Characterization of cellular furin content as a potential factor determining the susceptibility of cultured human and animal cells to coronavirus infectious bronchitis virus infection

Abstract

In previous studies, the Beaudette strain of coronavirus infectious bronchitis virus (IBV) was adapted from chicken embryo to Vero, a monkey kidney cell line, by serial propagation for 65 passages. To characterize the susceptibility of other human and animal cells to IBV, 15 human and animal cell lines were infected with the Vero-adapted IBV and productive infection was observed in four human cell lines: H1299, HepG2, Hep3B and Huh7. In other cell lines, the virus cannot be propagated beyond passage 5. Interestingly, cellular furin abundance in five human cell lines was shown to be strongly correlated with productive IBV infection. Cleavage of IBV spike protein by furin may contribute to the productive IBV infection in these cells. The findings that IBV could productively infect multiple human and animal cells of diverse tissue and organ origins would provide a useful system for studying the pathogenesis of coronavirus.

Fig. 1

Infection of 15 human and animal cell lines with IBV. (a) Western blot analysis of the expression of IBV N protein in IBV-infected HCT116, H1299, HepG2, Huh7 and Hep3B cells. Cells were infected with passages 1–3 of IBV and harvested at 24 h post-infection. Cell lysates were prepared and separated on SDS-10% polyacrylamide gels. The expression of IBV N protein was analyzed by Western blot with anti-N antibodies, after the proteins were separated by SDS-PAGE. The membrane was also probed with anti-actin monoclonal antibody as a loading control. (b) Western blot analysis of the expression of IBV N protein in IBV-infected DLD-1, CHO, MRC-5, DLD-1A and A549 cells. Cells were infected with passages 1–3 of IBV and harvested at 24 h post-infection. Cell lysates were prepared and separated on SDS-10% polyacrylamide gels. The expression of IBV N protein was analyzed by Western blot with anti-N antibodies, after the proteins were separated by SDS-PAGE. The membrane was also probed with anti-actin monoclonal antibody as a loading control. (c) Western blot analysis of the expression of IBV N protein in IBV-infected HeLa, 293T, Cos-7, U937 and BHK cells. Cells were infected with passages 1–3 of IBV and harvested at 24 h post-infection. Cell lysates were prepared and separated on SDS-10% polyacrylamide gels. The expression of IBV N protein was analyzed by Western blot with anti-N antibodies, after the proteins were separated by SDS-PAGE. The membrane was also probed with anti-actin monoclonal antibody as a loading control.

Fig. 2

Viral structural protein expression and syncytium formation in productive IBV infection of five human and animal cell lines. (a) Western blot analysis of the expression of IBV S, M and N proteins in IBV-infected Vero cells. Vero cells were infected with IBV at a multiplicity of infection of approximately 1 and harvested at 0, 4, 8, 12 and 24 hours post-infection, respectively. Cell lysates were prepared and separated on SDS-10% polyacrylamide gels. The expression of IBV S, N and M proteins was analyzed by Western blot with anti-S, -N and -M antibodies, respectively, after the proteins were separated by SDS-PAGE. The full-length glycosylated (S*), the full-length unglycosylated (S) and the S1/S2 cleavage forms of the S protein are labeled. Also indicated are the full-length N protein (N) and several more-rapidly-migrating bands (N^). Both glycosylated and unglycosylated M protein were labeled as M. The membrane was also probed with anti-actin monoclonal antibody as a loading control. (b) Analysis of IBV infection in H1299, Huh7, HepG2, Hep3B and HCT116. Cells were infected with IBV and harvested at 0, 12, 16, 24, 36, and 48 h post-transfection. Cell lysates were prepared and separated on SDS-10% polyacrylamide gels. The expression of IBV S and N proteins was analyzed by Western blot with anti-S and -N antibodies, respectively, after proteins were separated by SDS-PAGE. The membranes were also probed with anti-actin monoclonal antibody as loading controls. (c) Cytopathic effect of Vero, Huh7, H1299, Hep3B, HepG2 and HCT116 cells infected with the Vero-adapted IBV. Cells were infected with IBV at a multiplicity of infection of approximately 0.1 and observed with phase-contrast microscopy at 12–24 h post-infection.

