Identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from human (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor-binding domain of the S protein being potentially important for SARS progression and tropism. Using the single-round infection assay, we found that a two-amino acid substitution (N479K/T487S) of a huSARS-CoV for those of pcSARS-CoV almost abolished its infection of human cells expressing the SARS-CoV receptor ACE2 but no effect upon the infection of mouse ACE2 cells. Although single substitution of these two residues had no effects on the infectivity of huSARS-CoV, these recombinant S proteins bound to human ACE2 with different levels of reduced affinity, and the two-amino acid-substituted S protein showed extremely low affinity. On the contrary, substitution of these two amino acid residues of pcSARS-CoV for those of huSRAS-CoV made pcSARS-CoV capable of infecting human ACE2-expressing cells. These results suggest that amino acid residues at position 479 and 487 of the S protein are important determinants for SARS-CoV tropism and animal-to-human transmission.

Fig. 1

Sequence homology and diversity of the receptor-binding domain (RBD, amino acids 318–510) of the spike gene in palm civet and human SARS-CoV. The sequences of all HP03 are identical in RBD except several earliest cases (GZ01/GZ02, ZS-A, ZS-B, and ZS-C) at position 344 and some late cases (Singapore isolates) at residue 353. Thus, GZ02, BJ01, or TOR2 was used to represent general HP03. In the diagram, PC represents palm civet and HP represents human patient. Both of them were suffixed with “03”or“04” to indicate the time when they were isolated. The positions with variation were indicated at the top (344, 360, 472, 479, 480, and 487).

Fig. 2

The infectivity of pseudotyped viruses bearing a panel of huSARS-CoV S proteins with individual or combinatorial substitution of K344R, F360S, N479K, D480G, and T487S. These mutated S genes were constructed based on the S gene of BJ01 isolate. The substituted positions are marked with asterisks. All pseudotyped virus' stocks were normalized by HIV core antigen p24, and 10 ng/well was used for infection assay. Luciferase activity was determined 48 h after infection, and they were displayed as means ± S.D. of four parallel wells. Three independent experiments were done to confirm the results. A, the infectivity of various pseudotyped viruses to HeLa-F5 cells and HeLa cells. Pseudotyped virus bearing BJ01 S protein was used as positive control (first column). env-, pseudotyped virus without envelope protein. B, the infectivity of these pseudotyped viruses to HeLa-mACE2 cells and HeLa cells. C, relative human and mouse ACE2 mRNA expression examined using quantitative RT-PCR. The amount of mRNA expression of human ACE2 or mouse ACE2 was divided by that of β-actin in each sample to normalize the value. Quantitative RT-PCR was performed three times. The cDNA of HeLa cells was used as a negative control.

Fig. 3

The binding affinity of selected mutants of huSARS-CoV S proteins to human ACE2.A, Western blotting analysis of mutated S fragments (13–510) fused with human Fc. Proteins were submitted to run 10% SDS-PAGE and blotted with anti-human-Fc-alkaline phosphatase antibody. B, FACS analysis of S fragments binding to HeLa-F5 cells and HeLa cells (top right corner). Cells were incubated with 100 nm S fragments, and the binding affinity of S proteins was detected through FACS analysis using fluorescein isothiocyanate-conjugated anti-human IgG-Fc antibody (solid line, BJ01; gray, mutated S fragments; and dotted line, mock). C, binding affinity curves of various S fragments. The mean fluorescence intensity in the y-axis was determined by a series of FACS analyses in which HeLa F5 cells were incubated with serially diluted S fragments (▪, S_BJ01; ○, S_F360S; ▾, S_N479K; ▴, S_T487S; and □, S_N479K/T487S) from 1 μm to 2 nm.

Fig. 4

The infectivity of pseudotyped viruses bearing SZ3 S proteins and its variants.A, Western blotting analysis of S protein incorporated in pseudotyped virus. S protein variants associated with HIV particles were detected by Western blotting using rabbit anti-Spike protein serum. B, the infectivity of various pseudotyped viruses to HeLa-F5 cells and HeLa cells. All pseudotyped virus stocks were normalized by HIV core antigen p24, and 10 ng/well was used for the infection assay. Luciferase activity was determined 48 h after infection, and they were displayed as means ± S.D. of four parallel wells. Three independent experiments were done to confirm the results. Pseudotyped virus bearing BJ01 S protein was used as positive control (first column).