Differentially conserved amino acid positions may reflect differences in SARS-CoV-2 and SARS-CoV behaviour

Abstract

Motivation: SARS-CoV-2 is a novel coronavirus currently causing a pandemic. Here, we performed a combined in-silico and cell culture comparison of SARS-CoV-2 and the closely related SARS-CoV.

Results: Many amino acid positions are differentially conserved between SARS-CoV-2 and SARS-CoV, which reflects the discrepancies in virus behaviour, i.e. more effective human-to-human transmission of SARS-CoV-2 and higher mortality associated with SARS-CoV. Variations in the S protein (mediates virus entry) were associated with differences in its interaction with ACE2 (cellular S receptor) and sensitivity to TMPRSS2 (enables virus entry via S cleavage) inhibition. Anti-ACE2 antibodies more strongly inhibited SARS-CoV than SARS-CoV-2 infection, probably due to a stronger SARS-CoV-2 S-ACE2 affinity relative to SARS-CoV S. Moreover, SARS-CoV-2 and SARS-CoV displayed differences in cell tropism. Cellular ACE2 and TMPRSS2 levels did not indicate susceptibility to SARS-CoV-2. In conclusion, we identified genomic variation between SARS-CoV-2 and SARS-CoV that may reflect the differences in their clinical and biological behaviour.

Supplementary information: Supplementary data are available at Bioinformatics online.

Fig 1

SARS-CoV-2 and SARS-CoV replication in cell culture. (A) Cytopathogenic effect (CPE) formation 48 h post-infection in MOI 0.01-infected Caco2, CL14, DLD-1 and HT29 cells. Representative images showing immunostaining for double-stranded RNA (indicates virus replication) and quantification of virus genomes by qPCR are presented in Supplementary Figure S3. (B) CPE formation in SARS-CoV and SARS-CoV-2 (MOI 0.01)-infected ACE2-negative 293 cells and 293 cells stably expressing ACE2 cells (293/ACE2) 48 h post-infection. Immunostaining for double-stranded RNA and quantification of virus genomes by qPCR is shown in Supplementary Figure S4. (C) Western blots indicating cellular ACE2 and TMPRSS2 protein levels in uninfected cells. Uncropped blots are provided in Supplementary Figure S5. (D) A sequence view of the DCPs in the vicinity of the S two cleavage sites and an image of the R815 cleavage site and closely located DCPs. S is cleaved and activated by TMPRSS2. (E) Concentration-dependent effects of the TMPRSS2 inhibitors camostat and nafamostat on SARS-CoV-2- and SARS-CoV-induced cytopathogenic effect (CPE) formation determined 48 h post-infection in Caco2 infected at an MOI of 0.01 using a phase contrast microscope. Similar effects were observed in CL14 cells (Supplementary Fig.S6). Values are presented as means ± S.D. (n = 3)

Fig 2

SARS-CoV-2 and SARS-CoV S interaction with ACE2. (A–D)Differentially conserved positions in the Spike protein. (A) A sequence view of the DCPs present in the Spike protein, with an inset showing the receptor binding domain. (B) The S interface with ACE2 (cyan). The ACE2 interface is shown in blue spheres, DCPs in red. (C) The V404 = K417 DCP. (D) The R426 = N439 DCP, the left image shows SARS-CoV S R426, the image on the right show the equivalent N439 in SARS-CoV-2 S. (E) SARS-CoV residues associated with altering ACE2 affinity and the residues at these positions in SARS-CoV-2 S. (F) Cytopathogenic effect (CPE) formation in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected Caco2 cells in the presence of antibodies directed against ACE2 or DPP4 (MERS-CoV receptor) 48 h post-infection