Journal: mBio

Article Title: A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance

doi: 10.1128/mbio.01060-23

Figure Lengend Snippet: MHV helicase substitution A335V occurs at a conserved, surface-exposed residue of the CoV helicase RecA1 domain. ( A ) Schematic of the MHV genome with sites of nonsynonymous mutations selected during MHV passage with GS-441524 (black arrows). Below, a schematic of the MHV nsp13-HEL showing the helicase domains: (i) the Nidovirus-specific zinc-binding domain (ZBD; pink), (ii) the stalk (light purple), (iii) 1B beta barrel (blue), (iv) RecA1 (purple), and (v) RecA2 (magenta). A335V is in the RecA1 domain (yellow star). Figure made with Biorender. ( B ) The previously described cryoEM structure of the SARS-CoV-2 nsp13-HEL (PDB: 6XEZ) in complex with nsp7/8 2 /12 (not shown) and RNA (cyan) was used to map the location of the A336 residue (yellow spheres). Protein domains are colored to match the MHV nsp13-HEL domains shown in the schematic in ( A ). ( C ) Alignment between the PyMOL model of the MHV nsp13, generated using ColabFold v1.5.2 (green) and the SARS-CoV-2 structure used in ( C ) (purple). The RMSD value of the alignment is 3.364. ( D ) Amino acid conservation of A335 and surrounding region across divergent betacoronaviruses. Sequence identity to MHV is shown by dots.

Article Snippet: Purified SARS-CoV-2 nsp13-HEL was first buffer-exchanged into helicase buffer (20 mM HEPES pH 7.5, 40 mM KCl, 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM DTT, 0.5 mM glutathione, 0.01% Triton X-100, and 0.1 mg/mL bovine serum albumin) using a Zeba Spin Desalting column (7K MWCO; Thermofisher) and then diluted in assay buffer.

Techniques: Residue, Binding Assay, Generated, Sequencing

Journal: mBio

Article Title: A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance

doi: 10.1128/mbio.01060-23

Figure Lengend Snippet: SARS-CoV-2 helicase with the A336V substitution remains capable of association with other members of the RTC. ( A ) SDS-PAGE of purified WT nsp13-HEL and A336V nsp13-HEL. ( B ) Gel filtration traces for WT and A336V nsp13-HEL. nsp13-HEL peaks observed at 67 kDa. ( C ) WT and mutant nsp13-HEL were pre-incubated with ADP-AIF3 and dsRNA alone and with purified nsps 7/8 2 /12. Native electrophoretic mobility shift assay (EMSA) visualizing the formation of the nsp 7/8 2 /12 + RNA (lane 4). When WT or mutant nsp13-HEL is added, a stable complex of nsps 7/8 2 /12/13 is upshifted similarly. Complexes were separated on a 4.5% polyacrylamide gel and stained with Gel Red to detect RNA.

Article Snippet: Purified SARS-CoV-2 nsp13-HEL was first buffer-exchanged into helicase buffer (20 mM HEPES pH 7.5, 40 mM KCl, 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM DTT, 0.5 mM glutathione, 0.01% Triton X-100, and 0.1 mg/mL bovine serum albumin) using a Zeba Spin Desalting column (7K MWCO; Thermofisher) and then diluted in assay buffer.

Techniques: SDS Page, Purification, Filtration, Mutagenesis, Incubation, Electrophoretic Mobility Shift Assay, Staining

Journal: mBio

Article Title: A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance

doi: 10.1128/mbio.01060-23

Figure Lengend Snippet: SARS-CoV-2 helicase with the A336V substitution has a reduced unwinding rate. ( A ) Michaelis-Menten curve of helicase unwinding rate with dsDNA and dsRNA substrates. Initial unwinding velocities were graphed across varying concentrations of DNA (0–4 M) for 10 nM of WT or A336V nsp13-HEL. Points and error bars indicate the average and SD, respectively. n = 4 (DNA) and n = 3 (RNA). ( B ) Table of kinetic values derived from fitting unwinding rates to Michaelis-Menten kinetics. Values shown represent the calculated 95% confidence intervals for each parameter unless denoted N.D. (not determined).

Article Snippet: Purified SARS-CoV-2 nsp13-HEL was first buffer-exchanged into helicase buffer (20 mM HEPES pH 7.5, 40 mM KCl, 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM DTT, 0.5 mM glutathione, 0.01% Triton X-100, and 0.1 mg/mL bovine serum albumin) using a Zeba Spin Desalting column (7K MWCO; Thermofisher) and then diluted in assay buffer.

Techniques: Derivative Assay

Journal: mBio

Article Title: A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance

doi: 10.1128/mbio.01060-23

Figure Lengend Snippet: SARS-CoV-2 helicase with the A336V substitution has reduced ATPase activity. ( A ) Michaelis-Menten curve of ATPase activity of WT and A336V nsp13-HEL. Points and error bars indicate the average and SD, respectively. n = 2. ( B ) Table of kinetic values derived from fitting ATPase rates to Michaelis-Menten kinetics. Values shown represent the calculated 95% confidence intervals for each parameter.

Article Snippet: Purified SARS-CoV-2 nsp13-HEL was first buffer-exchanged into helicase buffer (20 mM HEPES pH 7.5, 40 mM KCl, 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM DTT, 0.5 mM glutathione, 0.01% Triton X-100, and 0.1 mg/mL bovine serum albumin) using a Zeba Spin Desalting column (7K MWCO; Thermofisher) and then diluted in assay buffer.

Techniques: Activity Assay, Derivative Assay