tetano
Editor, Senior Moderator
Mol Ther Nucleic Acids
. 2025 Jan 16;36(1):102452.
doi: 10.1016/j.omtn.2025.102452. eCollection 2025 Mar 11. Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7
Deepa Singh[SUP] 1 [/SUP], Tushar Kushwaha[SUP] 1 [/SUP], Rajkumar Kulandaisamy[SUP] 1 [/SUP], Vikas Kumar[SUP] 1 [/SUP], Kamal Baswal[SUP] 1 [/SUP], Saras H Tiwari[SUP] 1 [/SUP], Arkadyuti Ghorai[SUP] 1 [/SUP], Manoj Kumar[SUP] 1 [/SUP], Saroj Kumar[SUP] 1 [/SUP], Soumya De[SUP] 2 [/SUP], Aparoy Polamarasetty[SUP] 3 [/SUP], Deepak Sehgal[SUP] 4 [/SUP], Madhumohan R Katika[SUP] 5 [/SUP], Suresh Gadde[SUP] 6 [/SUP], Marceline Côté[SUP] 7 [/SUP], Sarala R Kayampeta[SUP] 8 [/SUP], Mohan Babu Appaiahgari[SUP] 8 9 [/SUP], Krishna K Inampudi[SUP] 1 [/SUP]
Affiliations
RdRp is a critical component of an RNA virus life cycle. Among coronaviruses, NSP12, along with one copy of NSP7 and two copies of NSP8, forms the RdRp holoenzyme and exhibits polymerase activity. While coronavirus RNA replication is sufficiently understood, the interplay among these NSPs and its influence on RNA binding and nascent strand synthesis remains poorly understood. Here, we reconstituted a functional RdRp holoenzyme using recombinant SARS-CoV-2 NSP12, NSP7, and NSP8 in vitro. Molecular interactions among NSPs and their effect on the polymerase activity were investigated, wherein NSP12 alone exhibited notable activity, which was further enhanced by the presence of both NSP7 and NSP8. The presence of only one cofactor, either NSP7 or NSP8, completely inhibited NSP12 activity and led to RNA template detachment. Computational analyses of different NSP12 complexes suggested that binding of NSP7 or NSP8 alone to NSP12 constricts the RNA entry channel, which was higher in the presence of NSP8, making it inappropriate for RNA entry/binding. We conclude that NSP7 and NSP8 together synergize to enhance the NSP12 activity, but antagonize when alone. These findings have implications for novel drug development, and compounds inhibiting NSP7 or NSP8 interactions with NSP12 can be lethal to coronavirus replication.
Keywords: MT: Oligonucleotides: Therapies and Applications; NSP12; NSP7; NSP8; RNA entry channel; RdRp; SARS-CoV-2; constriction; holoenzyme; replication shut-off; replication-transcription complex.
. 2025 Jan 16;36(1):102452.
doi: 10.1016/j.omtn.2025.102452. eCollection 2025 Mar 11. Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7
Deepa Singh[SUP] 1 [/SUP], Tushar Kushwaha[SUP] 1 [/SUP], Rajkumar Kulandaisamy[SUP] 1 [/SUP], Vikas Kumar[SUP] 1 [/SUP], Kamal Baswal[SUP] 1 [/SUP], Saras H Tiwari[SUP] 1 [/SUP], Arkadyuti Ghorai[SUP] 1 [/SUP], Manoj Kumar[SUP] 1 [/SUP], Saroj Kumar[SUP] 1 [/SUP], Soumya De[SUP] 2 [/SUP], Aparoy Polamarasetty[SUP] 3 [/SUP], Deepak Sehgal[SUP] 4 [/SUP], Madhumohan R Katika[SUP] 5 [/SUP], Suresh Gadde[SUP] 6 [/SUP], Marceline Côté[SUP] 7 [/SUP], Sarala R Kayampeta[SUP] 8 [/SUP], Mohan Babu Appaiahgari[SUP] 8 9 [/SUP], Krishna K Inampudi[SUP] 1 [/SUP]
Affiliations
- PMID: 39944792
- PMCID: PMC11816038
- DOI: 10.1016/j.omtn.2025.102452
RdRp is a critical component of an RNA virus life cycle. Among coronaviruses, NSP12, along with one copy of NSP7 and two copies of NSP8, forms the RdRp holoenzyme and exhibits polymerase activity. While coronavirus RNA replication is sufficiently understood, the interplay among these NSPs and its influence on RNA binding and nascent strand synthesis remains poorly understood. Here, we reconstituted a functional RdRp holoenzyme using recombinant SARS-CoV-2 NSP12, NSP7, and NSP8 in vitro. Molecular interactions among NSPs and their effect on the polymerase activity were investigated, wherein NSP12 alone exhibited notable activity, which was further enhanced by the presence of both NSP7 and NSP8. The presence of only one cofactor, either NSP7 or NSP8, completely inhibited NSP12 activity and led to RNA template detachment. Computational analyses of different NSP12 complexes suggested that binding of NSP7 or NSP8 alone to NSP12 constricts the RNA entry channel, which was higher in the presence of NSP8, making it inappropriate for RNA entry/binding. We conclude that NSP7 and NSP8 together synergize to enhance the NSP12 activity, but antagonize when alone. These findings have implications for novel drug development, and compounds inhibiting NSP7 or NSP8 interactions with NSP12 can be lethal to coronavirus replication.
Keywords: MT: Oligonucleotides: Therapies and Applications; NSP12; NSP7; NSP8; RNA entry channel; RdRp; SARS-CoV-2; constriction; holoenzyme; replication shut-off; replication-transcription complex.