuc RNA). Six hours post-transfection (hpt), cells were lysed, and FLuc and RLuc activities were measured. (A) Expression of the ANDV His-N protein was confirmed by western blotting, using a monoclonal mouse anti-His antibody. GAPDH was used as a loading control. (B) The FLuc and RLuc activities were used to determine the relative translation efficiency (RTA), FLuc/RLuc, for each mRNA. Values shown are the mean (+/- SEM) of at least three independent experiments, each conducted in triplicate. Statistical analysis was performed by a two-way ANOVA with Sidak’s multiple comparisons test (P<0.05).