Fig 1.
The KR mt enhances SARS-CoV-2 replication.
(A) Amino acid frequencies for nucleocapsid residues 203–205 in SARS-CoV-2 sequences reported to the GISAID, binned by month of collection and graphed as percent of total sequences reported during that period. (B-C) Viral titer from Vero E6 (B) or Calu-3 2b4 cells (C) infected with WA-1 (black) or the alpha variant (red) at an MOI of 0.01 (n≥6). (D). Schematic of the SR domain of SARS-CoV-2 nucleocapsid. Variable residues are displayed as red text within the sequence of their corresponding lineages. Phosphorylated residues are indicated by a †. (E) Schematic of the SARS-CoV-2 genome, showing the creation of the KR mutation and the replacement of ORF7 with the mNG reporter protein. (F-G) Viral titer of Vero E6 (F) or Calu-3 2b4 (G) cells infected with WT (black) or the KR mt (red) at a MOI of 0.01 (n = 9). (H) Competition assay between WT (gray) and KR mt (red) in Vero E6 or Calu-3 2b4 cells infected at a 1:1 input ratio with an MOI of 0.01 (n = 6). Titer data are the mean ± s.d. For competition, individual replicates are graphed as points, while the mean percentage of each virus is displayed as a bar graph. Statistical significance was determined by two-tailed student’s T-test with p≤0.05 (*), p≤0.01 (**), and p≤ 0.001 (***). Grey dotted lines are equal to LOD. Schematic in (D) generated using Biorender.com.
Fig 2.
The KR mt enhances SARS-CoV-2 pathogenesis.
(A) Schematic of the infection of hamsters with SARS-CoV-2. (B-E) Three- to four-week-old male hamsters were mock-infected (gray) or inoculated with 104 PFU of WT SARS-CoV-2 (black) or the KR mt (red). Animals were then monitored for weight loss (B). On days 2 and 4 post infection, viral titers in the lung (C), trachea (D), and from nasal washes (E) were determined. For weight loss data, mean percent weight loss was graphed ± s.e.m. For titer data, individuals were graphed with means ± s.d. indicated by lines. Statistical significance between WT and the KR mt was determined by student’s T-Test with 0p≤0.05 (*), p≤0.01 (**), and p≤ 0.001 (***). Grey dotted lines are equal to LOD. Schematic in (A) was generated by authors using Biorender.com.
Fig 3.
Lung histopathology in hamsters infected with WT and KR mt SARS-CoV-2.
Lung tissue was harvested, fixed, and 5 μm sections cut from mock, WT SARS-CoV-2, or KR mt-infected hamsters and stained with hematoxylin and eosin. (A) Normal bronchus, pulmonary artery, and alveoli in mock infection on day 4 (20X). (B) Bronchiolitis, peribronchiolitis, interstitial pneumonia, and edema surrounding branch of the pulmonary artery at day 4 in hamsters infected with WT virus (10X). (C) Severe bronchiolar cytopathic effect, interstitial pneumonia, cytopathic alveolar pneumocytes, alveoli containing mononuclear cells and red blood cells at day 4 in hamsters infected with KR mt. This lesion extended over numerous fields (10X). (D) Normal respiratory bronchiole, alveolar ducts, and alveolar sacs in mock infection on day 10 (20X). (E) Interstitial pneumonia adjacent to a bronchus at day 10 in hamsters infected with WT (20X). (F) Bronchiolar epithelial cytopathic effect, peribronchiolitis, focal interstitial pneumonia, branch of pulmonary artery with surrounding edema and mononuclear cell infiltration of endothelium at day 10 in a hamster infected with the KR mt (20X). Shown are representative images typical of data gathered from 5 animals from each group.
Fig 4.
The KR mt enhances SARS-CoV-2 fitness in vivo.
(A) Schematic of competition/transmission experiment. (B-C) Three- to four-week-old male donor hamsters were inoculated with 104 PFU of WT SARS-CoV-2 and the KR mt at a 1:1 ratio. On day 1 of the experiment, donor and recipient hamsters were co-housed for 8 hours, then separated, and the donor hamsters underwent nasal washing. On day 2, recipient hamsters were nasal washed. Hamsters were then monitored and nasal washes and lung tissue harvested on days 4 (donors) and 5 (recipients). The ratio of WT (gray) to KR mt (red) was then determined by NGS of all donor (B) and recipient (C) samples. Individual replicates are graphed as points, while bars represent the mean. Schematic in (A) generated using Biorender.com.
