Nsp3-N interactions are critical for SARS-CoV-2 fitness and virulence

Fig. 1.. Nsp3-S676T substitution attenuates SARS-CoV-2 virulence in mice. (A) Schematic showing the generation of SARS2-N501YMA30-Nsp3-S676T in mice. SARS2-N501YMA30-Nsp3-S676T was generated by passage through mouse lungs as previously described (11). After 30 serial passages, the virus was collected from the lungs and then plaque purified three times on Vero E6 cells. Individual plaques were isolated, and viral genomes were sequenced by Nanopore sequencing. (B) Schematic diagram of SARS-CoV-2 genome (①), adaptive mutations in SARS2-N501YMA30-Nsp3-S676T (②), and SUD (③). The viral proteins containing mutations associated with mouse adaptation are shown in the middle panel (②). SUD-M, SUD-N, and SUD-C are colored purple, teal, and pink. Lower panel: The serine to threonine substitution at position 676 in Nsp3 is shown (③). (C) Percentage of initial weight and survival of BALB/c mice infected with 5,000 PFU of SARS2-N501YMA30 (n = 9 mice) or SARS2-N501YMA30-Nsp3-S676T (n = 7 mice). (D) Predicted structures of SUD of SARS-CoV-2 (Left) and SARS-CoV (Right). Globular domains, including SUD-M, SUD-N, and SUD-C, are colored in purple, teal, and pink, respectively. Linker regions between the…

Fig. 2.. The effect of Nsp3-S676T on viral replication and pathogenesis in mice. BALB/c mice were infected with 5,000 PFU of rMA30 or rMA30Nsp3-S676T. (A) At 2 and 4 dpi, lungs were harvested to evaluate viral titers, levels of subgenomic RNA (sgRNA, N gene) and genomic RNA [gRNA, ORF1 (nsp12)]. LOD: limit of detection. rMA30 (n = 6); rMA30Nsp3-S676T (n = 7). For statistical analysis, in samples with undetectable amounts of virus, we assigned a value of 10 PFU/mL. (B) Gross lung pathology and (C) histopathological analysis of the lungs, harvested at 4 dpi. (n = 3 mice/group). (Scale bars, 430 [Upper] and 86 [Lower] µm, respectively.) (D) Lung edema was quantified as described in Materials and Methods (n = 3 each group). Asterisks indicate pulmonary edema. P values were calculated by a two-tailed, unpaired t test with Welch’s correction. (A) Data are combined from two independent experiments.

Fig. 3.. Nsp3-S676T effect on viral replication in cultured cells. (A) Vero E6 or Calu-3 cells were infected with rWuH-1 or rWuH-1Nsp3-S676T at MOI = 8, and supernatants were harvested at the indicated time points to determine infectious viral titer by plaque assay (Representative of two independent experiments with four replicates/experiment). LOD: limit of detection. (B) Human primary airway epithelial cells (HAEs) from 13 donors were infected with 0.1 MOI of rWuH-1Nsp3-S676T or rWuH-1. At 24 and 48 hpi, PBS was used to elute the released viruses at 37 °C for 30 min. Viral titers were quantified by plaque assay (n = 13). (C) Human MDMs differentiated from human PBMCs (four donors) were infected with rWuH-1 or rWuH-1Nsp3-S676T at an MOI of 1 for 12 or 24 h. The levels of viral sgRNA and gRNA were determined by qRT-PCR (n = 4). A two-tailed, unpaired t test with Welch’s correction (A and C) and a two-tailed, Wilcoxon matched-pairs test (B) were used to assess statistical significance.

Fig. 4.. Nsp3-S676T diminishes the translational stimulatory ability of SUD. (A) Schematic diagram of luciferase reporters. Human beta-globin (HBB), SARS-CoV-2 5′ UTR, or 5′ leader was positioned upstream of the Renilla luciferase gene in pRL-TK. The SARS-CoV-2 3′ UTR was inserted downstream of the Renilla luciferase in plasmids containing the 5′ UTR or 5′ leader. (B and C) Quantification of Renilla luciferase expression. HEK-293T cells were transfected with HBB-5’ UTR or SARS-CoV-2 5′ UTR Renilla-Luc and vector, SUD or S676T-SUD. After 24 h, cells were lysed in 1× lysis buffer for measuring luciferase activity (B, n = 5 replicates, representative of three independent experiments) or in TRIzol to evaluate mRNA levels of luciferase using qRT-PCR (C, n = 4 replicates, representative of two independent experiments). (D) HEK293 cells were transfected with vector, myc-SUD, or myc-S676T-SUD, together with V5-PABP1 and Flag-Paip1. As a control, cells were transfected with vector, myc-SUD, and V5-PABP1. After 24 h, transfected cells were lysed and immunoprecipitated with Pierce™ Anti-Flag Affinity Resin. Intensity of PABP1 or SUD was normalized to Paip1 in the same immunoprecipitation. Intensity…

