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. 2014 Apr;88(8):4251-64.
doi: 10.1128/JVI.03571-13. Epub 2014 Jan 29.

Attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity

Vineet D Menachery  1 , Boyd L Yount Jr, Laurence Josset, Lisa E Gralinski, Trevor Scobey, Sudhakar Agnihothram, Michael G Katze, Ralph S Baric
Affiliations

Attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity

Vineet D Menachery et al. J Virol. 2014 Apr.

Abstract

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and, more recently, Middle Eastern respiratory syndrome CoV (MERS-CoV) underscores the importance of understanding critical aspects of CoV infection and pathogenesis. Despite significant insights into CoV cross-species transmission, replication, and virus-host interactions, successful therapeutic options for CoVs do not yet exist. Recent identification of SARS-CoV NSP16 as a viral 2'-O-methyltransferase (2'-O-MTase) led to the possibility of utilizing this pathway to both attenuate SARS-CoV infection and develop novel therapeutic treatment options. Mutations were introduced into SARS-CoV NSP16 within the conserved KDKE motif and effectively attenuated the resulting SARS-CoV mutant viruses both in vitro and in vivo. While viruses lacking 2'-O-MTase activity had enhanced sensitivity to type I interferon (IFN), they were not completely restored in their absence in vivo. However, the absence of either MDA5 or IFIT1, IFN-responsive genes that recognize unmethylated 2'-O RNA, resulted in restored replication and virulence of the dNSP16 mutant virus. Finally, using the mutant as a live-attenuated vaccine showed significant promise for possible therapeutic development against SARS-CoV. Together, the data underscore the necessity of 2'-O-MTase activity for SARS-CoV pathogenesis and identify host immune pathways that mediate this attenuation. In addition, we describe novel treatment avenues that exploit this pathway and could potentially be used against a diverse range of viral pathogens that utilize 2'-O-MTase activity to subvert the immune system.

Importance: Preventing recognition by the host immune response represents a critical aspect necessary for successful viral infection. Several viruses, including SARS-CoV, utilize virally encoded 2'-O-MTases to camouflage and obscure their viral RNA from host cell sensing machinery, thus preventing recognition and activation of cell intrinsic defense pathways. For SARS-CoV, the absence of this 2'-O-MTase activity results in significant attenuation characterized by decreased viral replication, reduced weight loss, and limited breathing dysfunction in mice. The results indicate that both MDA5, a recognition molecule, and the IFIT family play an important role in mediating this attenuation with restored virulence observed in their absence. Understanding this virus-host interaction provided an opportunity to design a successful live-attenuated vaccine for SARS-CoV and opens avenues for treatment and prevention of emerging CoVs and other RNA virus infections.

