Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication

Mateusz Wilamowski; Michal Hammel; Wellington Leite; Qiu Zhang; Youngchang Kim; Kevin L. Weiss; Robert Jedrzejczak; Daniel J. Rosenberg; Yichong Fan; Jacek Wower; Jan C. Bierma; Altaf H. Sarker; Susan E. Tsutakawa; Sai Venkatesh Pingali; Hugh M. O'Neill; Andrzej Joachimiak; Greg L. Hura

doi:10.1016/j.bpj.2021.06.006

Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication

Mateusz Wilamowski, Michal Hammel, Wellington Leite, Qiu Zhang, Youngchang Kim, Kevin L. Weiss, Robert Jedrzejczak, Daniel J. Rosenberg, Yichong Fan, Jacek Wower, Jan C. Bierma, Altaf H. Sarker, Susan E. Tsutakawa, Sai Venkatesh Pingali, Hugh M. O'Neill, Andrzej Joachimiak, Greg L. Hura

Research output: Contribution to journal › Article › peer-review

38 Scopus citations

Abstract

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.

Original language	English
Pages (from-to)	3152-3165
Number of pages	14
Journal	Biophysical Journal
Volume	120
Issue number	15
DOIs	https://doi.org/10.1016/j.bpj.2021.06.006
State	Published - Aug 3 2021

Funding

Inspiration for the finalizing of this work came from the story shared by Lawrence Riley's family, representing the hardships faced throughout this pandemic. Research was supported by the U.S. Department of Energy (DOE) Office of Science through the National Virtual Biotechnology Laboratory, a consortium of DOE national laboratories focused on response to coronavirus disease 2019, with funding provided by the Coronavirus CARES Act. The Advanced Light Source is a DOE Office of Science user facility run under contract No. DE-AC02-05CH11231 with the SAXS SIBYLS (12.3.1) beamline supported by the DOE Biological and Environmental Research Integrated Diffraction Analysis Technologies (DOE-BER-IDAT) program, National Cancer Institute Structural Biology of DNA Repair (NCI-SBDR) P01 CA092584, National Institute for General Medical Sciences ALS-ENABLE (P30 GM124169) and United States-Israel Binational Science Foundation (BSF) Grant 2016070. J.W. was supported by the Hatch program of the National Institute of Food and Agriculture, U.S. Department of Agriculture. Bio-SANS is supported by Oak Ridge National Laboratory's (ORNL) Center for Structural Molecular Biology funded by the DOE-BER. EQ-SANS is supported by the Scientific User Facilities Division, DOE Basic Energy Science (DOE-BES). Neutron scattering experiments were performed using the High Flux Isotope Reactor and Spallation Neutron Source, a DOE Office of Science User Facilities operated by ORNL. Preliminary protein expression and purification studies were supported by the ORNL Laboratory Directed Research and Development Program. Further funding for this research was provided by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract HHSN272201700060C. The use of the Structural Biology Center (SBC) beamlines at the Advanced Photon Source is supported by DOE Office of Science and operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Research was supported by the U.S. Department of Energy (DOE) Office of Science through the National Virtual Biotechnology Laboratory , a consortium of DOE national laboratories focused on response to coronavirus disease 2019, with funding provided by the Coronavirus CARES Act . The Advanced Light Source is a DOE Office of Science user facility run under contract No. DE-AC02-05CH11231 with the SAXS SIBYLS (12.3.1) beamline supported by the DOE Biological and Environmental Research Integrated Diffraction Analysis Technologies (DOE-BER-IDAT) program, National Cancer Institute Structural Biology of DNA Repair (NCI-SBDR) P01 CA092584 , National Institute for General Medical Sciences ALS-ENABLE ( P30 GM124169 ) and United States-Israel Binational Science Foundation (BSF) Grant 2016070 . J.W. was supported by the Hatch program of the National Institute of Food and Agriculture, U.S. Department of Agriculture . Bio-SANS is supported by Oak Ridge National Laboratory’s (ORNL) Center for Structural Molecular Biology funded by the DOE-BER . EQ-SANS is supported by the Scientific User Facilities Division, DOE Basic Energy Science (DOE-BES) . Neutron scattering experiments were performed using the High Flux Isotope Reactor and Spallation Neutron Source, a DOE Office of Science User Facilities operated by ORNL. Preliminary protein expression and purification studies were supported by the ORNL Laboratory Directed Research and Development Program . Further funding for this research was provided by the National Institute of Allergy and Infectious Diseases, National Institutes of Health , Department of Health and Human Services , under Contract HHSN272201700060C . The use of the Structural Biology Center (SBC) beamlines at the Advanced Photon Source is supported by DOE Office of Science and operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357 .

