FIG 1.

In vitro enzymatic activities of RVFV L protein. (A) SDS–PAGE profile of Rift Valley fever virus (RVFV) L protein. (B) Size-exclusion chromatogram of RVFV L protein. Absorbance curves for the sample at wavelengths of 260 nm and 280 nm. (C) Endonuclease assay with high concentration protein. 0.1 μΜ RVFV L protein was incubated at 30°C for 1 h with 0.45 μM fluorescently labeled 30 nt PolyA RNA substrate and 25 μM MnCl₂. The band in the bottom of the gel was the degradation product. (D) Endonuclease assay. RVFV L protein (0.06 μΜ) was incubated with 0.45 μM fluorescently labeled 30 nt PolyA RNA substrate at 30°C for 40 min in the presence of 10, 25, or 50 μM MnCl₂ or CaCl₂. Reactions without protein, in the presence of 50 μM EDTA or the known endonuclease-specific inhibitor 2,4-dioxo-4-phenylbutanoic acid (DPBA), were negative controls in the presence of 25 μM MnCl₂. All inputs are shown as red arrows in panels C, D, and E. (E) In vitro replication initiation assay. The endonuclease and polymerase inactivation site double mutant (D111A/D1133A) was added in the left lane as a negative control. Mutant (D111A) was added in the middle and right lanes. These three lanes were incubated with the conserved 5′ 20 nt of the M segment (5′ M: 5′-ACACAAAGACGGUGCAUUAA-3′) or/and a 20 nt template RNA named RNA* (5′-UGUGUUUCUGGCCACGUUGA-3′). Nucleotide incorporation assay was detected by fluorescence (fluorescein-12-UTP). (F) Effect of metal ions (Mn²⁺ or Mg²⁺) and reducing agent (DTT or TCEP (Tris[2-carboxyethyl]phosphine)) on nucleotide incorporation assay by endonuclease active site mutant (D111A). The assay was performed with 5 mM MnCl₂/MgCl₂ and 2.5 mM DTT/TCEP for 40 min at 30°C. (G) The schematic diagram of the template and product in the polymerase experiment, “T” represents the template, and ‘’P2-P4’ represents the dinucleotide product to the tetranucleotide product, respectively.