SARS-CoV-2 NSP12 associates with TRiC and the P323L substitution acts as a host adaption

Abstract

SARS-CoV-2 has caused a worldwide health and economic crisis. During the course of the pandemic, genetic changes occurred in the virus, which have resulted in new properties of the virus-particularly around gains in transmission and the ability to partially evade either natural or vaccine-acquired immunity. Some of these viruses have been labeled Variants of Concern (VoCs). At the root of all VoCs are two mutations, one in the viral spike protein that has been very well characterized and the other in the virus polymerase (NSP12). This is the viral protein responsible for replicating the genome. We show that NSP12 associates with host cell proteins that act as a scaffold to facilitate the function of this protein. Furthermore, we found that different variants of NSP12 interact with host cell proteins in subtle and different ways, which affect function.

Keywords: NSP12; P323L; SARS-CoV-2; TRiC; phosphatase.

Fig 1

Expression of EGFP, EGFP-NSP12_P323, NSP12_P323-EGFP, EGFP-NSP12_L323, and NSP12_L323-EGFP in HEK-293T cells. (A) Schematic diagram of the general methodology and approach used in this study. The NSP12 fusion proteins were expressed in HEK-293T cells and immunoprecipitated using a GFP-Trap. LC-MS/MS was used to identify the interactome, and western blot was used to validate key interactions. The functional implications of these findings were investigated using inhibitors or ablating the protein/function of interest, and the effect on viral biology was quantified. (B) Expression of EGFP, EGFP-NSP12_P323, NSP12_P323-EGFP, EGFP-NSP12_L323, and NSP12_L323-EGFP in HEK-293T cells was confirmed by immunofluorescence microscopy with DAPI (blue) staining the nucleus. Scale bar, 90 μM. (C) Immunoblot analysis of input (I) and elute (E) EGFP, EGFP-NSP12_P323, NSP12_P323-EGFP, EGFP-NSP12_L323, and NSP12_L323-EGFP by western blot. (D) Eluted samples for the mass spectrometry analysis were run on SDS-PAGE and stained with a silver stain, showing a difference in protein profile.

Fig 2

Analysis of selected significant cellular proteins that potentially interact with NSP12_L323. Potential protein-protein interaction networks of NSP12_L323 were derived using Cytoscape from the mass spectrometry interactome data. Multiple proteins were found to be associated with NSP12_L323. In this simplified figure, the highly enriched chaperonin-containing TCP1 (TRiC/CCT complex) is involved in the folding and assembly of proteins associated with the NSP12_L323 interactome. A few proteins were identified to be enhanced in the NSP12_L323 interactome (STRN3 and PP2R1A, which are part of the PP2A phosphatase family). Full interactome data for NSP12_P323 and NSP12_L323 are presented in Fig. S1–S4 and Tables S1 to S4.

Fig 3

Validation of protein-protein interactions between EGFP tagged NSP12_P323 or NSP12_L323 and the host proteins in HEK-293T cells. (A) Transfection and pulldowns were repeated with EGFP, EGFP tagged NSP12_P323, and NSP12_L323 at the C- and N-terminus for the detection of CCT1, CCT5, CCT7, CCT8, PP2Aa, and STRN3 (I = input, E = elute). (B) Reverse pulldowns for CCT1, CCT5, CCT7, and STRN3 and detection of EGFP, EGFP-NSP12_P323, NSP12_P323-EGFP, EGFP-NSP12_L323, and NSP12_L323-EGFP with a specific antibody against SARS-CoV-2 (SCV2) NSP12. (C) Agarose gel electrophoresis showing that the treatment with RNase (+RNase) removed host RNA from cell lysate compared to untreated (−RNase). (D) Samples from panel (C) were run on SDS-PAGE for the detection of host proteins in the absence of RNA using a forward pull-down approach.

Fig 4

Determining the effect of TRiC/CCT inhibitor HSF1A on the recombinant virus P323 and L323 replication. (A and C) ACE2-A549 cells were pre-treated with either DMSO (control) or HSF1A (25, 50, or 100 µM) for 4 hours before being mock-infected or infected with recombinant viruses expressing either NSP12_P323 or NSP12_L323 viruses (MOI of 0.1) and then maintained in DMSO or HSF1A. At 24 hours after infection, gRNA and sgRNA levels were measured by qRT-PCR and normalized to β-actin. At the same time, whole cell lysate samples were collected, and the expression of nucleocapsid and CCT proteins was detected using western blot analysis with specific antibodies. GAPDH was used as a loading control. ImageJ was used to assess the expression of the nucleocapsid protein. Viral protein expressions were normalized to GAPDH. (B and D) ACE2-A549 cells were pre-treated for 4 hours with DMSO (control) or for 4 hours with HSF1A (25, 50, and 100 µM) before being mock-infected or infected with reverse genetically created P323 or L323 viruses at an MOI of 0.1. Infected cells were maintained in media containing 0.05% DMSO or the previous concentrations of HSF1A for 24 hours. Viral titers were determined by plaque assay. For the plaque assays, representative image data shown are from one experiment. (E) HEK-293T cells were transfected with a plasmid containing EGFP as a control, NSP12_P323-EGFP, or NSP12_L323-EGFP, respectively. Cells were then treated at 4 hours post-transfection with either DMSO (0.05%) or HSF1A (50 µM). Whole cell lysates were collected, and western blot analysis was performed to detect the abundance of EGFP, NSP12_P323-EGFP, and NSP12_L323-EGFP proteins. GAPDH was used as a loading control. **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. All error bars represent the standard deviation. Data shown in panels A–D represent the mean of three independent experiments.

Fig 5

The impact of STRN3 and PP2Aa suppression on the replication of recombinant P323 and L323 viruses. (A and C) ACE2-A549 cells were mock-transfected or transfected with non-targeting siRNA (NTC), si-GAPDH (to monitor transfection efficacy), si-STRN3, or si-PP2Aa for 48 hours. Subsequently, the ACE2-A549 cells were infected with recombinant viruses expressing either NSP12_P323 or NSP12_L323 (MOI = 1 for 24 hours). RNA samples were extracted, and gRNA and sgRNA levels of the recombinant viruses were determined by qRT-PCR and normalized to β-actin. At the same time, whole cell lysate samples were collected, and the expression of nucleocapsid, STRN, and PP2Aa proteins was detected using western blot analysis. GAPDH was used as a loading control. ImageJ was used to assess the expression of the nucleocapsid protein. Viral protein expression was normalized to GAPDH. (B and D) Supernatants, from the experiments described in panels A and C, were collected and viral titers were determined by plaque assay. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Dunnett’s multiple comparison test. Data shown in panels A–D represent the mean of three independent experiments. All error bars represent the standard deviation. For the plaque assays, representative image data shown are from one experiment.