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Characterization of SARS2 Nsp15 Nuclease Activity Reveals it's Mad About U

TL;DR: In this article, the authors used cryo-EM to capture structures of Nsp15 bound to RNA in pre-and post-cleavage states, and determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp 15 has a strong preference for purines 3 of the cleaved uridine.
Abstract: Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3 of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3 of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.

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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Content maybe subject to copyright    Report

Characterization of SARS2 Nsp15 Nuclease Activity Reveals
it’s Mad About U
Meredith N. Frazier
1
, Lucas B. Dillard
2
, Juno M. Krahn
2
, Lalith Perera
2
, Jason G. Williams
3
, Isha M.
Wilson
1
, Zachary D. Stewart
1
, Monica C. Pillon
1
, Leesa J. Deterding
3
, Mario J. Borgnia
2
, Robin E.
Stanley
1*
1
Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National
Institutes of Health, Department of Health and Human Services, 111 T. W. Alexander Drive, Research
Triangle Park, NC 27709, USA
2
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health
Sciences, National Institutes of Health, Department of Health and Human Services, 111 T. W.
Alexander Drive, Research Triangle Park, NC 27709, USA
3
Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences,
National Institutes of Health, Department of Health and Human Services, 111 T. W. Alexander Drive,
Research Triangle Park, NC 27709, USA
*To whom correspondence should be addressed. Tel: 01 984 287 3568; Email: robin.stanley@nih.gov
Present Address: Monica C. Pillon, Department of Biochemistry and Molecular Biology, Baylor
College of Medicine, Houston, TX, 77030, USA
ABSTRACT
Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and
evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae
revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to
RNase A. Beyond a preference for cleaving RNA 3′ of uridines, it is unknown if Nsp15 has any
additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to
RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical
assays revealed critical residues involved in substrate specificity, nuclease activity, and
oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage
and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3′ of the cleaved
uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA
and will aid in the development of new anti-viral therapeutics.
USC 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17
The copyright holder for this preprintthis version posted June 1, 2021. ; https://doi.org/10.1101/2021.06.01.446181doi: bioRxiv preprint

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).
Coronaviruses are members of the Nidovirus order, which encompasses large, positive-strand RNA
viruses with genomes that range in size from 12-41 kb (2). The 30 kb SARS-CoV-2 genome encodes
for 4 structural proteins that are part of the mature viral particle, 8 accessory proteins, and 15 non-
structural proteins (Nsps) (3). The Nsps are encoded in two open reading frames found in the first
two-thirds of the viral genome. These proteins are translated by host ribosomes as two long
polyproteins and are cleaved into functional proteins by the viral proteases (4). The Nsps play
important roles in viral replication and pathogenicity and many of them are promising drug targets
(3,4).
Nsp15 is a uridine specific endoribonuclease conserved across the Coronaviridae family (5).
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, positive-
strand RNA viruses (5,6). 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). For example, in studies of porcine endemic diarrhea virus (PEDV), Nsp15-deficient virus resulted
in higher levels of type I and III interferon responses in cells, and piglets infected with the mutant virus
had much higher survival rates than those infected with WT PEDV (8). A similar effect was also seen
in studies of mouse hepatitis virus (MHV); mice immunized with Nsp15 nuclease deficient virus were
able to successfully clear WT virus, with commonly affected organs showing no pathology (7). Recent
work also revealed a similar trend in the chicken infection bronchitis virus (IBV), where animals
infected with nuclease-deficient virus had reduced mortality and viral shedding (12). Therefore, Nsp15
is a promising therapeutic target for coronaviruses.
One of the major outstanding questions about the function of Nsp15 is what is its RNA target for
cleavage (5). Recent studies have begun to shed light on this important question (9,13). One study
found a link between Nsp15 activity and the length of the polyuridine sequence at the 5¢ end of the
template negative strand. When Nsp15-mutant MHV infected cells, there was a greater amount of
polyuridine (PUN) RNA compared to cells infected with WT virus, suggesting Nsp15 cleaves the PUN
RNA produced in the negative strand intermediate state (9). The PUN RNA was found to trigger the
pathogen associated molecular pattern (PAMP) receptor MDA5, which mediates interferon response
(9). Another study used cyclic phosphate RNA sequencing to identify Nsp15 cleavage products within
MHV infected bone marrow-derived macrophages (13). This analysis revealed that Nsp15 cleaves
numerous targets throughout the positive strand with a preference for cleaving between U^A and C^A
sequences (13). More recent work with IBV demonstrated that Nsp15 nuclease activity prevents the
accumulation of both dsRNA and cytoplasmic stress granules which have established anti-viral
USC 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17
The copyright holder for this preprintthis version posted June 1, 2021. ; https://doi.org/10.1101/2021.06.01.446181doi: bioRxiv preprint

