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Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis [1]

['Bryan A. Johnson', 'Department Of Microbiology', 'Immunology', 'University Of Texas Medical Branch', 'Galveston', 'Texas', 'United States Of America', 'Yiyang Zhou', 'Department Of Biochemistry', 'Molecular Biology']

Date: 2022-08 

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.

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.

Funding: Research was supported by grants from NIAID of the NIH (R01-AI153602 and R21-AI145400 to VDM; R24-AI120942 (WRCEVA) to SCW). ALR was supported by the Centers for Disease Control and Prevention (Contract 200-2021-11195). Both ALR and BAJ were supported by the Institute of Human Infection and Immunity at UTMB COVID-19 Research Fund. Research was also supported by STARs Award provided by the University of Texas System to VDM. Trainee funding provided by NIAID of the NIH to MNV (T32-AI060549) and to CS (T32-AI007526). BAJ was supported by the James W. McLaughlin Fellowship Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we utilize our reverse genetic system [ 8 , 9 ] to generate the KR nucleocapsid mutation in the ancestral WA-1 strain of SARS-CoV-2. This change alone was sufficient to increase viral replication in respiratory cells and exhibited enhanced fitness in direct competition studies with wild type (WT) SARS-CoV-2. In the hamster model, the KR mutant (mt) enhanced pathogenesis and outcompeted WT in direct competition. We subsequently found that the KR mt corresponds with increased viral RNA both in vitro and in vivo. Notably, we observed that the KR mt resulted in augmented nucleocapsid phosphorylation relative to WT SARS-CoV-2; similar increases in N phosphorylation were also seen in the alpha and kappa variants. Importantly, the KR mt was more resistant to GSK-3 kinase inhibition relative to WT SARS-CoV-2, suggesting that the KR mt alters interactions with N targeting kinases. Finally, an analogous alanine double substitution mutant at position 203+204 (AA mt) also increased fitness and altered phosphorylation relative to WT SARS-CoV-2. Together, these results suggest that disruption of the ancestral “RG” motif in nucleocapsid augments infection, fitness, and pathogenesis of SARS-CoV-2.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS)-CoV-2 is the most significant infectious disease event of the 21st century [ 1 , 2 ]. Since its initial expansion, SARS-CoV-2 has continued to adapt for human infection and transmission, resulting in several variants of concern [ 3 ]. While most mutations occur within a single lineage, a small number are shared across multiple variants [ 4 ]. Spike mutations have dominated SARS-CoV-2 variant research, owing to concerns that they enhance replication, augment transmission, or allow escape from immunity [ 4 ]. However, less attention has been focused on mutations outside spike, despite the existence of other “mutational hotspots” in the genome [ 4 ]. The SARS-CoV-2 nucleocapsid (N) gene is one hotspot for coding mutations, particularly at amino acid residues 203–205 within its serine rich (SR) domain [ 5 ]. Three prominent mutations occur in this region including R203K+G204R, a double substitution (KR mt) present in the alpha, gamma, and omicron variants; T205I present in the beta variant; and R203M that occurs in the kappa and delta variants [ 6 , 7 ]. Together, this genetic variation and convergent evolution in residues 203–205 suggests positive selection in this motif of N.

Results

The KR and R203M mts alone are sufficient to increase viral replication Because the alpha variant exhibited increased replication in Calu-3 2b4 cells, we hypothesized that the KR mt may enhance infection. To study the effects of the KR mt in isolation, we utilized a SARS-CoV-2 reverse genetic system to recreate the KR mt in a WA-1 background (Fig 1E) [8,9]. In addition, due to gain-of-function concerns, the accessory protein ORF7 was replaced with mNeonGreen (mNG), which reduces but does not eliminate disease in golden Syrian hamsters (S2 Fig). After recovery of recombinant virus, we evaluated the KR mt’s effects on SARS-CoV-2 replication. Both Vero E6 and Calu-3 2b4 cells were infected at a low MOI (0.01) with either SARS-CoV-2 WA-1 harboring the mNG reporter (herein referred to as WT) or the KR mt and viral titer monitored for 48 hpi. Like the alpha variant, the KR mt grew to a lower titer at 24 hpi, but had an equivalent endpoint titer in Vero E6 cells (Fig 1F). Notably, in Calu-3 2b4 cells, the KR mt had increased viral titer at both 24 and 48 hpi compared to WT SARS-CoV-2 (Fig 1G). These data suggest the KR mt in nucleocapsid alone is sufficient to enhance viral replication. Next, we wanted to examine the ability of other variant mutations in the 203–205 motif of nucleocapsid to enhance SARS-CoV-2 replication. Using our reverse genetic system, we recreated the R203M mutation in a WA-1 mNG background and evaluated its replication in Vero E6 and Calu-3 2b4 cells (S3 Fig). Interestingly, like the KR mt, in Vero E6 cells the R203M mutant grew to lower titer at 24 hpi but had a similar endpoint titer compared to WT SARS-CoV-2 (S3B Fig). In Calu-3 2b4 cells, the R203M mutant again mimicked the KR mt, growing to a higher titer than WT at 24 and 48 hpi (S3C Fig). These data suggest that like the KR mt, the R203M mutation alone is sufficient to enhance SARS-CoV-2 replication.