Fig. 3

Growth curves of passages 1, 5 and 20 of IBV in H1299 and Huh7 cells. (a) Vero cells were infected with IBV and harvested at 0, 4, 8, 12, 16, 24 and 36 h post-infection, respectively. Cells were lysed by freezing and thawing three time and viral titers were determined by TCID50 assay on Vero cells. (b) H1299 cells were infected with H1299-p1, H1299-p5 and H1299-p20, respectively, and harvested at 0, 6, 10, 16, 24 and 36 h post-infection, respectively. Cells were lysed by freezing and thawing three times and viral titers were determined by TCID50 assay on Vero cells. (c) Huh7 cells were infected with Huh7-p1, Huh7-p5 and Huh7-p20, respectively, and harvested at 0, 6, 10, 16, 24 and 36 h post-infection. Cells were lysed by freezing and thawing three times and viral titers were determined by TCID50 assay on Vero cells.

Fig. 4

Correlation between cellular furin content and productive IBV infection in five human cell lines. (a) Western blot analysis of the furin abundance in 293 T, A549, H1299, HeLa and Huh7 cells. Total lysates were prepared and separated on SDS-12% polyacrylamide gels. The expression of furin was analyzed by Western blot with anti-furin antibodies. The furin band (furin) and an unknown band (*) are indicated. The membrane was also probed with anti-actin monoclonal antibody as a loading control. The relative furin abundance in these cells is shown as the fraction of the furin abundance in Huh7 cells, which was arbitrarily defined as 1. (b) Productive IBV infection in 293T, A549, H1299, HeLa and Huh7 cells. 2×10⁶ cells in duplicate were plated and infected with IBV-Luc at a multiplicity of infection of approximately 2. Cells were harvested at 24 h post-infection and virus stocks were prepared from one set of cells by freezing and thawing and used to infect fresh cells. The luciferase activities were measured from the other set of cells and the readings from IBV-infected Huh7 cells were considered as 100% infection efficiency for each passage. The readings in other cells were shown as percentages of that in Huh7 cells. (c) Western blot analysis of the furin abundance in Hep3B, HCT116, HepG2 and Huh7 cells. Total lysates were prepared and separated on SDS-12% polyacrylamide gels. The expression of furin was analyzed by Western blot with anti-furin antibodies. The furin band (furin) and an unknown band (*) are indicated. The membrane was also probed with anti-actin monoclonal antibody as a loading control. The relative furin abundance in these cells is shown as the fraction of the furin abundance in Huh7 cells, which was arbitrarily defined as 1.

Fig. 5

Effects of furin-knockdown in Huh7 and H1299 cells on productive IBV infection. (a) Knockdown of furin by siRNA in H1299 and Huh7 cells. Cells were transfected with siRNA duplexes targeting either EGFP or Furin and were harvested 48 h post-transfection. Total lysates were prepared and separated on SDS-12% polyacrylamide gels. The expression of furin was analyzed by Western blot with anti-furin antibodies. The membrane was also probed with anti-actin monoclonal antibody as a loading control. (b) Effect of furin-knockdown on syncytium formation in IBV-infected H1299 cells. H1299 cells were transfected with siRNA duplexes targeting either EGFP or Furin, and infected with IBV at a multiplicity of infection of approximately 1 at 24 h post-transfection. Cells were observed under microscopy at 24 h post-infection. The cells were then fixed and stained with anti-IBV S antibodies. (c) Effect of furin-knockdown on productive IBV infection in H1299 and Huh7 cells. H1299 and Huh7 cells were transfected with siRNA duplexes targeting either EGFP or Furin, and infected with IBV at a multiplicity of infection of approximately 1 at 24 h post-transfection. Total cell lysates prepared from p1 and p2 infected cells were separated on SDS-12% polyacrylamide gels and analyzed by Western blot with ant-IBV S antibodies. The membrane was also probed with anti-actin monoclonal antibody as a loading control.