properties (14). Collectively these studies confirm that Nsp15 nuclease activity is critical to prevent the
accumulation of viral ds-RNA and activation of the immune response.
Numerous structures of Nsp15 have been determined from several Coronaviridae family members,
however there are no structures of Nsp15 with more than a di-nucleotide bound, which has hindered
our understanding of how Nsp15 recognizes its RNA targets. Crystal structures of Nsp15 revealed
that Nsp15 assembles into a hexameric complex, formed from back-to-back trimers with the EndoU
domains facing outward (15-19). The active site of Nsp15 shares considerable similarity to the well-
studied endoribonuclease RNase A, and is composed of a catalytic triad including two histidines and
lysine (18,20,21) . These residues support a two-step reaction of transesterification and hydrolysis,
however due to an altered position of one active site histidine, Nsp15 accumulates products from the
transesterification reaction which contain a cyclic phosphate (20). 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). However, beyond
the preference for uridines, it is unclear if Nsp15 has any additional specificity requirements. RNA
sequencing suggests there is a preference for adenine 3¢ to the uridine, but the structural basis for
this is unknown (13). In contrast, RNase A is known to have additional non-catalytic sites that affect
substrate preference (22,23), prompting further characterization of how Nsp15 engages RNA.
Here we used cryo-EM, molecular dynamics simulations, and in vitro RNA cleavage assays to probe
the substrate specificity of SARS-CoV-2 Nsp15. We determined cryo-EM reconstructions of Nsp15
with RNA bound in the pre- and post-cleavage states. The structures revealed that, in contrast to
RNase A, Nsp15 does not contain any additional well-ordered sites for RNA binding and recognition.
This observation was further supported by molecular dynamics simulations with tri-nucleotide
substrates. We probed RNA specificity by determining how the nucleotide 5¢ and 3¢ of the uridine
affects cleavage and found that Nsp15 has a preference for purines 3′ of the cleaved pyrimidine.
Finally, we looked at Nsp15’s ability to cleave SARS-CoV-2 viral RNA substrates, such as the PUN
and the transcriptional regulatory sequence (TRS). Collectively our work suggests that SARS CoV-2
Nsp15 is able to cleave a broad spectrum of RNA substrates and that this activity is driven by
recognition of uridine within the active site.
MATERIAL AND METHODS
Protein expression and purification
Wild type (WT) and mutant Nsp15 constructs were created as described previously (20). Nsp15 was
overexpressed in E. coli C41 (DE3) competent cells in Terrific Broth with 100 mg/L ampicillin. At an
optical density (600 nm) between 0.8-1.0, cultures were cooled at 4˚C for 1 hour prior to induction with
0.2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were harvested after overnight
expression at 16˚C and stored at -80˚C until use. Nsp15 purification was done as described
previously (20). Briefly, cells were resuspended in Lysis Buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5%
glycerol, 5 mM b-ME, 5 mM imidazole) supplemented with cOmplete EDTA-free protease inhibitor
USC 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17
The copyright holder for this preprintthis version posted June 1, 2021. ; https://doi.org/10.1101/2021.06.01.446181doi: bioRxiv preprint

tablets (Roche) and disrupted by sonication. The lysate was clarified at 26,915 x g for 50 minutes at
4˚C and then incubated with TALON metal affinity resin (Clontech). His-Nsp15 was eluted from the
resin with 250 mM imidazole, and buffer exchanged into Thrombin Cleavage Buffer (50 mM Tris pH
8.0, 150 mM NaCl, 5% glycerol, 2 mM b-ME, 2 mM CaCl
2
) for cleavage at room temperature for 3
hours. The cleavage reaction was repassed over TALON resin and quenched with 1 mM
phenylmethylsulfonyl fluoride (PMSF) prior to gel filtration using a Superdex-200 column equilibrated
in SEC buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM MnCl
2
, 5 mM b-ME).
Cryo-EM sample preparation
Purified Nsp15 was diluted in a low-salt buffer (20 mM Hepes pH 7.5, 100 mM NaCl, 5 mM MnCl
2
, 5
mM b-ME) to 0.75 µM and incubated with excess RNA substrates (1 mM AU
f
A or AUA, see Table S1)
for 1 hour at 4˚C. UltrAuFoil R1.2/1.3 300 mesh gold grids (Quantifoil) 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 (Leica).
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. Particles were selected by
template-based particle picking, downsampled by a factor of 4, extracted with a box size of 64 and
subjected to an initial round of 2D classification. Full resolution particle projections from good classes
were re-extracted using a box size of 256. Ab initio reconstruction was used to generate an initial
model. Three independent 3D refinement cycles were performed while applying C1, C3, and D3
symmetry respectively. Although previous apo and UTP-bound datasets had D3 symmetry, the longer
RNA bound in both datasets here resulted in particles that no longer had D3 symmetry, perhaps due
to incomplete or mixed occupancy. Inspection of the C1 map did not reveal any asymmetric
differences, although active site density was difficult to interpret for one half of the pre-cleavage state
map. Therefore, C3 symmetry was used for model building and analysis for both datasets. Maps were
re-scaled to optimize RMS fit to core domain residues of reference structure PDBID 6WLC (21).
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¢ A. For the post-cleavage state,
the 5¢ A could be fit in the density along with the U. A combination of rigid body and real-space
refinement in Phenix as well as iterative rounds of building in COOT (28) were used to improve the fit
of the model. Molprobity (29) was used to evaluate the model (Table 1). Figures were prepared using
Chimera (30) and Chimera X (31).
USC 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17
The copyright holder for this preprintthis version posted June 1, 2021. ; https://doi.org/10.1101/2021.06.01.446181doi: bioRxiv preprint

