Characterization of SARS2 Nsp15 Nuclease Activity Reveals it's Mad About U
Summary (4 min read)
INTRODUCTION
- The novel SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) emerged in late 2019 and became a worldwide pandemic that is still ongoing and has infected millions worldwide (1) .
- Recent studies have begun to shed light on this important question (9, 13) .
- Recent structures determined of Nsp15 bound to uridine nucleotides uncovered the molecular basis for uridine specificity, which is driven by a well-conserved serine residue within the uridine binding pocket (20, 21) .
- The structures revealed that, in contrast to RNase A, Nsp15 does not contain any additional well-ordered sites for RNA binding and recognition.
- Finally, the authors looked at Nsp15's ability to cleave SARS-CoV-2 viral RNA substrates, such as the PUN and the transcriptional regulatory sequence (TRS).
Protein expression and purification
- Wild type (WT) and mutant Nsp15 constructs were created as described previously (20) .
- Cells were harvested after overnight expression at 16˚C and stored at -80˚C until use.
- Nsp15 purification was done as described previously (20) .
- The lysate was clarified at 26,915 x g for 50 minutes at 4˚C and then incubated with TALON metal affinity resin .
Cryo-EM sample preparation
- UltrAuFoil R1.2/1.3 300 mesh gold grids were plasma cleaned (Pie Scientific) before use.
- The Nsp15/RNA mixture (3 µL) was deposited onto the grids, back-blotted for 3 seconds, and vitrified using an Automatic Plunge Freezer .
Data collection and processing
- Nsp15 images were collected using a Krios electron microscope at 300 keV with a Gatan K2 detector in super-resolution mode.
- Beam-induced motion and drift were corrected using MotionCor2 (24) and aligned dose-weighted images were used to calculate CTF parameters using CTFFIND4 (25) .
- CryoSPARC v2 (26) was used in all subsequent image processing.
- Full resolution particle projections from good classes were re-extracted using a box size of 256.
- Three independent 3D refinement cycles were performed while applying C1, C3, and D3 symmetry respectively.
Model building
- A SARS-CoV-2 Nsp15 crystal structure (PDBID 6WLC) was used as a starting model and fit into the cryo-EM maps using rigid body docking in Phenix (27) .
- For the pre-cleavage state, which was captured with an AU f A tri-nucleotide, the density for the 5¢.
- A was weaker than the density for the U, so only the C5¢ group was modeled; no density was observed for the 3¢.
- For the post-cleavage state, the 5¢ A could be fit in the density along with the U. Molprobity (29) was used to evaluate the model (Table 1 ).
FRET endoribonuclease assay
- Nsp15 cleavage was monitored in real-time as described previously (19, 20) .
- Briefly, 6-mer substrates were labeled with 5′-fluorescein (FI) and 3′-TAMRA, where TAMRA quenches FI and cleavage is measured by increasing FI fluorescence (5′-FI-AAxxxA-TAMRA-3′; x nucleotides varied among substrates) (see Supplementary Table 1 ).
- Fluorescence was measured every 2.5 minutes using a POLARstar Omega plate reader (BMG Labtech) set to excitation and emission wavelengths of 485 ± 12 nm and 520 nm, respectively.
- Three technical replicates were performed for each condition, and the assay was repeated with at least two independent protein preparations.
- Prism was used to calculate significant differences using Dunnett's T3 multiple corrections test.
Urea-PAGE endoribonuclease assay
- Due to the expected size of cleavage products and the size of bromophenol blue, loading buffer without dye was used.
- To monitor the gel front, a control lane of protein only with bromophenol blue was run.
Mass spectrometry of RNA cleavage products
- Mass spectrometry was performed as previously described (20) .
- For mass spectrometry analysis, the reaction was chromatographically separated with a gradient of buffer A (400 mM hexafluoro-2-propanol, 3 mM triethylamine, pH 7.0) and buffer B .
Molecular dynamics simulations
- Based on the RNA bound cryo-EM hexamer structure of Nsp15, the initial structure of Nsp15-AUA hexamer complex was prepared by manually introducing an adenine nucleotide at the B-2 position.