The KR mt enhances SARS-CoV-2 fitness during direct competition We next determined if the KR mt increases SARS-CoV-2 fitness using competition assays, which offer increased sensitivity compared to individual culture experiments [13]. WT SARS-CoV-2 and the KR mt were directly competed by infecting Vero E6 and Calu-3 2b4 cells at a 1:1 plaque forming unit ratio. Twenty-four hpi, total cellular RNA was harvested and the ratio of WT to KR mt genomes determined by next generation sequencing (NGS) [14]. Consistent with the kinetic data, WT outcompeted the KR mt at a ratio of ~4:1 in Vero E6 cells (Fig 1H). In contrast, the KR mt outcompeted WT at a ratio of ~10:1 in Calu-3 2b4 cells (Fig 1H). These data indicate that the KR mt has a fitness advantage over WT SARS-CoV-2 in Calu-3 2b4, but not Vero E6 cells.

The KR mt increases viral RNA and antigen levels The CoV N protein has previously been shown to play a role in the transcription of viral RNA [16–19]. To evaluate changes in viral RNA levels during infection with the KR mt, we performed RT-qPCR to measure levels of SARS-CoV-2 transcripts following infection of Calu-3 2b4 cells (S4 Fig). Compared to WT infected cells, the KR mt had a >32-fold increase in levels of all viral transcripts, demonstrating a broad increase in SARS-CoV-2 RNA levels (Fig 5A). This finding is not surprising considering the increased viral titer observed in Calu-3 2b4 cells (Fig 1G). We subsequently examined the levels of full-length viral RNA in the lungs of infected hamsters (Fig 5B), finding a significant increase at 2- and 4- dpi. In contrast, the virus lung titer at both time points had no significant difference (Fig 2C), indicating that the KR mt increases the levels of viral RNA despite not increasing titer. Further extending our analysis, we explored in vivo SARS-CoV-2 N antigen staining in the lungs of infected animals (Fig 5C). KR mt infected animals showed increased viral antigen staining and substantially larger lesion size compared to WT. Additionally, while lesions in WT infected animals were mostly limited to cells adjacent to the airways, KR mt infected animals showed staining throughout the parenchyma. Together, these data indicate despite having no effect on lung titer, the KR mt leads to increased viral RNA accumulation and greater virus spread in the lung compared to WT SARS-CoV-2. PPT PowerPoint slide

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TIFF original image Download: 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 (**). https://doi.org/10.1371/journal.ppat.1010627.g005