Keywords

Crystallography
SANS
SARS-CoV-2
SAXS
Transcription

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.bpj.2021.06.006

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Wilamowski, M., Hammel, M., Leite, W., Zhang, Q., Kim, Y., Weiss, K. L., Jedrzejczak, R., Rosenberg, D. J., Fan, Y., Wower, J., Bierma, J. C., Sarker, A. H., Tsutakawa, S. E., Pingali, S. V., O'Neill, H. M., Joachimiak, A., & Hura, G. L. (2021). Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication. Biophysical Journal, 120(15), 3152-3165. https://doi.org/10.1016/j.bpj.2021.06.006

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title = "Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication",

abstract = "The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.",

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author = "Mateusz Wilamowski and Michal Hammel and Wellington Leite and Qiu Zhang and Youngchang Kim and Weiss, {Kevin L.} and Robert Jedrzejczak and Rosenberg, {Daniel J.} and Yichong Fan and Jacek Wower and Bierma, {Jan C.} and Sarker, {Altaf H.} and Tsutakawa, {Susan E.} and Pingali, {Sai Venkatesh} and O'Neill, {Hugh M.} and Andrzej Joachimiak and Hura, {Greg L.}",

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doi = "10.1016/j.bpj.2021.06.006",

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Wilamowski, M, Hammel, M, Leite, W , Zhang, Q, Kim, Y, Weiss, KL, Jedrzejczak, R, Rosenberg, DJ, Fan, Y, Wower, J, Bierma, JC, Sarker, AH, Tsutakawa, SE, Pingali, SV , O'Neill, HM, Joachimiak, A & Hura, GL 2021, 'Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication', Biophysical Journal, vol. 120, no. 15, pp. 3152-3165. https://doi.org/10.1016/j.bpj.2021.06.006

TY - JOUR

T1 - Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication

AU - Wilamowski, Mateusz

AU - Hammel, Michal

AU - Leite, Wellington

AU - Zhang, Qiu

AU - Kim, Youngchang

AU - Weiss, Kevin L.

AU - Jedrzejczak, Robert

AU - Rosenberg, Daniel J.

AU - Fan, Yichong

AU - Wower, Jacek

AU - Bierma, Jan C.

AU - Sarker, Altaf H.

AU - Tsutakawa, Susan E.

AU - Pingali, Sai Venkatesh

AU - O'Neill, Hugh M.

AU - Joachimiak, Andrzej

AU - Hura, Greg L.

PY - 2021/8/3

Y1 - 2021/8/3

N2 - The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.

AB - The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.

KW - Crystallography

KW - SANS

KW - SARS-CoV-2

KW - SAXS

KW - Transcription

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U2 - 10.1016/j.bpj.2021.06.006

DO - 10.1016/j.bpj.2021.06.006

M3 - Article

C2 - 34197805

AN - SCOPUS:85110415656

SN - 0006-3495

VL - 120

SP - 3152

EP - 3165

JO - Biophysical Journal

JF - Biophysical Journal

IS - 15

ER -

Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication

Abstract

Funding

Keywords

UN SDGs

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