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.
Highlights
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has plagued every populated continent and has been implicated in 3,000,000 deaths at the time of writing
The remdesivir triphosphate is incorporated into newly synthesized RNA by RNA-dependent RNA polymerase (RdRp) and either stalls or interrupts viral RNA polymerization
We propose that Nsp8 is involved in recognizing single-stranded RNA (ssRNA) over ssDNA and guiding ssRNA to or from the replication transcription complex (RTC) complex
Summary
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has plagued every populated continent and has been implicated in 3,000,000 deaths at the time of writing. In the US far, a single drug, remdesivir, has been conditionally approved for targeting the virus. It targets the RNA-dependent RNA polymerase (RdRp) [1]. SARS-CoV-2 RdRp, referred to as nonstructural protein 12 (Nsp12), is the catalytic center of the macromolecular complex frequently referred to as the replication transcription complex (RTC). The RTC is essential for virus replication because it makes copies of genomic and subgenomic RNAs and polymerizes antisense RNA. After maturation, these RNAs serve as mRNAs to produce virus Nsps, which are structural and accessory proteins. The remdesivir triphosphate is incorporated into newly synthesized RNA by RdRp and either stalls or interrupts viral RNA polymerization
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