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MERS-CoV NSP16 necessary for IFN resistance and viral pathogenesis
Abstract
Coronaviruses encode a mix of highly conserved and novel genes as well as genetic elements 2 necessary for infection and pathogenesis, raising the possibility for common targets for 3 attenuation and therapeutic design. In this study, we focus on the highly conserved non-4 structural protein (NSP) 16, a viral 2'O methyl-transferase (MTase) that encodes critical 5 functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif 6 in the conserved KDKE motif of MERS NSP16 (D130A) and evaluated the effect on viral 7 infection and pathogenesis. While the absence of 2'O MTase activity had only marginal impact 8 on propagation and replication in Vero cells, the MERS dNSP16 mutant demonstrated 9 significant attenuation relative to control both in primary human airway cultures and in vivo.
10 Further examination indicated the MERS dNSP16 mutant had a type I IFN based attenuation 11 and was partially restored in the absence of IFIT molecules. Importantly, the robust attenuation 12 permitted use of MERS dNSP16 as a live attenuated vaccine platform protecting from challenge 13 with a mouse adapted MERS-CoV strain. These studies demonstrate the importance of the 14 conserved 2'O MTase activity for CoV pathogenesis and highlight NSP16 as a conserved 15 universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.
16 All rights reserved. No reuse allowed without permission. Significance 1 Coronavirus emergence in both human and livestock represents a significant threat to global 2 public health, as evidenced by the sudden emergence of SARS-CoV, MERS-CoV, PEDV and 3 swine delta coronavirus in the 21 st century. These studies describe an approach that effectively 4 targets the highly conserved 2'O methyl-transferase activity of coronaviruses for attenuation. 5 With clear understanding of the IFN/IFIT based mechanism, NSP16 mutants provide a suitable 6 target for a live attenuated vaccine platform as well as therapeutic development for both current 7 and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-8 coronavirus functions have not yet proven effective against MERS-CoV, illustrating the broad 9 applicability of targeting viral 2'O MTase function across coronaviruses.
Similarly, a number of highly conserved viral proteins for structure, replication, and fidelity are 17 also maintained in the CoV backbone (7). Among these, MERS-CoV NSP16 provides a potent 25 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https: //doi.org/10.1101/173286 doi: bioRxiv preprint Using reverse genetics to target residues in the highly conserved active site, we 1 evaluated MERS-CoV infection outcomes in the context of an inactive NSP16 (dNSP16).
2 Consistent with previous studies in SARS-CoV (10), the dNSP16 MERS-CoV mutant 3 maintained no significant attenuation in terms of replication or the initial host immune response. Importantly, the dNSP16 mutant also provided robust protection against a lethal MERS-CoV 8 challenge and maintained attenuation in the mouse adapted backbone. Together, the results 9 illustrate the broad conservation and necessity of NSP16 in CoV pathogenesis and highlight 10 targeting this protein as a rapid response platform for future emergent CoV strains.
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint
A combination of structural and biochemical approaches has established a critical role for CoV 2 NSP16 in 2'O methyltransferase activity (Fig. 1A) . Stabilized by interactions with NSP10 3 (orange), NSP16 relies on a highly conserved KDKE motif (red) to mediate its activity (11).
Previous alteration of this motif in both group 2b SARS-CoV (10) and group 2a MHV (9) 5 disrupted 2'O methyltransferase activity and attenuated varying aspects of infection. Based on 6 high conservation across the CoV family (Fig. 1B) , we hypothesized that disruption of the KDKE 7 motif would also attenuate other emerging CoV families, including the group 2c MERS-CoV.
Utilizing a MERS-CoV reversed genetic system (12), we disrupted the KDKE motif by mutating 9 two nucleotides to produce a D130A change (Fig. 1A) . The resulting disrupted NSP16 mutant 10 (dNSP16) had no significant defect noted in stock titer generation (not shown); similarly, low 11 MOI infection of both Vero cells and Calu3 cells, a respiratory epithelial cell line, demonstrated 12 only modest attenuation at late time points (Fig. 1C & D) . Together, these results indicate that 13 NSP16 activity is not required for replication capacity.
In vitro host response similar between SARS and MERS dNSP16 mutants.
