Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6

Figures

• 

  Fig 1. Recent Omicron variants have higher replication than early ones. A and B) Consensus non-Spike (A) and Spike (B) mutations (>95% of sequences at time of emergence) in all dominant SARS-CoV-2 variants post-Delta. BA.2 fixed mutations are indicated in red. C) RNA replication measurement of each variant paired with its concordant Spike expression vector using SARS-CoV-2 Spike replicons. The data is plotted as mean +/- SD of two independent biological replicates conducted in triplicate. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by two-sided Student’s T-test. NTD: N-terminal domain; RBD: receptor binding domain; FCS: furin cleavage site; VAT: Vero cells stably expressing ACE2 and TMPRSS2; A549-ACE2h: A549 cells stably expressing high levels of ACE2.

• 

  Fig 2. RNA replication and entry negatively correlate for Omicron variants. A) Schematic of replicon experiments to measure RNA replication and entry. B and C) Normalized RNA replication and normalized entry for all variants were measured in VAT (B) and A549-ACE2h (C) cells and graphed on a scatter plot. Data is plotted as mean +/- SD of two independent biological replicates each conducted in triplicate. VAT: Vero cells stably expressing ACE2 and TMPRSS2; A549-ACE2h: A549 cells stably expressing high levels of ACE2.

• 

  Fig 3. NSP6 L260F mutation enhances RNA replication and reduces LD content in infected cells. A) NSP6 mutations in Omicron variants. BA.2 fixed mutations are indicated in red. B) RNA replication measurement of indicated variants in VAT cells. Data is plotted as mean +/- SD of three independent biological replicates each conducted in duplicate. C) Quantification of the relative LD mean fluorescent intensity (MFI) per dsRNA positive cells in images shown in S2 Fig using box and whiskers plot, and comparisons were made as indicated by two-sided Student’s T-test. D) Immunofluorescence staining of HuH-7.5 cells stably expressing indicated NSP6 proteins and stained for LD, PLIN2, and NSP6. E, F, and G) Quantification of the relative LD MFI, LD number, and average LD size per NSP6-positive cells in images shown in D using box and whiskers plot, and comparisons were made as indicated by two-sided Student’s T-test. For panels using box and whiskers plots, interquartile range (IQR) of boxplot is between 25th and 75th percentiles and center line indicates median value. Whiskers of boxplot is extended to the maxima and minima. Maxima is the largest value and minima is the smallest value in the…

• 

  Fig 4. NSP6 L260F is critical for viral infection in vitro and pathogenesis in vivo. A) Plaque morphology of indicated viruses in VAT cells at 48 hours post-infection. The images were pseudocolored to black and white for optimal visualization. B) Particle production and intracellular viral RNA abundance were measured by plaque assay and RT-qPCR, respectively, at 24 and 48 hours post-infection in A549-ACE2h cells for indicated viruses. Data is plotted as mean +/- SD for three independent biological replicates each conducted in duplicate. C) Particle production was measured by plaque assay at 24, 48, and 72 hours post-infection in VAT cells for indicated viruses. Data is plotted as mean +/- SD of one representative biological replicate conducted in triplicate. D) Schematic representation of the in vivo experiment to measure the replication and pathogenesis of BA.5 and BA.5 NSP6 F260L viruses (105 PFU) in K18-hACE2 mice. n=10 per virus group per time-point. Clip art was created with BioRender.com with permission. E) Animal weight is plotted over the study time course as mean +/- SEM. BA.5 group animals all succumbed to infection at 7 days post-infection and therefore weight data are only…