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Figures

FIG 1
FIG 1
Construction of SARS-CoV NSP16 mutant viruses. (A) SARS-CoV NSP16 protein (black) bound to NSP10 (orange) based on published crystal structure (21). The individual residues of the conserved KDKE motif required for MTase function are highlighted in complex, and the insets (on the right side) demonstrate wild-type and constructed NSP16 mutants with alanine substitution at circled residues. (B and C) Endpoint virus titers of wild-type icSARS-CoV and icSARS NSP16 mutants in Vero cells (multiplicity of infection [MOI] = 0.01) (B) and Calu3 airway epithelial cells (MOI = 0.01) (C).
FIG 2
FIG 2
dNSP16 mutant viruses attenuation in vitro and in vivo. (A and B) Virus titers after infection of Calu3 cells (A) or human airway epithelial (HAE) cells (B) with SARS-CoV WT or dNSP16 at an MOI of 0.01. Time zero represents input titers. (C to F) Weight loss (n > 5 for WT and dNSP16 groups) (C), lung virus titer (n = 3 per group) (D), airway resistance (E), and expiratory flow (F) after infection of female BALB/c mice with 105 PFU of SARS-CoV WT or dNSP16. P values based on the Student t test are marked (*, P < 0.05; ***, P < 0.001).
FIG 3
FIG 3
dNSP16 is more sensitive to IFN but is not restored by type I IFN deficiency. (A) Mock-treated (solid lines) or IFN-β pretreated (dotted lines) Vero cells infected with either SARS-CoV WT (black) or dNSP16 (red) at an MOI of 5. (B) Percentage reduction in log virus titer derived from panel A for WT (black) or dNSP16 (red). (C and D) RNA expression of IFNb1 from Calu3 cells (C) or C57BL/6 (D) mice after infection with WT or dNSP16 SARS-CoV. (E and F) Viral lung titers (n > 3 per group) (E) and weight loss (n > 6 per group) (F) after infection of female, 10-week-old IFNAR−/− mice with SARS-CoV WT (black) or dNSP16 (red). P values based on the Student t test are marked (*, P < 0.05; ***, P < 0.001).
FIG 4
FIG 4
Kinetic expression of MDA5 and IFIT proteins correspond to dNSP16 attenuation. (A to C) RNA (solid line) and protein (dashed line) expression of IFIT1 (A), IFIT2 (B), and MDA5 (C) in Calu3 cells after infection with WT icSARS-CoV. (D to F) RNA (solid) and protein (dashed) expression of IFIT1 (D), IFIT2 (E), and MDA5 (F) after infection of C57BL/6 mice.
FIG 5
FIG 5
ISG expression kinetics and effector function after in vitro infection with dNSP16. (A to C) In vitro RNA expression of IFIT1 (A), IFIT2 (B), and MDA5 (C) after infection at an MOI of 5 of Calu3 cells with WT SARS-CoV (black) or dNSP16 (red), as measured by microarray. (D to F) In vivo RNA expression of IFIT1 (D), IFIT2 (E), and MDA5 (F) after infection of C57BL/6 mice with WT SARS-CoV (black) or dNSP16 (red). (G to H) Vero cells expressing shRNA targeting IFIT1 (blue), IFIT2 (purple), or no shRNA (black) were pretreated with IFN-β and infected with WT SARS-CoV (G) or dNSP16 (H). P values based on Student t test and are marked as indicated (*, P < 0.05; ***, P < 0.001).
FIG 6
FIG 6
Pathogenesis after infection of MDA5−/− or IFIT1−/− mice with dNSP16. (A to H) Male and female C57BL/6 (black, n > 13 per group), MDA5−/− (green, n > 13 per group), and IFIT1−/− (blue, n > 8 per group) mice were infected with SARS-CoV WT (solid lines) or dNSP16 (dashed line) and monitored for weight loss (A to C), virus titer in the lung (D), expiratory flow (E and F), and airway resistance (G and H). For panels E to H, data from mock-infected IFIT1−/− (two mice) and MDA5−/− (one mouse) animals were combined to provide a relative baseline for respiratory function. P values based on Student t test and are marked as indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 7
FIG 7
Histological analysis of C57BL/6, IFIT1−/−, and MDA5−/− mice after infection with WT-SARS-CoV and dNSP16. (A to F) Representative images show broad lung cross-sections (first column, 10×), alveolar edema (second column, 40×), and vascular cuffing (third column, 40×) after WT-SARS-CoV (A, C, and E) or dNSP16 (B, D, and F) infection of C57BL/6 (A and B), IFIT1−/− (C and D), and MDA5−/− (E and F) mice. (G and H) Blinded histology scoring of alveolar exudates (G) and vasculature disease (edema and perivascular cuffing) (H) after WT (solid) or dNSP16 (hatched) infection of C57BL/6 (black), IFIT1−/− (blue), and MDA5−/− (green) mice 7 days postinfection. P values based on Student t test and are marked as indicated (*, P < 0.05).
FIG 8
FIG 8
Vaccination with dNSP16 protects from lethal challenge. (A and B) Weight loss (A) and lethality (B) after infection of 10-week-old female BALB/c mice vaccinated with 100 PFU of dNSP16 or PBS (n = 6), monitored for 28 days, and then challenged with SARS-CoV WT at 105 PFU. (C) Neutralization titer for WT-SARS-CoV from serum of dNSP16-vaccinated mice or mock-vaccinated mice 28 days postinfection. P values are based on the Student t test and are marked as indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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