Having established replication competence in both Vero and Calu3 cells, we next evaluated 16 induction of host pathways following infection. Calu3 cells infected at MOI 5 demonstrated no 17 differences in replication (not shown) and only modest differences in host induction (0 genes 18 fold expression g >1.5 log2). Unlike previous studies with SARS-CoV, rapid cytopathic effect 19 (CPE) by 24 hours limited analysis to early time points. Further DAVID based-analysis 20 compared network host responses between MERS-CoV and SARS-CoV dNSP16 (Fig. 2) .
Over the first 24 hours of infection, both MERS-CoV and SARS-CoV dNSP16 showed no 22 significant functional enrichment of any categories relative to corresponding wild-type (WT) 23 infections, consistent with the lack of replication attenuation. However, at late times (>24 hours 24 post infection), SARS-CoV produces robust changes in several host pathways including 25 cytokine responses, inflammation, and extracellular activity. Similarly, changes in apoptosis, 26 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint transcription repression, and regulation of phosphorylation indicated a host response more 1 hostile to viral infection. While more rapid CPE following MERS-CoV dNSP16 infection 2 precluded an equivalent finding at late time points, the SARS-CoV results suggest that the 3 absence of NSP16 activity eventually initiates host response changes that contribute to 4 attenuation at late time points.
MERS-CoV dNSP16 attenuated in primary and in vivo models.
To further examine the replicative capacity of dNSP16, we infected both human airway models 7 and mice expressing human dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV.
Primary human airway cultures (HAE) were challenged with wild-type and dNSP16 MERS-CoV 9 at a low MOI (Fig. 3A) . While robust replication was observed following wild-type infection, 10 dNSP16 MERS mutant had significant attenuation that corresponded well to previous results 11 seen with SARS-CoV dNSP16 (10). We next examined MERS dNSP16 replication phenotypes The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint
Having established a deficit in MERS NSP16 mutant replication in relevant in vitro and in vivo 1 models, we next sought to evaluate the mechanism of attenuation. Previous work by our lab and 2 others had established increased susceptibility of NSP16 mutants to type I IFN (9, 10). While 3 both viruses were sensitive to IFN treatment, the MERS dNSP16 mutant had a significant 4 reduction in viral replication relative to control virus (Fig. 4A) . These results are consistent with 5 reports for NSP16 mutants in other coronaviruses (8). Extending this analysis further, we The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint vaccine platform that not only induces high levels of neutralizing antibodies, but provides 1 compete protection from lethal MERS-CoV challenge.
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint
In the context of the ongoing MERS-CoV outbreak, the development of universal 2 platform strategies to attenuate emerging and contemporary coronaviruses is a significant 3 priority. In this study, we demonstrate the critical importance of NSP16 function to MERS-CoV The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.
Together, these results suggest that targeting NSP16 may be the most broadly applicable 20 platform for CoV attenuation. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint is paramount in its pursuit as a platform. Similarly, the existence of numerous CoVs in animal 1 populations raises the concerns for heterologous challenge from emergent viruses (30, 31).
Prior reports had also demonstrated vaccine induced disease following heterologous challenge 3 with related SARS-like viruses (29, 32) . With this in mind, NSP16 vaccinated mice will need to 4 be examined in the context of heterologous challenge to determine if vaccine induced pathology 5 occurs. Together, these two factors represent important checkpoints in the pursuit of NSP16 as 6 a universal CoV vaccine platform.
In addition to aging and heterologous challenge, reversion and baseline pathogenesis 8 also represent important risks that must be evaluated in the context of an NSP16 vaccine. While 9 the NSP14 SARS vaccine was absent sterilizing immunity in immunodeficient mice, the lack of
Overall, the current study demonstrates that targeting 2'O MTase activity is a robust 21 strategy to attenuate MERS-CoV and other emergent coronaviruses. In the absence of NSP16 22 activity, the MERS-CoV mutant is sensitive to type I IFN in an IFIT dependent manner providing 23 clear mechanism for attenuation. Importantly, unlike other conserved CoV platforms, the NSP16 24 mutant is both viable and robust enough to be utilized as an effective live-attenuated vaccine.
While further vaccine characterization is required, the results indicate that disruption of CoV 26 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint NSP16 activity can be the basis for therapeutic strategies for both current and future emergent 1 CoV infection both in human and animal populations.