Fig 5.
The KR mt increases levels of viral RNA and antigen.
(A) Full-length and sub-genomic transcript levels 24 hours post infection from Calu-3 2b4 cells infected at an MOI of 0.01 with WT SARS-CoV-2 or the KR mt (n = 3). Transcript levels were normalized to 18S ribosomal RNA and graphed as the fold change in the KR mt relative to WT (B-C) Three- to four-week old male hamsters were inoculated with PBS (mock) or 104 PFU of WT or the KR mt. On days 2 and 4 post infection, lung tissue was harvested. The levels of full-length SARS-CoV-2 RNA in WT and KR mt infected animals (n = 5) (B). Representative SARS-CoV-2 antigen staining (anti-Nucleocapsid) of lung tissue from mock, WT, or KR mt infected animals (n = 5) (C). For in vitro transcripts, bars are mean titer ± s.d. For in vivo RNA, individual replicates are graphed with means ± s.d. indicated by lines. Significance was determined by student’s T-Test with p≤0.05 (*) and p≤0.01 (**).
Fig 6.
The KR mt increases N phosphorylation to enhance infection.
(A) Schematic of phosphate-affinity (PA) SDS-PAGE. (B) Whole cell lysates from Calu-3 2b4 cells infected with SARS-CoV-2 WA-1-mNG (WT), the KR mt, WA-1, alpha, beta, and kappa variants were analyzed by PA SDS-Page (top) or standard SDS-PAGE (bottom) followed by blotting with an N-specific antibody (n = 3). (C) Whole cell lysates from Vero E6 cells infected with WT or the KR mt and analyzed by PA-SDS-Page (top) or standard SDS-Page (bottom) followed by blotting with an N-specific antibody (n = 3). (D) Schematic of phosphorylation by GSK-3 of the SR domain of SARS-CoV-2 N. Residues targeted by GSK-3 are indicated with arrows and priming residues designated by a ‘*’. (E) Viral titer 48 hours post infection from Calu-3 2b4 cells infected with WT SARS-CoV-2 (gray) or the KR mt (red) at an MOI of 0.01. Cells were treated with the indicated concentrations of kenpaullone 1 hour prior to and during infection. Brackets and numbers indicate the mean log-titer difference between WT and the KR mt. Bars are the mean titer ± s.d. (n = 4). Significance indicates a change in mean titer difference compared to untreated cells and was determined by student’s T-Test with p≤0.05 (*) and p≤0.01 (**). Schematic in (A) and (D) generated using Biorender.com.
Fig 7.
The AA mt mimics the KR mt’s enhancement of SARS-CoV-2 infection.
(A-B). Viral titer from Vero E6 (A) or Calu-3 2b4 (B) cells infected at an MOI of 0.01 with WT (black) or the AA mt (green) (n = 9). (C) Competition assay between WT (gray) and the AA mt (green) in Vero E6 and Calu-3 2b4 cells at a 1:1 input ratio and an MOI of 0.01 (n = 6). (D) Full-length and subgenomic transcript levels 24 hours post infection from Calu-3 2b4 cells infected with WT or the AA mt. Transcripts were normalized to 18S ribosomal RNA and graphed as fold change in the AA mt relative to WT (n = 3). (E) Whole cell lysates from Calu-3 2b4 cells infected with WT or the AA mt and analyzed by PA SDS-Page (top) and standard SDS-Page (bottom) followed by blotting with an N-specific antibody (n = 3). (F) Viral titer 48 hours post infection from Calu-3 2b4 cells infected with WT (gray) or the AA mt (green) at an MOI of 0.01. Cells were treated with the indicated concentrations of kenpaullone prior to and during infection (n = 4). Brackets and numbers indicate the mean log-titer difference between WT and the KR mt. Bars represent mean titer ± s.d. Significance was determined by two-tailed student’s t-test with p≤0.01 (**) and p≤ 0.001 (***). Grey dotted lines are equal to LOD.