- After introducing all protons using the TLeap module of Amber.18 (32) , the Nsp15-AUA hexamer system was solvated in 68,849 water molecules, while 203 sodium ions and 125 chloride ions provided the 100 mM salt concentration and the charge neutralization.
- The monomer assembly was solvated with 24,545 water molecules.
- For each system, two additional 500 ns simulations were performed.
- When calculating the residues interaction energies, only the values from 50 ns segments with bound trinucleotides were selected.
Cryo-EM reconstructions of Nsp15 bound to RNA in pre-and post-cleavage states reveal substrate binding interactions.
- Given the similar active site arrangement and chemistry between Nsp15 and RNase A, the authors hypothesized that analogous to RNase A, there may be additional base specific binding pockets in Nsp15 (23, 33) .
- The lack of well resolved density for either adenine base suggests that in contrast to RNase A, beyond the uridine recognition site Nsp15 does not have strong secondary base binding sites.
- Inspection of the C1 map revealed unambiguous density for RNA in all 6 active sites, however the RNA density was better resolved in one of the two trimers, so C3 symmetry was applied.
- While the 3¢-PO4 remains in the same place as the pre-cleavage state, the uracil has moved to pi-stack with W333 instead of interacting with Y343 and S294, a >10 Å movement of the base .
- While the adenine density is poor the Nsp15 side chain density was well-resolved, and the authors observed unexpected density near H15 and C291 in close proximity to the active site .
Molecular dynamics simulations and energy calculations support cryo-EM structural observations
- The authors cryo-EM structure provided partial density for the uncleaved trinucleotide RNA substrate, with the nucleotides adjacent to the uridine seemingly highly dynamic.
- Therefore, the authors turned to molecular dynamics simulations to further characterize the behavior of the nucleotides near the active site.
- This is consistent with their previous molecular dynamics simulations revealing that the hexamer is important for protein stability (20) .
- Residues interacting with the B+1 base overall feature larger energy values than the B-2 base (with the exception of W333), suggesting that while both bases are not as fixed as U, the B+1 base has more favorable interactions with Nsp15 active site residues (Supplementary Tables 2 and 3 ).
Nsp15 active site mutants exhibit reduced or abrogated RNA cleavage
- To determine the significance of SARS-CoV-2 Nsp15 active residues in mediating RNA interactions and supporting cleavage the authors made a series of single point mutations.
- All four active site variants were purified as stable hexamers, indicating that they did not disrupt the oligomerization of Nsp15 .
- The authors measured RNA cleavage with a FRET-based assay (19,20) using 6mer RNA substrates with 5¢ fluorescein and 3¢ TAMRA labels.
- Given that the authors noticed extra density extending from the C291 side chain, they tested the importance of this residue in mediating cleavage .
- Mutating C291A did not significantly affect the oligomerization or activity of Nsp15, which is not surprising given that it is not well conserved .
In vitro FRET endoribonuclease assay reveals Nsp15 prefers a purine in the position following the uridine
- While the structures of Nsp15 in the pre-and post-cleavage states did not reveal strong secondary base binding sites, RNA cleavage assays revealed that Nsp15 has a preference for a purine 3¢ to the uridine in the cleavage site.
- Using the FRET endoribonuclease assay described above, the authors studied the importance of the bases in the -2 and +1 position (B-2, B+1) relative to the uridine being cleaved (B-1) using a series of oligomers with a single nucleotide change .
- In contrast, substitutions in the B-2 position did not significantly affect Nsp15 cleavage.
- The authors further analyzed the cleavage products of the unmodified double U RNAs by mass spectrometry which showed the accumulation of cleavage products following the uridine nucleotides at all three positions .
- Therefore, the differences observed in cleavage with the double U constructs suggests that the position of the U relative to the 5¢ or 3¢ end may also impact cleavage.
Cleavage of biologically relevant substrates shows Nsp15 has a clear preference for U^A versus U^C
- The authors FRET-based cleavage assays revealed that Nsp15 demonstrates selectivity beyond uridine recognition in small 6-mer substrates leading us to ask whether this specificity is conserved in a) longer and b) more biologically relevant RNA substrates.
- The authors synthesized ~20-mer oligos containing the consensus TRS-B and flanking sequence for the nucleoprotein (N) as well as the spike (S) protein sub-genomic RNAs with labels on both the 5' and 3' ends (Table S1 ).