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.
CAACCTCAATACAAGCAGAC. The two resulting products were digested with SapI
(underlined) and ligated overnight with T4 DNA ligase. This product was then digested with 25 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint 1 similarly digested. Thereafter, plasmids containing wild-type and mutant MERS-CoV genome 2 fragments were amplified, excised, ligated, and purified. In vitro transcription reactions were 3 then preformed to synthesize full-length genomic RNA, which was transfected into Vero E6 4 cells. The media from transfected cells were harvested and served as seed stocks for 5 subsequent experiments. Viral mutants were confirmed by sequence analysis prior to use.
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint Figure 1. NSP16 highly conserved across coronavirus family. A) NSP16-NSP10 complex for MERS-CoV. NSP16 (gray) highlighting the conserved KDKE motif (red) required for 2'O methyltransferase activity. Also shown, required NSP10 scaffold for MERS-CoV (orange). Inset displays conserved KDKE (right) as well as D130A (left) mutation that disrupts function. Homology models were created using Modeller in the Max-Planck Institute's Bioinformatics Toolkit. The known crystal structure for the NSP10/16 complex (3R24 in the RCSB protein data bank) was used as the template structure (Chen et al., 2011) . Homology models were then manipulated using MacPyMol. B) Heat maps were constructed from a set of representative coronaviruses from all four genogroups using alignment data paired with neighbor-joining phylogenetic trees built in Geneious (v.9.1.5) and visualized in EvolView (evolgenius.info). Trees show the degree of genetic similarity of NSP16 across CoV families. C & D) Viral replication of MERS dNSP16 mutant (red) relative to wild-type (WT) MERS-CoV (black) in (C) Vero cells and (D) Calu3 2B4 cells following MOI 0.01 infection. P-value representative of Student's Ttest with values representing *** <0.001.
Changes in func-onal host gene clusters based on RNA expression following MOI 5 infec-on of Calu3 cells with MERS-CoV dNSP16 (leG) or SARS-CoV dNSP16 (right) rela-ve to wild-type (WT) control virus. Heat-map plots significant enrichment of clustered func-onal categories (as determined by DAVID analysis) for each mutant over -me. Only marginal changes noted during the first 24 hours for both SARS and MERS-CoV dNSP16 mutants. AGer 24 hours (right) significant changes noted for SARS-CoV; MERS-CoV had significant cytopathic effect aGer 24 hours post infec-on precluding analysis. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint Figure 3 . MERS dNSP16 aAenuated in primary cultures and in vivo. A) Primary human airway epithelial cells infected with wild-type MERS-CoV (black) or dNSP16 mutant (red) at MOI 0.01 and monitored over -me course. B) Day 2 and 4 lung -ters from adenovirus transduced mice expressing human DPP4 infected with wild-type MERS-CoV (black) or dNSP16 mutant (red). C) Day 2 and 4 lung -ter from 288-330 +/+ CRISPR mice infected with WT (Black) or dNSP16 (Red). P-value representa-ve of Student's T-test with values represen-ng *< 0.05 ** <0.01 *** <0.001. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint Figure 5 . MERS dNSP16 mutants protects from lethal challenge. A) Weight loss, B) day 4 viral -ter, and C) hemorrhage score following challenge of 288-330+/+ CRISPR/Cas9 mice vaccinated with wild-type MERS dNSP16 (red) or mock (black) with 10^6 pfu passaged mouse adapted . D) Plaque reduc-on neutraliza-on with sera from WT (black) or dNSP16 (red) vaccinated mice. P-value representa-ve of Student's T-test with values represen-ng *< 0.05 ** <0.01 *** <0.001. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint Figure 6 . MERS dNSP16 mutant aAenuated in virulent mouse adapted MERS-CoV strain. A) Weight loss, B) lung -ters, and C) hemorrhage score following infec-on of 288-330 +/+ CRISPR/Cas9 mice infected with 10^6 pfu MERS-CoV MA1 (black) or dNSP16 MA1 (red) at day 2 and 4. C) P-value representa-ve of Student's T-test with values represen-ng *< 0.05 ** <0.01 *** <0.001. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/173286 doi: bioRxiv preprint
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