Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PL...
Saved in:
| Main Authors: | , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2021-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0253364 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849337487369961472 |
|---|---|
| author | Lee A Armstrong Sven M Lange Virginia Dee Cesare Stephen P Matthews Raja Sekhar Nirujogi Isobel Cole Anthony Hope Fraser Cunningham Rachel Toth Rukmini Mukherjee Denisa Bojkova Franz Gruber David Gray Paul G Wyatt Jindrich Cinatl Ivan Dikic Paul Davies Yogesh Kulathu |
| author_facet | Lee A Armstrong Sven M Lange Virginia Dee Cesare Stephen P Matthews Raja Sekhar Nirujogi Isobel Cole Anthony Hope Fraser Cunningham Rachel Toth Rukmini Mukherjee Denisa Bojkova Franz Gruber David Gray Paul G Wyatt Jindrich Cinatl Ivan Dikic Paul Davies Yogesh Kulathu |
| author_sort | Lee A Armstrong |
| collection | DOAJ |
| description | Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle. |
| format | Article |
| id | doaj-art-7e4e63d2ba344215bb01a64466ff2cc4 |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-7e4e63d2ba344215bb01a64466ff2cc42025-08-20T03:44:40ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01167e025336410.1371/journal.pone.0253364Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.Lee A ArmstrongSven M LangeVirginia Dee CesareStephen P MatthewsRaja Sekhar NirujogiIsobel ColeAnthony HopeFraser CunninghamRachel TothRukmini MukherjeeDenisa BojkovaFranz GruberDavid GrayPaul G WyattJindrich CinatlIvan DikicPaul DaviesYogesh KulathuOf the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.https://doi.org/10.1371/journal.pone.0253364 |
| spellingShingle | Lee A Armstrong Sven M Lange Virginia Dee Cesare Stephen P Matthews Raja Sekhar Nirujogi Isobel Cole Anthony Hope Fraser Cunningham Rachel Toth Rukmini Mukherjee Denisa Bojkova Franz Gruber David Gray Paul G Wyatt Jindrich Cinatl Ivan Dikic Paul Davies Yogesh Kulathu Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies. PLoS ONE |
| title | Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies. |
| title_full | Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies. |
| title_fullStr | Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies. |
| title_full_unstemmed | Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies. |
| title_short | Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies. |
| title_sort | biochemical characterization of protease activity of nsp3 from sars cov 2 and its inhibition by nanobodies |
| url | https://doi.org/10.1371/journal.pone.0253364 |
| work_keys_str_mv | AT leeaarmstrong biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT svenmlange biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT virginiadeecesare biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT stephenpmatthews biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT rajasekharnirujogi biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT isobelcole biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT anthonyhope biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT frasercunningham biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT racheltoth biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT rukminimukherjee biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT denisabojkova biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT franzgruber biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT davidgray biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT paulgwyatt biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT jindrichcinatl biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT ivandikic biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT pauldavies biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies AT yogeshkulathu biochemicalcharacterizationofproteaseactivityofnsp3fromsarscov2anditsinhibitionbynanobodies |