A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance
ABSTRACT Coronaviruses (CoVs) encode nonstructural proteins 1–16 (nsps 1–16) which form replicase complexes that mediate viral RNA synthesis. Remdesivir (RDV) is an adenosine nucleoside analog antiviral that inhibits CoV RNA synthesis. RDV resistance mutations have been reported only in the nonstruc...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-08-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mbio.01060-23 |
| _version_ | 1797730240148013056 |
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| author | Samantha L. Grimes Young J. Choi Anoosha Banerjee Gabriel Small Jordan Anderson-Daniels Jennifer Gribble Andrea J. Pruijssers Maria L. Agostini Alexandra Abu-Shmais Xiaotao Lu Seth A. Darst Elizabeth Campbell Mark R. Denison |
| author_facet | Samantha L. Grimes Young J. Choi Anoosha Banerjee Gabriel Small Jordan Anderson-Daniels Jennifer Gribble Andrea J. Pruijssers Maria L. Agostini Alexandra Abu-Shmais Xiaotao Lu Seth A. Darst Elizabeth Campbell Mark R. Denison |
| author_sort | Samantha L. Grimes |
| collection | DOAJ |
| description | ABSTRACT Coronaviruses (CoVs) encode nonstructural proteins 1–16 (nsps 1–16) which form replicase complexes that mediate viral RNA synthesis. Remdesivir (RDV) is an adenosine nucleoside analog antiviral that inhibits CoV RNA synthesis. RDV resistance mutations have been reported only in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp). We here show that a substitution mutation in the nsp13-helicase (nsp13-HEL A335V) of the betacoronavirus murine hepatitis virus (MHV) that was selected during passage with the RDV parent compound confers partial RDV resistance independently and additively when expressed with co-selected RDV resistance mutations in the nsp12-RdRp. The MHV A335V substitution did not enhance replication or competitive fitness compared to WT MHV and remained sensitive to the active form of the cytidine nucleoside analog antiviral molnupiravir (MOV). Biochemical analysis of the SARS-CoV-2 helicase encoding the homologous substitution (A336V) demonstrates that the mutant protein retained the ability to associate with the core replication proteins nsps 7, 8, and 12 but had impaired helicase unwinding and ATPase activity. Together, these data identify a novel determinant of nsp13-HEL enzymatic activity, define a new genetic pathway for RDV resistance, and demonstrate the importance of surveillance for and testing of helicase mutations that arise in SARS-CoV-2 genomes. IMPORTANCE Despite the development of effective vaccines against COVID-19, the continued circulation and emergence of new variants support the need for antivirals such as RDV. Understanding pathways of antiviral resistance is essential for surveillance of emerging variants, development of combination therapies, and for identifying potential new targets for viral inhibition. We here show a novel RDV resistance mutation in the CoV helicase also impairs helicase functions, supporting the importance of studying the individual and cooperative functions of the replicase nonstructural proteins 7–16 during CoV RNA synthesis. The homologous nsp13-HEL mutation (A336V) has been reported in the GISAID database of SARS-CoV-2 genomes, highlighting the importance of surveillance of and genetic testing for nucleoside analog resistance in the helicase. |
| first_indexed | 2024-03-12T11:41:24Z |
| format | Article |
| id | doaj.art-351eda1dc8534b2e94fffb7f9053450a |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-03-12T11:41:24Z |
| publishDate | 2023-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-351eda1dc8534b2e94fffb7f9053450a2023-08-31T15:04:20ZengAmerican Society for MicrobiologymBio2150-75112023-08-0114410.1128/mbio.01060-23A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistanceSamantha L. Grimes0Young J. Choi1Anoosha Banerjee2Gabriel Small3Jordan Anderson-Daniels4Jennifer Gribble5Andrea J. Pruijssers6Maria L. Agostini7Alexandra Abu-Shmais8Xiaotao Lu9Seth A. Darst10Elizabeth Campbell11Mark R. Denison12Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center , Nashville, Tennessee, USALaboratory of Molecular Biophysics, The Rockefeller University , New York, New York, USALaboratory of Molecular Biophysics, The Rockefeller University , New York, New York, USALaboratory of Molecular Biophysics, The