- The authors observed faster accumulation of the U6^A cleavage product over the U1^C and U4^C products.
- The authors assessed the ability of SARS-CoV-2 Nsp15 to cleave polyU sequences in two ways.
- These results are in excellent agreement with the earlier work showing that Nsp15 targets the PUN RNA sequence, however Nsp15 activity is not restricted to the PUN as it also cleaves additional uridines within the negative strand.
DISCUSSION
- How Nsp15 recognizes its RNA targets was poorly understood.
- The authors identified several NTD residues from the adjacent protomer that interact with the B-2 adenine in their model and are important for oligomerization and nuclease activity.
- SARS-CoV-1 Nsp15 does not cleave 2¢methylated RNA substrate efficiently and preferentially cleaves unpaired U's within a structured RNA substrate (36) .
- Beyond RNA modification and secondary structure, another potential mechanism of Nsp15 nuclease regulation is through compartmentalization.
- The authors data show that Nsp15 acts in a distributive fashion to catalyze cleavage following uridines.
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Frequently Asked Questions (14)
Q2. What are the contributions in "Characterization of sars2 nsp15 nuclease activity reveals it’s mad about u" ?
This work advances their understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics. ( which was not certified by peer review ) is the author/funder. This article is a US Government work.
Q3. How many samples were selected at each nanosecond?
Since the trinucleotide was not bound to the binding site residues during the entire half a microsecond production runs in most systems, the energy calculations were performed for each 50 ns segments (with 50 samples selected at each nanosecond) separately for each trajectory.
Q4. What other proteins may influence Nsp15 RNA targets and regulation in host cells?
other viral proteins may influence Nsp15 RNA targets and regulation in host cells, as Nsp15 is believed to localize within the replication-transcription complex of Nsps, including the RdRp complex (54,55).
Q5. What is the role of endoribonuclease in RNA clea?
Enzymatic activity occurs in the C-terminal EndoU domain, which is more broadly conserved across nidoviruses, suggesting that this endoribonuclease activity is critically important for large, positivestrand RNA viruses (5,6).
Q6. What is the role of Nsp15 in blocking dsRNA?
Nsp15 is a key player in blocking activation of host dsRNA sensors by preventing the accumulation of viral RNA and a promising therapeutic target (9,13,14).
Q7. What is the role of Nsp15 in the evasion of the host immune system?
Work in animals and cell culture has shown that Nsp15 function is not necessary for viral replication, however Nsp15 nuclease activity is critically important for evasion of the host immune response to the virus, specifically by preventing the activation of dsRNA sensors (7- 11).
Q8. What were the RMSDs used to establish the stability of the simulated systems?
Rootmean square deviations (RMSDs) were used to establish the stability of the simulated systems (Supplementary Figure 4A) in which the isolated monomer systems displayed elevated dynamics (as assessed by RMSDs) compared to the protomers assembled into the hexamer.
Q9. How did the authors capture the post-cleavage state of Nsp15?
The authors captured the post-cleavage state by incubating Nsp15 with excess unmodified RNA (AUA) prior to vitrification and cryo-EM data collection.
Q10. What substrates were used to cleave MHV Nsp15?
The authors incubated WT Nsp15 with the TRS-N and TRS-S containing RNA substrates and then resolved the cleavage products on denaturing urea gels.
Q11. What is the consensus motif for Nsp15 cleavage?
Based on these endoribonuclease assay results, the authors define the consensus motif for Nsp15 cleavage as (N)(U)^(R>U>>C) (where N is any nucleotide and R is a purine).
Q12. What substrate did the Nsp15 variant produce more C cleavage products?
Consistent with their assay results with a six nucleotide substrate, the N278A Nsp15 variant cleaved more slowly and produced more C cleavage products than WT Nsp15 (Fig. 7).
Q13. What is the way to test the ability of Nsp15 to degrade polyU?
the authors also looked at Nsp15’s ability to degrade polyU sequences and found that Nsp15 efficiently degrades polyU containing RNAs in vitro.
Q14. What did the authors do to characterize the behavior of the nucleotides near the active?
the authors turned to molecular dynamics simulations to further characterize the behavior of the nucleotides near the active site.