The KR mt increases phosphorylation of SARS-CoV-2 N Having confirmed a role in viral RNA transcription, we next considered how mutations in residues 203–205 of nucleocapsid’s SR domain might provide an advantage for SARS-CoV-2. Nsp3, the multi-faceted viral protease, interacts with the SR domain to increase viral transcription [18–22]. Importantly, this interaction is governed by phosphorylation of the SR domain, which is targeted by the SRPK, GSK-3, and Cdk1 kinases [5,22–28] Given the proximity to key priming residues required for GSK-3 mediated phosphorylation (Fig 6D) [28], we hypothesized that the KR mt alters nucleocapsid phosphorylation. To overcome the lack of phospho-specific antibodies for nucleocapsid, we used phosphate-affinity SDS-PAGE (PA SDS-PAGE). PA SDS-PAGE utilizes a divalent Zn2+ compound (Phos-Tag) within acrylamide gels that selectively binds to phosphorylated serine, threonine, and tyrosine residues; the bound Zn2+ decreases electrophoretic mobility of a protein proportionally with the number of phosphorylated amino acids [29]. Importantly, if a protein exhibits multiple phosphorylation states, this will cause a laddering effect, with each phospho-species appearing as a distinct band (Fig 6A). PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1010627.g006 To assess the KR mt’s effects on N-phosphorylation, we infected Calu-3 2b4 cells at a MOI of 0.01 and harvested whole cell lysates 48 hpi. Lysates then underwent PA SDS-PAGE followed by western blotting with an N-specific antibody. When analyzed by PA SDS-PAGE, WT SARS-CoV-2 displayed a two-band pattern consisting of a faint upper and prominent lower band, corresponding to a highly phosphorylated and a less phosphorylated species, respectively (Fig 6B, lane 1). In contrast, the KR mt displayed four dark bands of progressively slower mobility, indicating a substantially different phosphorylation pattern (Fig 6B, lane 2). Importantly, all four bands migrated more slowly than the prominent WT band, indicating an overall increase in phosphorylation in the KR mt, which corresponds to increased virus replication (Fig 1G). We next examined N phosphorylation in Vero E6 cells; cells were infected at a MOI of 0.01 and whole cell lysates taken 24 hpi. In Vero E6 cells, WT SARS-CoV-2 exhibited 2-bands of equal strength, indicating a relative increase in phosphorylation compared to Calu-3 2b4 cells (Fig 6C, lane 1). In contrast, the KR mt displayed 3 dark bands with faster mobility relative to WT, indicating a decrease in overall phosphorylation (Fig 6C, lane 2). This reduced phosphorylation corresponds with the replication attenuation seen in this cell type (Fig 1F). Together, these data indicate that the relative level of SARS-CoV-2 nucleocapsid phosphorylation plays a role in virus replication. Given the KR mt’s effects, we next determined if SARS-CoV-2 variants had altered N- phosphorylation. Calu-3 2b4 cells were infected at a MOI of 0.01 with WA-1 or the alpha, beta, or kappa variants and whole cell lysates harvested at 48 hpi. When analyzed by PA SDS-PAGE, WA-1 had a two-band pattern similar to WT SARS-CoV-2, while the alpha variant displayed a four-band pattern with slower mobility similar to that of the KR mt (Fig 6B, lanes 3–4). Interestingly, the mobilities of the alpha variant bands were decreased compared to the KR mt indicating an even higher level of phosphorylation, potentially due to the alpha variant’s additional nucleocapsid mutations at D3L and S235F [6, 7]. While both the beta (T205I) and kappa (R203M) variants also displayed slower electrophoretic mobility compared to WA-1, the beta variant displayed a two-band pattern reminiscent of WA-1 while kappa displayed a laddered pattern similar to the KR mt (Fig 6B, lanes 5–6). Together, these data suggest variant mutations at residues 203–205 result in increased N phosphorylation.

The KR mt does not alter phosphorylation of virion-associated N While CoV N proteins are hyperphosphorylated intracellularly, they are believed to lack phosphorylation within the mature virion [30,31]. Nevertheless, given the ability of the KR mt to augment phosphorylation, we were curious if it influenced virion-associated SARS-CoV-2 N. To examine this, Calu-3 2b4 cells were infected with WT SARS-CoV-2 or the KR mt. 48 hpi, Viral supernatants were taken, clarified, and virions pelleted on a 20% sucrose cushion by ultracentrifugation. Protein recovered from the pellets was then analyzed by PA SDS-PAGE followed by western blotting with an N-specific antibody. Curiously, for both WT and the KR mt, a light upper and dark lower band were detected, indicating some level of N phosphorylation is present in the SARS-CoV-2 virion, albeit at a lower level than intracellular N (S5 Fig). However, the KR mt had no effect on the banding pattern, indicating the KR mt does not affect phosphorylation of mature virions.

The KR mt is more resistant to GSK-3 inhibition Our results indicate that changes in N phosphorylation correlate with differences in virus replication; thus, we sought to modulate N phosphorylation using kinase inhibitors. Prior work has identified two consensus sites for GSK-3 phosphorylation within the SR domain and inhibition of GSK-3 has been shown to reduce SARS-CoV-2 replication (Fig 6D) [28]. Importantly, the KR mt is proximal to the priming residue of the C-terminus GSK-3 consensus site, suggesting it may impact GSK-3 mediated N phosphorylation. Therefore, we examined the impact of GSK-3 inhibition on both WT and the KR mt. Using kenpaullone, a GSK-3 inhibitor, we showed a dose dependent inhibitory effect on both WT and KR mt titer at 48 hpi (Fig 6E). Importantly, GSK-3 inhibition had a greater impact on WT, significantly increasing the mean titer difference between WT and the KR mt from ~4-fold (0.6 log-titer) to ~38-fold (1.6 log-titer) at 10 μM kenpaullone. This suggests that the KR mt is more resistant to GSK-3 inhibition, and that the change at position 203–204 increases affinity of the KR mt for GSK-3.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010627

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