Rockefeller University , New York, New York, USADepartment of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center , Nashville, Tennessee, USADepartment of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center , Nashville, Tennessee, USADepartment of Pediatrics, Vanderbilt University Medical Center , Nashville, Tennessee, USADepartment of Pediatrics, Vanderbilt University Medical Center , Nashville, Tennessee, USADepartment of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center , Nashville, Tennessee, USADepartment of Pediatrics, Vanderbilt University Medical Center , Nashville, Tennessee, USALaboratory of Molecular Biophysics, The Rockefeller University , New York, New York, USALaboratory of Molecular Biophysics, The Rockefeller University , New York, New York, USADepartment of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center , Nashville, Tennessee, USAABSTRACT Coronaviruses (CoVs) encode nonstructural proteins 1–16 (nsps 1–16) which form replicase complexes that mediate viral RNA synthesis. Remdesivir (RDV) is an adenosine nucleoside analog antiviral that inhibits CoV RNA synthesis. RDV resistance mutations have been reported only in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp). We here show that a substitution mutation in the nsp13-helicase (nsp13-HEL A335V) of the betacoronavirus murine hepatitis virus (MHV) that was selected during passage with the RDV parent compound confers partial RDV resistance independently and additively when expressed with co-selected RDV resistance mutations in the nsp12-RdRp. The MHV A335V substitution did not enhance replication or competitive fitness compared to WT MHV and remained sensitive to the active form of the cytidine nucleoside analog antiviral molnupiravir (MOV). Biochemical analysis of the SARS-CoV-2 helicase encoding the homologous substitution (A336V) demonstrates that the mutant protein retained the ability to associate with the core replication proteins nsps 7, 8, and 12 but had impaired helicase unwinding and ATPase activity. Together, these data identify a novel determinant of nsp13-HEL enzymatic activity, define a new genetic pathway for RDV resistance, and demonstrate the importance of surveillance for and testing of helicase mutations that arise in SARS-CoV-2 genomes. IMPORTANCE Despite the development of effective vaccines against COVID-19, the continued circulation and emergence of new variants support the need for antivirals such as RDV. Understanding pathways of antiviral resistance is essential for surveillance of emerging variants, development of combination therapies, and for identifying potential new targets for viral inhibition. We here show a novel RDV resistance mutation in the CoV helicase also impairs helicase functions, supporting the importance of studying the individual and cooperative functions of the replicase nonstructural proteins 7–16 during CoV RNA synthesis. The homologous nsp13-HEL mutation (A336V) has been reported in the GISAID database of SARS-CoV-2 genomes, highlighting the importance of surveillance of and genetic testing for nucleoside analog resistance in the helicase.https://journals.asm.org/doi/10.1128/mbio.01060-23coronavirusmurine hepatitis virus (MHV)SARS-CoV-2nsp13helicaseremdesivir |
| spellingShingle | Samantha L. Grimes Young J. Choi Anoosha Banerjee Gabriel Small Jordan Anderson-Daniels Jennifer Gribble Andrea J. Pruijssers Maria L. Agostini Alexandra Abu-Shmais Xiaotao Lu Seth A. Darst Elizabeth Campbell Mark R. Denison A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance mBio coronavirus murine hepatitis virus (MHV) SARS-CoV-2 nsp13 helicase remdesivir |
| title | A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance |
| title_full | A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance |
| title_fullStr | A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance |
| title_full_unstemmed | A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance |
| title_short | A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance |
| title_sort | mutation in the coronavirus nsp13 helicase impairs enzymatic activity and confers partial remdesivir resistance |
| topic | coronavirus murine hepatitis virus (MHV) SARS-CoV-2 nsp13 helicase remdesivir |
| url | https://journals.asm.org/doi/10.1128/mbio.01060-23 |
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