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Cited by 142SARS-CoV-2 and COVID-19 ResearchAnimal Virus Infections StudiesCOVID-19 Clinical Research Studies

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SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients

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Klimstra, William B., Tilston-Lunel, Natasha L., Nambulli, Sham, Boslett, James, McMillen, Cynthia M., Gilliland, Theron, Dunn, Matthew D., Sun, Chengun, Wheeler, Sarah E., Wells, Alan, Hartman, Amy L., McElroy, Anita K., Reed, Douglas S., Rennick, Linda J., Duprex, W. Paul·2020·Journal of General Virology
DOI 10.1099/jgv.0.001481PMC PMC7879561

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

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Fig. 1. Virus growth and characterization. (a, b) Representative images showing plaques generated by infection of Vero E6 cells with different passages of the Wash1 (a) and Munich (b) virus stocks. Chromatograms under each plaque image show the nucleotide sequence (genome positions 23603–23647) coding for the furin cleavage signal (RRAR) region (amino acid positions 681–695) of the S protein for each virus passage. Red and black lines indicate the putative S1 and S2 subunits, respectively, generated after cleavage. Grey lines indicate the nucleotide triplet coding for S686. (c) Immunodetection of plaques (blue), using an anti-SARS-CoV-2 N antibody and HRP-conjugated secondary, confirming that plaques produced in Vero E6 monolayers represent SARS-CoV-2 infection. The scale bar represents 1 cm. (d) Change in genome copies-to-p.f.u. ratio for virus stocks over two passages in Vero E6 cells (Wash1 : P4–P6; Munich : P1–P3). RNA was extracted from cell free supernatant virus stock and genome copies were enumerated by RT-qPCR. Virus stock titres were determined by plaque assay. Error bars represent one standard error of the mean.
Fig. 1. Virus growth and characterization. (a, b) Representative images showing plaques generated by infection of Vero E6 cells with different passages of the Wash1 (a) and Munich (b) virus stocks. Chromatograms under each plaque image show the nucleotide sequence (genome positions 23603–23647) coding for the furin cleavage signal (RRAR) region (amino acid positions 681–695) of the S protein for each virus passage. Red and black lines indicate the putative S1 and S2 subunits, respectively, generated after cleavage. Grey lines indicate the nucleotide triplet coding for S686. (c) Immunodetection of plaques (blue), using an anti-SARS-CoV-2 N antibody and HRP-conjugated secondary, confirming that plaques produced in Vero E6 monolayers represent SARS-CoV-2 infection. The scale bar represents 1 cm. (d) Change in genome copies-to-p.f.u. ratio for virus stocks over two passages in Vero E6 cells (Wash1 : P4–P6; Munich : P1–P3). RNA was extracted from cell free supernatant virus stock and genome copies were enumerated by RT-qPCR. Virus stock titres were determined by plaque assay. Error bars represent one standard error of the mean.CC BY · PubMed Central
Fig. 2. Virus adaptation. (a) Coverage of MinION sequencing reads. Full genome coverage was achieved for both Wash1 : P6 (top) and Munich : P3 (bottom) virus stocks. Sequencing reads were aligned against the published Wash1 genome sequence (MN985325). The number of mapped reads are depicted on the y-axis with the nucleotide position within the SARS-CoV-2 assembly on the x-axis. (b) The S1/S2 region (genome positions 23603–23647; S amino acid positions 681–695) of the S protein including the furin cleavage signal (RRAR). Red and black lines indicate the putative S1 and S2 subunits, respectively, generated after cleavage. (c) Alignment showing representative, individual, mapped reads from the Wash1 : P6 MinION sequencing (genome positions 23561–23660) to illustrate the diversity present in the nucleotide sequence coding for the furin cleavage signal region of the S protein. The reference sequence is shaded yellow and variable regions are shaded pink. The sequence logo at the bottom depicts the consensus sequence (letters) and its frequency (bar height). (d) Alignment showing the amino acid translations (S amino acid positions 667–699) of the nucleotide sequences in (c) to illustrate the diversity present in the amino acid sequence of the furin cleavage signal (RRAR) region of the S protein. Red and black lines indicate the putative S1 and S2 subunits, respectively, generated after cleavage. The reference sequence is shaded yellow and variable regions are shaded pink. The sequence logo at the bottom depicts the consensus sequence (letters) and its frequency (bar height). (e) Table showing the frequencies of each individual variant present in all mapped reads covering genome positions 23561–23660 in the Wash1 : P6 MinION sequencing. The first row contains the wild-type sequences and red and black text indicates the putative S1 and S2 subunits, respectively, generated after cleavage. Lower case nucleotide sequences indicate point mutations and – indicates nucleotide/amino acid deletions. Several different nucleotide variants gave rise to the same amino acid variant, denoted by (1).