Fig. 5.. The effect of a compensatory N-S194L mutation on viral virulence. (A) Location of S194L mutation in the N protein. NTD: N-terminal domain. CTD: C-terminal domain. SR: Serine/Arginine rich region. (B) Viral genomic RNA sequence analysis showing the proportion of the C28854T base (N-194L) in three mice after five passages. Three BALB/c mice were challenged with 5,000 PFU of rMA30Nsp3-S676T. At 2 dpi, supernatants from lung homogenates from each mouse were serially passaged four more times in uninfected mice. The passaged virus was then propagated in Calu-3 cells. At 24 hpi, supernatants from infected Calu-3 cells were harvested to isolate viral RNA. Viral genomic RNA from each group was sequenced by Nanopore sequencing and compared to unpassaged rMA30Nsp3-S676T. (C) Weight and survival of BALB/c mice infected with 5,000 PFU of rMA30, rMA30Nsp3-S676T or rMA30Nsp3-S676T;N-S194L (n = 3 for rMA30, n = 4 for rMA30Nsp3-S676T, n = 5 for rMA30Nsp3-S676T; N-S194L) (D) Viral titers and sgRNA levels from BALB/c mice after infection with 5,000 PFU rMA30 (n = 5), rMA30Nsp3-S676T (n = 5) or rMA30Nsp3-S676T;N-S194L (n = 5) infection. (E) Histopathological analysis of mouse lungs at 4 dpi after…

Fig. 6.. N-S194L enhances N protein expression. (A) Expression of N protein. HEK-293T cells were transfected with plasmids encoding Flag-N, Flag-N-S194L, or vector, and cell lysates were subject to analysis by western blotting at 24 h posttransfection. (B) Vero E6 or Calu-3 cells were inoculated with rWuH-1, rWuH-1Nsp3-S676T, rWuH-1Nsp3-S676T;N-S194L, or rWuH-1N-S194L at MOI = 8. Supernatants were collected from cells at 8 and 24 hpi to determine virus titers by plaque assay (n = 4 replicates). (C) Vero E6 or Calu-3 cells infected with rWuH-1, rWuH-1Nsp3-S676T, rWuH-1Nsp3-S676T;N-S194L, or rWuH-1N-S194L were lysed at 8 and 24 hpi and then subject to western blotting to detect N protein. (D) Vero E6 cells were transfected with NC siRNA or siRNA targeting eIF4G or eIF4E; after 48 h, the transfected cells were infected with 0.05 PFU/cell rWuH-1, rWuH-1Nsp3-S676T, rWuH-1Nsp3-S676T;N-S194L, or rWuH-1N-S194L. Supernatants were harvested at 24 h to measure virus titers by plaque assay, and cell lysates were harvested for detecting viral N gene expression by qRT-PCR (n = 3 replicates). A one-way ANOVA test with Tukey’s multiple comparisons test was used for assessing statistical significances in…

Fig. 7.. Two putative models of the effect of Nsp3-S676T on virus replication and compensatory role of N-S194L. (A) Coronaviruses employ Nsp3 SUD to increase the binding between Paip1 and PABP1 to enhance viral mRNA translation. SARS-CoV-2 with the Nsp3-S676T mutation decreases translational stimulation, resulting in diminished virus infectivity. The N-S194L mutation augments N protein expression to compensate for the loss of viral fitness caused by S676T and reverts virus virulence. (B) Nsp3 and nsp4 are the minimal components required for the formation of the DMV spanning pore (30). The N-terminal domains of nsp3, including Ubl-1, HVR, Mac, and SUD, are critical for forming the crown-like shaped gate of the pore at the convex side of DMV. The SUD is a major component of this gate (30). N protein, serving as an RNA chaperone in CoV, is located at the gate and binds to Ubl1 to regulate viral RNA export. T676 in SUD is postulated to increase linker flexibility (Fig. 4F), resulting in increased mobility of the SUD-M relative to the SUD-C and leading to instability of the gate and diminished viral mRNA transport. S194L augments expression of the N protein. Increased levels of N are…