Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis

Abstract

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo . Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection.

Author summary: Since its emergence, SARS-CoV-2 has continued to adapt for human infection resulting in the emergence of variants with unique genetic profiles. Most studies of genetic variation have focused on spike, the target of currently available vaccines, leaving the importance of variation elsewhere understudied. Here, we characterize a highly variable motif at residues 203-205 in nucleocapsid. Recreating the prominent nucleocapsid R203K+G204R mutation in an early pandemic background, we show that this mutation is alone sufficient to enhance SARS-CoV-2 replication and pathogenesis. We also link augmentation of SARS-CoV-2 infection by the R203K+G204R mutation to its modulation of nucleocapsid phosphorylation. Finally, we characterize an analogous alanine double substitution at positions 203-204. This mutant was found to mimic R203K+G204R, suggesting augmentation of infection occurs by disrupting the ancestral sequence. Together, our findings illustrate that mutations outside of spike are key components of SARS-CoV-2's adaptation to human infection.

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.

Fig 2.. The KR mt enhances SARS-CoV-2 pathogenesis and fitness.

(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. (F) Schematic of competition/transmission experiment. (G-H) 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 (G) and recipient (H) samples. 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. For competition studies, individual replicates are graphed as points, while bars represent the mean. Statistical significance was between WT and the KR mt was determined by student’s T-Test with p≤0.05 (*), p≤0.01 (**), and p≤ 0.001 (***). Grey dotted lines are equal to LOD.

Fig 3.. The KR mt increases levels of viral RNA and antigen.

(A) Full-length and subgenomic 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 4.. 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 prior to and during infection. Bars are the mean titer ± s.d. (n=4). Significance indicates a change in mean titer difference and was determined by student’s T-Test with p≤0.05 (*) and p≤0.01 (**).

Fig 5.. 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). Graphs 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.