Fig. 2. Virus adaptation. (a) Coverage of MinION sequencing reads. Full genome coverage was achieved for both Wash1 : P6 (top) and Munich : P3 (bottom) virus stocks. Sequencing reads were aligned against the published Wash1 genome sequence (MN985325). The number of mapped reads are depicted on the y-axis with the nucleotide position within the SARS-CoV-2 assembly on the x-axis. (b) The S1/S2 region (genome positions 23603–23647; S amino acid positions 681–695) of the S protein including the furin cleavage signal (RRAR). Red and black lines indicate the putative S1 and S2 subunits, respectively, generated after cleavage. (c) Alignment showing representative, individual, mapped reads from the Wash1 : P6 MinION sequencing (genome positions 23561–23660) to illustrate the diversity present in the nucleotide sequence coding for the furin cleavage signal region of the S protein. The reference sequence is shaded yellow and variable regions are shaded pink. The sequence logo at the bottom depicts the consensus sequence (letters) and its frequency (bar height). (d) Alignment showing the amino acid translations (S amino acid positions 667–699) of the nucleotide sequences in (c) to illustrate the diversity present in the amino acid sequence of the furin cleavage signal (RRAR) region of the S protein. Red and black lines indicate the putative S1 and S2 subunits, respectively, generated after cleavage. The reference sequence is shaded yellow and variable regions are shaded pink. The sequence logo at the bottom depicts the consensus sequence (letters) and its frequency (bar height). (e) Table showing the frequencies of each individual variant present in all mapped reads covering genome positions 23561–23660 in the Wash1 : P6 MinION sequencing. The first row contains the wild-type sequences and red and black text indicates the putative S1 and S2 subunits, respectively, generated after cleavage. Lower case nucleotide sequences indicate point mutations and – indicates nucleotide/amino acid deletions. Several different nucleotide variants gave rise to the same amino acid variant, denoted by (1).CC BY · PubMed Central
Fig. 3. Antibody studies. (a, b) Western blot analysis of SARS-CoV-2-infected Vero E6 cell lysates. Vero E6 cells were infected with Wash1 : P5 (lane 3) or P6 (lane 4) stock or Munich : P2 (lane 5) or P3 (lane 6) stock. Mock cell lysate prepared from cells transfected with a pCAGGS vector expressing EGFP (lane 1) and a lysate prepared from cells transfected with a pCAGGS vector expressing a codon-optimized S protein (lane 2) were included as a negative and positive control, respectively. SARS-CoV-2 S protein and the S2 subunit (a), and the N protein (b), were detected with a SARS coronavirus spike protein polyclonal antibody or SARS coronavirus N protein monoclonal antibody, respectively, and HRP-conjugated secondary. The predicted full-length S, S2 subunit and full-length N are indicated based on the marker sizes. Loading of cell lysates was adjusted to show equal levels of virus proteins. Equal amounts of protein were loaded for the pCAGGS EGFP control and pCAGGS S protein expression vectors. (c–e) Virus protein distribution in Munich : P3-infected Vero E6 cells at 24 h post-infection, visualized by immunodetection of N protein (c; green) or S protein (d and e; green) in permeabilized (c, e) or non-permeabilized (d) cells. Filamentous (f)-actin (d, e) and/or nuclei (c–e) were counterstained with phalloidin (red) and DAPI (blue), respectively. Areas marked by white boxes are shown at higher magnification in the insets (d, e). An arrow marks S protein distributed across the cell membrane of an infected cell (d). Scale bars represent 100 µm. (f) Virus protein distribution in permeabilized, Munich : P3-infected Vero E6 cells at 24 h post-infection, visualized by immunodetection with human serum (patient 3, day 14; Table 1; green). Nuclei were counterstained with DAPI (blue). The scale bar represents 100 µm. (g) Development of neutralizing antibodies against SARS-CoV-2 Munich : P3 virus stock in sequential human serum samples collected during the acute phase of COVID-19. Mean PRNT80 values are joined by lines.
Fig. 3. Antibody studies. (a, b) Western blot analysis of SARS-CoV-2-infected Vero E6 cell lysates. Vero E6 cells were infected with Wash1 : P5 (lane 3) or P6 (lane 4) stock or Munich : P2 (lane 5) or P3 (lane 6) stock. Mock cell lysate prepared from cells transfected with a pCAGGS vector expressing EGFP (lane 1) and a lysate prepared from cells transfected with a pCAGGS vector expressing a codon-optimized S protein (lane 2) were included as a negative and positive control, respectively. SARS-CoV-2 S protein and the S2 subunit (a), and the N protein (b), were detected with a SARS coronavirus spike protein polyclonal antibody or SARS coronavirus N protein monoclonal antibody, respectively, and HRP-conjugated secondary. The predicted full-length S, S2 subunit and full-length N are indicated based on the marker sizes. Loading of cell lysates was adjusted to show equal levels of virus proteins. Equal amounts of protein were loaded for the pCAGGS EGFP control and pCAGGS S protein expression vectors. (c–e) Virus protein distribution in Munich : P3-infected Vero E6 cells at 24 h post-infection, visualized by immunodetection of N protein (c; green) or S protein (d and e; green) in permeabilized (c, e) or non-permeabilized (d) cells. Filamentous (f)-actin (d, e) and/or nuclei (c–e) were counterstained with phalloidin (red) and DAPI (blue), respectively. Areas marked by white boxes are shown at higher magnification in the insets (d, e). An arrow marks S protein distributed across the cell membrane of an infected cell (d). Scale bars represent 100 µm. (f) Virus protein distribution in permeabilized, Munich : P3-infected Vero E6 cells at 24 h post-infection, visualized by immunodetection with human serum (patient 3, day 14; Table 1; green). Nuclei were counterstained with DAPI (blue). The scale bar represents 100 µm. (g) Development of neutralizing antibodies against SARS-CoV-2 Munich : P3 virus stock in sequential human serum samples collected during the acute phase of COVID-19. Mean PRNT80 values are joined by lines.CC BY · PubMed Central

