SARS-CoV-2 variant spike and accessory gene mutations alter pathogenesis

Abstract

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the spike protein. Although these differences in spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome that may also affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through interference with innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other nonspike mutations on SARS-CoV-2 pathogenesis, we synthesized both viruses possessing deletions in the accessory genes as well as viruses where the WA-1 spike is replaced by each variant spike gene in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to both WA-1 and the full variant viruses. Our work has revealed that the accessory proteins contribute to SARS-CoV-2 pathogenesis and the nonspike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host. This work suggests that while spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may alter clinical disease presentation.

Keywords: SARS-CoV-2; coronavirus; mouse; pathogenesis; variant.

Fig. 1.

Assembly of infectious clone genomes of SARS-CoV-2. (A) The genome of WA1 was assembled from sequence-validated overlapping (colored ends) DNA fragments (1a-1, 1a-2, 1a-3, 1b-1, 1b-2, S, AP; blue lines) by TAR in yeast. The infectious clone genomes can be maintained in yeast and E. coli by a YCpBAC vector. The infectious clone genome, which is flanked by I-SceI sites, is driven by a CMV promoter (P_cmv) and has a hepatitis delta virus ribozyme sequence (Hdz) as well as a BGH terminator (Term) at the 3′ end of the genome. SARS-CoV-2 WA1 genomes containing either spike variants or accessory ORF deletions were assembled from a mix of unmodified and appropriate modified DNA fragments. (B) PCR amplifications of assembly junctions (J1to J10) to confirm a full-length genome in E. coli. M, 2-log marker.

Fig. 2.

WA-1 accessory deletion viruses in 12-wk-old K18-hACE2 mice. (A) Supernatant titers of VeroE6 cells infected with an M.O.I. of 0.01 of accessory deletion viruses with supernatant pulled at 0, 6, 24, 48, 72, and 96 h and titered by plaque assay. (B) Supernatant titers of A549-ACE2 cells infected with an M.O.I. of 0.01 of accessory deletion viruses with supernatant pulled at 0, 6, 24, 48, 72, and 96 h and titered by plaque assay. (C) Percent starting weight on days 0 to 4 of K18-hACE2 mice infected with 1e3 pfu of each accessory deletion virus. (D) Lung viral titers of mice euthanized on day 2 and day 4 by pfu/g lung. (E) Brain viral titers of mice euthanized on day 2 and day 4 by pfu/g brain. (F) Lung viral loads of mice euthanized on day 2 and day 4 by qPCR for Rdrp. (G) Brain viral loads of mice euthanized on day 2 and day 4 by qPCR for Rdrp. LOD = Limit of Detection. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005. Error bars are standard deviation.

Fig. 3.

Lung pathology of 12-wk-old K18-hACE2 mice infected with accessory deletion viruses of WA-1. (A) H&E-stained sections of the lungs. (B) Pathological scoring of the lungs. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005. Error bars are standard deviation.

Fig. 4.

Variant spikes in WA-1 viruses in 12-wk-old K18-hACE2 mice. (A) Supernatant titers of VeroE6 cells infected with an M.O.I. of 0.01 of variant spikes in WA-1 and parent variant viruses, with supernatant pulled at 0, 6, 24, 48, 72, and 96 h and titered by plaque assay. (B) Percent starting weight on days 0 to 4 of K18-hACE2 mice infected with 1e3 pfu of each variant spike in WA-1 virus or the parent variant virus. (C) Lung viral titers of mice euthanized on day 2 and day 4 by pfu/g lung. (D) Brain viral titers of mice euthanized on day 2 and day 4 by pfu/g brain. (E) Lung viral loads of mice euthanized on day 2 and day 4 by qPCR for Rdrp. (F) Brain viral loads of mice euthanized on day 2 and day 4 by qPCR for Rdrp. LOD = Limit of Detection. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005. Error bars are standard deviation.

Fig. 5.

Lung pathology of 12-wk-old K18-hACE2 mice infected with variant spikes in WA-1 and parent variants. (A) H&E-stained lung sections. (B) Pathological scoring of the lungs.

Fig. 6.

Variant spikes in WA-1 viruses in 12-wk-old BALB/c mice. (A) Percent starting weight on day 0 to 4 of BALB/c mice infected with 1e4 pfu of each variant spike in WA-1 virus. (B) Lung viral titers of mice euthanized on day 2 and day 4 by pfu/g lung. (C) Lung viral titers of mice euthanized on day 2 and day 4 by qPCR for Rdrp. LOD = Limit of Detection. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005. Error bars are standard deviation.

Fig. 7.

Lung pathology of 12-wk-old BALB/c mice infected with variant spike in WA-1 viruses and parent variants. (A) H&E-stained lung sections. (B) Pathological scoring of the lungs. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005. Error bars are standard deviation.

Fig. 8.

Transcriptional response of mouse lungs after infection with wild-type and accessory deletion viruses. (A) Overview heatmap showing all genes differentially expressed in at least one condition from murine lungs infected with accessory deletion viruses, relative to WA-1 at day 2 (Left) or 4 (Right). Blue indicates higher expression relative to WA-1; red indicates reduced relative expression. A table with all gene names and fold changes is included in SI Appendix, Table S8A; this table includes values for all other heatmaps in this figure. (B) Venn diagrams showing the number of genes for each variant and combinations that are significantly different from WA-1 at day 2 (Top) and day 4 (Bottom). Up-regulated genes are shown in the blue-shaded diagrams (Left) and down-regulated in red (Right). (C) Fold changes of murine ISGs in accessory deletion viruses relative to WA-1. A table with all gene names and fold changes is included in SI Appendix, Table S8B. Conditions with a significant change in mean ISG expression compared to wild-type infection (mean fold change not equal to 0) are indicated by an asterisk (P < 0.05, one-way ANOVA followed by one-way T-test with Bonferroni correction). Error bars are standard deviation. (D) Heatmap showing IL-17–regulated gene expression relative to WA-1. Genes that met our significance threshold (at least twofold change in expression, adjusted P value < 0.05) in a particular condition are outlined in black. (E) Expression of genes in the wound healing signaling pathway in knockout infections relative to WA-1. Genes that met our significance threshold (at least twofold change in expression, adjusted P value < 0.05) in a particular condition are outlined in black.