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

Abstract

The RNA 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, base pairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interface 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 (RdRp)). 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 multi-angle light scattering coupled SAXS (SEC-MALS-SAXS) we defined which combination of components form transient or stable complexes. We used contrast matching neutron scattering 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.

Development of Monoclonal Antibodies to Detect for SARS-CoV-2 Proteins

“…To further characterize these mAbs, we focused on the IgGs binding to the key viral proteins Nsp1, Nsp8, Nsp12 and Orf3b for their direct involvement into host translation shutdown [6] , viral replication [3] , [12] and immune antagonism [13] . We performed western blot (WB) analysis to further evaluate the ability of the mAbs to specifically detect each respective protein.…”

“…Two-thirds of the 5’ end of the viral genome encode Non-structural proteins (Nsp) as a polyprotein, which self-cleaves by viral proteases and contributes to virus replication [1] . For example, Nsp1 has a role in translational l shutdown and evasion of host immune response [2] , and Nsp12 is the RNA-dependent RNA polymerase, which is essential for virus replication [3] . The last one-third of the genome encodes the structural envelope (E), membrane (M), and nucleocapsid (N) proteins, in addition to the non-essential accessory proteins.…”

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins

“…33717). Recombinant nsp7 protein was detected of the expected size (10 kDa) and of higher molecular weight, probably due to its reported dimerization [17]. Analysis of antibodies raised against ORF3a showed binding to the ORF3a peptide conjugated to the bovine serum albumin (BSA) carrier, and thus, protein band corresponding to the molecular weight of ORF3a could not be detected.…”

Structural biology of SARS-CoV-2: open the door for novel therapies

“… 207 , 208 The C-terminus of Nsp8 comprises five α-helices and one four-stranded antiparallel β-sheet, and its topology resembles a golf club. 209 The SARS-CoV-2 Nsp7-Nsp8 complex forms a hetero-tetramer [PDB ID 7JLT and 7DCD] 204 , 206 (Fig. 6b ), in contrast to the hexadecameric structure of SARS-CoV Nsp7-Nsp8.…”