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


OBJECTIVE
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.
Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina, USA.
Vineet D
VD
Menachery
Boyd L
BL
Yount
Laurence
L
Josset
Lisa E
LE
Gralinski
Trevor
T
Scobey
Sudhakar
S
Agnihothram
Michael G
MG
Katze
Ralph S
RS
Baric
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D000072640:Interferon-Induced Helicase, IFIH1; D008297:Male; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009154:Mutation; D016601:RNA-Binding Proteins; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology; D045169:Severe Acute Respiratory Syndrome / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014774:Virulence; D014779:Virus Replication
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C092573:Ifit1 protein, mouse; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; C558879:Nsp16 protein, SARS virus; C510592:Ifih1 protein, mouse; D053487:DEAD-box RNA Helicases; D000072640:Interferon-Induced Helicase, IFIH1