Journal: bioRxiv

Article Title: Structural basis and dynamics of Chikungunya alphavirus RNA capping by the nsP1 capping pores

doi: 10.1101/2022.08.13.503841

Figure Lengend Snippet: Panel A) Autoradiograph of a 20% urea gel with the products of the capping reactions performed in the presence of 32 PαGTP and SAM. 2 μM NsP1 was incubated with 5μM RNA (15mer CHIKV, 27mer CHIKV, 15mer VEEV and a control RNA beginning GAG) with a diphosphate (2p) or triphosphate (3p) at the 5’ end, or with a free hydroxyl (OH). The vaccinia capping enzyme (V) was used as a positive control. Panel B) Schematic of the different RNA substrates synthesised via in vitro transcription for this study, corresponding to the 15 and 27 nucleotide 5’UTR of CHIKV (strain S27), the 15 nucleotide 5’UTR of VEEV, and a 16 nucleotide control RNA sequence corresponding to the 5’UTR of Crimean Congo Hemorrhagic fever virus (CCHFV). RNA substrates were synthesized with a diphosphate or triphosphate 5’ end, and a 15mer for the CHIKV sequence with a free hydroxyl group at the 5’ end was purchased commercially. 27mer and 15mers with a cap0 structure were generated using a commercially available vaccinia capping enzyme for decapping experiments. Panel C) Autoradiograph of TLC for selected capping reactions following digestion with P1 nuclease, where ADP was also used as a control substrate. A CAP-Clip enzyme was used to confirm for the presence of a bona fide cap structure. Panel D) Autoradiograph of 20% urea PAGE gel of purified capped RNAs (estimated final concentration at 2 μM) incubated in the presence of increasing concentrations of nsP1 in the absence or presence of 2mM SAH. A decapping enzyme from S. pombe was used as a positive control reaction (labelled C). Panel E) Autoradiograph of TLC plate showing the products of selected decapping reactions. A reaction with and without nsP1 was digested with P1 nuclease as a control. Panel E) Structural overlay of an nsP1 monomer from the closed form of the rings (m 7 GMP covalently capped structure in grey) and the open form obtained from the decapping reaction (orange). There is a substantial movement in the first 130 residues of the capping domain and C-terminal helix, coloured in deep orange and labelled in bold typeface. Panel F) The structures of the open and closed ring forms overlaid, colored as in E. A tilting of the capping domains 8° away from the central pore results in an overall expansion of the pores in the rings obtained from the decapping reaction. Dimensions of the ring and central aperture are indicated with a solid line for the open conformation and a dashed line for the closed conformation. Panel G) Difference in charge distribution in the rings for the closed and open forms.

Article Snippet: Sample quality was assessed by 8M-urea PAGE, analysis of A 260 /A 280 and A 230 /A 260 ratios, and an aliquot was digested with P1 nuclease (NEB #M0660S) and analysed by LC-MS to identify the phosphorylation state of the nucleotide at the 5’ end.

Techniques: Autoradiography, Incubation, Positive Control, In Vitro, Sequencing, Synthesized, Generated, Purification, Concentration Assay