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Authors
Klimstra, W. B., Tilston-Lunel, N. L., Nambulli, S., Boslett, J., McMillen, C. M., Gilliland, T., Dunn, M. D., Sun, C., Wheeler, S. E., Wells, A., Hartman, A. L., McElroy, A. K., Reed, D. S., Rennick, L. J., & Duprex, W. P.
Affiliations
  • University of Pittsburgh (US)
  • Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
Corresponding author
  • Klimstra, William B.
  • Duprex, W. Paul
ORCID
Venue
Journal of General Virology
Publisher
Microbiology Society
Published
2020-11-01
Volume
101
Issue
11
Pages
1156-1169
ISSN
  • 0022-1317 (ISSN-L)
  • 1465-2099
DOI
10.1099/jgv.0.001481
PMC
PMC7879561
OpenAlex
W3081204841
Cited by
137
References
69
Citations by year
2026: 3 · 2025: 5 · 2024: 6 · 2023: 20 · 2022: 29 · 2021: 61 · 2020: 13
FWCI
4.03
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of General Virology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • Dr. Patrick Gallagher, Chancellor of the University of Pittsburgh
  • Center for Vaccine Research, University of Pittsburgh
  • University of Pittsburgh Clinical and Translational Science Institute
  • DSF Charitable Foundation
  • National Institutes of Health (AI060525)
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
  • Health Sciences › Medicine › Infectious Diseases › COVID-19 Clinical Research Studies
Keywords
SARS-CoV-2, COVID-19 pandemic, furin cleavage site, spike protein, viral growth, neutralizing antibodies, host range, SARS-CoV, angiotensin-converting enzyme 2

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