MERS-CoV NSP16 necessary for IFN resistance and viral pathogenesis

Coronaviruses encode a mix of highly conserved and novel genes as well as genetic elements necessary for infection and pathogenesis, raising the possibility for common targets for attenuation and therapeutic design. In this study, we focus on the highly conserved nonstructural protein (NSP) 16, a viral 2’O methyl-transferase (MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of MERS NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2’O MTase activity had only marginal impact on propagation and replication in Vero cells, the MERS dNSP16 mutant demonstrated significant attenuation relative to control both in primary human airway cultures and in vivo. Further examination indicated the MERS dNSP16 mutant had a type I IFN based attenuation and was partially restored in the absence of IFIT molecules. Importantly, the robust attenuation permitted use of MERS dNSP16 as a live attenuated vaccine platform protecting from challenge with a mouse adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2’O MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting. Significance Coronavirus emergence in both human and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of SARS-CoV, MERS-CoV, PEDV and swine delta coronavirus in the 21st century. These studies describe an approach that effectively targets the highly conserved 2’O methyl-transferase activity of coronaviruses for attenuation. With clear understanding of the IFN/IFIT based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-coronavirus functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2’O MTase function across coronaviruses.


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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

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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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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).

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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.

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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.

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In vitro host response similar between SARS and MERS dNSP16 mutants.

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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).

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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 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.

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MERS-CoV dNSP16 attenuated in primary and in vivo models.

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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.

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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

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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

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Together, these results suggest that targeting NSP16 may be the most broadly applicable 20 platform for CoV attenuation.       8 RNA isolation, microarray processing and identification of differential expression. RNA 9 isolation and microarray processing, quality control, and normalization from Calu-3 cells was 10 carried out as previously described (34) (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.

Figure 2. MERS dNSP16 infec6on produces minimal changes in early host responses.
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.