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). The substrate (0.8 µM) was incubated with Nsp15 (2.5 nM)
in RNA cleavage buffer (20 mM Hepes pH 7.5, 75 mM NaCl, 5 mM MnCl
2
, 5 mM DTT) at 25°C for 60
minutes. 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 (Graphpad) was used to calculate significant
differences using Dunnett’s T3 multiple corrections test.
Urea-PAGE endoribonuclease assay
Double fluorescently-labeled RNA substrates (5′-FI and 3′-Cy5, 500 nM) were incubated with Nsp15
(50 nM) in RNA cleavage buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM MnCl
2
, 5 mM DTT, 1
u/µL RNasin ribonuclease inhibitor) at room temperature for 30 minutes, with samples collected at 0,
1, 5, 10, and 30 minutes. The reaction was quenched with 2x urea loading buffer (8M urea, 20 mM
Tris pH 8.0, 1 mM EDTA). 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. To generate a ladder, alkaline hydrolysis of the RNA was
carried out for 15 min at 90˚C using 1 µM RNA in alkaline hydrolysis buffer (50 mM sodium carbonate
pH 9.2, 1 mM EDTA) and quenched with 2x urea loading buffer. The cleavage reactions were
separated using 15%-20% TBE-urea PAGE gels and visualized with a Typhoon RGB imager
(Amersham) using Cy2
ex
=488 nm, λ
em
=515-535 nm
) and Cy5
ex
=635 nm, λ
em
=655-685 nm)
channels.
Mass spectrometry of RNA cleavage products
Mass spectrometry was performed as previously described (20). Briefly, the FRET RNA substrate of
interest (0.8 µM) was incubated +/- Nsp15 (2.5 nM) in RNA cleavage buffer for 30 minutes at RT. 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 (methanol). Parallel
reaction monitoring (PRM) analyses were included in the MS analyses with included masses of m/z
914.14; 923.14; 1463.42.
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.
Except for H250, all histidine residues were selected to be N
ε
protonated. Since the ring nitrogen
atoms on H250 were found to make two strong hydrogen bonds with the phosphate backbone and the
USC 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17
The copyright holder for this preprintthis version posted June 1, 2021. ; https://doi.org/10.1101/2021.06.01.446181doi: bioRxiv preprint

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Journal ArticleDOI
TL;DR: The PHENIX software for macromolecular structure determination is described and its uses and benefits are described.
Abstract: Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. How­ever, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallo­graphic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.

18,531 citations

Journal ArticleDOI
TL;DR: MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions.
Abstract: MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions.

5,491 citations

Journal ArticleDOI
TL;DR: It is shown that stochastic gradient descent (SGD) and branch-and-bound maximum likelihood optimization algorithms permit the major steps in cryo-EM structure determination to be performed in hours or minutes on an inexpensive desktop computer.
Abstract: Single-particle electron cryomicroscopy (cryo-EM) is a powerful method for determining the structures of biological macromolecules. With automated microscopes, cryo-EM data can often be obtained in a few days. However, processing cryo-EM image data to reveal heterogeneity in the protein structure and to refine 3D maps to high resolution frequently becomes a severe bottleneck, requiring expert intervention, prior structural knowledge, and weeks of calculations on expensive computer clusters. Here we show that stochastic gradient descent (SGD) and branch-and-bound maximum likelihood optimization algorithms permit the major steps in cryo-EM structure determination to be performed in hours or minutes on an inexpensive desktop computer. Furthermore, SGD with Bayesian marginalization allows ab initio 3D classification, enabling automated analysis and discovery of unexpected structures without bias from a reference map. These algorithms are combined in a user-friendly computer program named cryoSPARC (http://www.cryosparc.com).

4,342 citations

Frequently Asked Questions (14)
Q1. What was used to improve the fit of the model?

A combination of rigid body and real-space refinement in Phenix as well as iterative rounds of building in COOT (28) were used to improve the fit of the model. 

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. 

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. 

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). 

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). 

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). 

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). 

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. 

The authors captured the post-cleavage state by incubating Nsp15 with excess unmodified RNA (AUA) prior to vitrification and cryo-EM data collection. 

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. 

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). 

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). 

the authors also looked at Nsp15’s ability to degrade polyU sequences and found that Nsp15 efficiently degrades polyU containing RNAs in vitro. 

the authors turned to molecular dynamics simulations to further characterize the behavior of the nucleotides near the active site.