Fig. 6

Effects of furin-knockin in A549 cells on productive IBV infection. (a) Selection of furin-knockin clones and the effect on productive IBV infection in A549 cells. Wild type A549 cells and five G418-selected clones with furin-knockin were selected and infected with IBV at a multiplicity of infection of approximately 1, and harvested at 24 h post-infection. Total cell lysates were prepared, separated on SDS-10% polyacrylamide gels and analyzed by Western blot with anti-furin and anti-IBV S antibodies. The same membranes were also probed with anti-actin antibodies. (b) Syncytium formation in furin-knockin A549 cells infected with IBV. Wild type A549 cells and the two furin-knockin cells (clones 4 and 5) were infected with IBV at a multiplicity of infection of approximately 1 and virus stocks were prepared by freezing/thawing the infected cells three times at room temperature at 24 h post-infection. Continuous propagation was carried out by infection of fresh cell monolayers with the virus stocks. The formation of syncytia was observed under the microscope at 24–48 h post-infection. (c) Effect of furin-knockin on productive IBV infection in A549 and furin-knockin clones. Total cell lysates prepared from p1 to p5 infected cells, separated on SDS-12% polyacrylamide gels and analyzed by Western blot with ant-IBV N antibodies. The membrane was also probed with anti-actin monoclonal antibody as a loading control. (d) Effect of overexpression of furin on IBV infection in 293 T cells. Cells were transfected with furin, and infected with IBV stocks prepared from IBV-infected Vero cells at a multiplicity of infection of approximately 1 at 14 h post-transfection. Cells were harvested at 24 h post-infection, lysates prepared, separated on SDS-10% polyacrylamide gels and analyzed by Western blot with anti-furin and anti-IBV N antibodies. The same membranes were also probed with anti-actin antibodies.

Fig. 7

Differential efficiency in proteolysis of IBV S protein at the two confirmed furin-cleavage sites in Huh7, H1299, HeLa, A549 and 293T cells. (a) Diagram of S(1–789)Fc construct and a recombinant IBV (IBV-Flag539) expressing a Flag tag inserted into IBV S protein at amino acid position 539. The positions of first (R₆₉₀/S) and second (R₆₉₀/S) furin sites, the sites for the Fc domain and the Flag tag, and the cleavage products are shown. (b) Expression and cleavage of S(1–789)Fc in Huh7, H1299, HeLa, A549 and 293 T cells. Cells were transfected with S(1–789)Fc construct, total lysates prepared and separated on SDS-12% polyacrylamide gels. The expression and processing of the S-Fc fusion protein were analyzed by staining the membrane with an anti-human IgG antibody. Products derived from cleavage of S-Fc fusion protein at R₅₃₇/S (cl-1C) R₆₉₀/S (cl-2C) as well as the full-length product (S(1–789)Fc are indicated. (c) Expression and proteolysis of S protein in Huh7, HeLa, A549 and Vero cells infected with IBVS-Flag539. Cells were infected with IBVS-Flag539 at a multiplicity of infection of approximately 2 and harvested at 18 h post-infection. Total cell lysates prepared from p1 and p2 infected cells were separated on SDS-15% polyacrylamide gels and analyzed by Western blot with ant-Flag antibodies. Products derived from single cleavage at R₅₃₇/S (cl-1C), R₆₉₀/S (cl-2N), dual cleavage (cl-dual) and the full-length Flag-tagged S protein (S-Flag539) are indicated. The percentages of the three cleavage products, cl-2N, cl-1C and cl-dual, were also calculated and shown.