The S2 Subunit of Infectious Bronchitis Virus Beaudette Is a Determinant of Cellular Tropism

Abstract

The spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulence in vivo, and is responsible for cellular tropism in vitro We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the in vitro cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2' cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.

Keywords: S2′; cellular tropism; coronavirus; infectious bronchitis virus; reverse genetic analysis.

FIG 1

Schematic diagram of the IBV S glycoprotein and identification of Beaudette-specific amino acids in the S2 subunit. (A) The S1 domain contains the receptor binding domain and the cleaved signal sequence. The S2 domain contains two heptad repeats (HR1 and HR2), a transmembrane domain, and a cytoplasmic domain. Beau-R also contains a Beaudette-specific sequence not found in M41. (B) The sequences of the ectodomains of the S2 subunit of IBV strains Beau-CK (GenBank accession number AJ311317), M41 (GenBank accession number X04722), 4/91 (UK) (GenBank accession number JN192154), and QX L1148 (GenBank accession number KY933090) are shown. There are 19 amino acid differences between the S2 subunits of Beau-R and M41-CK. The Beaudette-specific motif is labeled with a horizontal line. A black box surrounds each amino acid change made in the spike glycoproteins of the recombinant viruses generated in this study, and the amino acid positions are shown above.

FIG 2

Growth characteristics of rIBVs BeauR-M41(S1) and BeauR-M41(S2) P₃-CK on CK and Vero cells. (A and B) Chick kidney cells (A) and Vero cells (B) in 6-well plates were infected with Beau-R, M41-CK, BeauR-M41(S), BeauR-M41(S1) P₃-CK, and BeauR-M41(S2) P₃-CK at an MOI of 0.5. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean. (C to H) Vero cells were mock infected (C) or were infected with Beau-R (D), M41-CK (E), BeauR-M41(S) (F), BeauR-M41(S1) P₃-CK (G), or BeauR-M41(S2) P₃-CK (H). Infected cells were fixed at 24 h postinfection and immunolabeled with mouse anti-dsRNA and secondary antibody Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (green; Invitrogen). Nuclei were labeled with DAPI (blue). Bars, 50 μm.

FIG 3

Adaptation of rIBV BeauR-M41(S1) to Vero cells. Vero cells in 6-well plates were infected with Beau-R and BeauR-M41(S1) P₇-Vero at an MOI of 0.5. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean; some error bars are too small to be observed.

FIG 4

Growth characteristics of rIBVs BeauR-S-MM and BeauR-M41-S-BSM P₃-CK on CK and Vero cells. (A and D) Chick kidney cells (A) and Vero cells (D) in 6-well plates were infected with Beau-R, M41-CK, BeauR-M41(S), BeauR-S-MM P₃-CK, and BeauR-M41-S-BSM P₃-CK at an MOI of 0.5. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean. (B and C) Vero cells were infected with BeauR-M41-S-BSM P₃-CK (B) and BeauR-S-MM P₃-CK (C). Infected cells were fixed at 24 h postinfection and immunolabeled with mouse anti-dsRNA and secondary antibody Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (green; Invitrogen). Nuclei were labeled with DAPI (blue). Bars, 50 μm.

FIG 5

Adaptation of rIBV BeauR-M41-S-BSM to Vero cells. Vero cells in 6-well plates were infected with Beau-R and BeauR-M41-S-BSM P₇-Vero at an MOI of 0.5. The supernatant was harvested at 1, 12, 24, 48, 72, and 96 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean; some error bars are too small to be observed.

FIG 6

Growth characteristics of rIBVs with modified S2 subunits on CK and Vero cells. (A and B) Chick kidney cells (A) and Vero cells (B) in 6-well plates were infected with Beau-R, BeauR-M41(S), BeauR-M41-S-BSM P₃-CK, BeauR-M41-S-BSM-N₆₁₇S P₃-CK, BeauR-M41-S-BSM-L₅₇₈F-N₆₁₇S P₃-CK, BeauR-M41-S-BSM-I₁₀₀₀V P₃-CK, BeauR-M41-S-BSM-L₈₅₇F-I₁₀₀₀V P₃-CK, BeauR-M41-S-BSM-N₈₂₆S-L₈₅₇F-I₁₀₀₀V P₃-CK, and BeauR-M41-S-BSM-L₅₇₈F-N₆₁₇S-N₈₂₆S-L₈₅₇F-I₁₀₀₀V P₃-CK at an MOI of 0.05. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean. (C to H) Vero cells were infected with BeauR-M41-S-BSM-N₆₁₇S P₃-CK (C), BeauR-M41-S-BSM-L₅₇₈F-N₆₁₇S P₃-CK (D), BeauR-M41-S-BSM-I₁₀₀₀V P₃-CK (E), BeauR-M41-S-BSM-N₈₂₆S-L₈₅₇F-I₁₀₀₀V P₃-CK (F), BeauR-M41-S-BSM-N₈₂₆S-L₈₅₇F-I₁₀₀₀V P₃-CK (G), and BeauR-M41-S-BSM-L₅₇₈F-N₆₁₇S-N₈₂₆S-L₈₅₇F-I₁₀₀₀V P₃-CK (H). Infected cells were fixed at 24 h postinfection and immunolabeled with mouse anti-dsRNA and secondary antibody Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (green; Invitrogen). Nuclei were labeled with DAPI (blue). Bars, 50 μm.

FIG 7

Adaptation of rIBVs with modified S2 subunits to Vero cells. Vero cells in 6-well plates were infected with Beau-R, BeauR-M41-S-BSM P₇-Vero, BeauR-M41-S-BSM-N₆₁₇S P₇-Vero, BeauR-M41-S-BSM-L₅₇₈F-N₆₁₇S P₇-Vero, BeauR-M41-S-BSM-I₁₀₀₀V P₇-Vero, BeauR-M41-S-BSM-L₈₅₇F-I₁₀₀₀V P₇-Vero, BeauR-M41-S-BSM-N₈₂₆S-L₈₅₇F-I₁₀₀₀V P₇-Vero, and BeauR-M41-S-BSM-L₅₇₈F-N₆₁₇S-N₈₂₆S-L₈₅₇F-I₁₀₀₀V P₇-Vero at an MOI of 0.005. The supernatant was harvested at 1, 12, 24, 48, and 72 h postinfection and titrated on CK cells. Three replicates were performed, and the averages were taken. Error bars indicate the standard error of the mean.

FIG 8

Proteolytic cleavage of rIBVs at the S2′ site is dependent on the Beaudette-specific motif. Chick kidney cells in 6-well plates were mock infected (lane 1) or infected with Beau-R (lane 2), M41-CK (lane 3), BeauR-M41(S) (lane 4), BeauR-S-MM P₃-CK (lanes 5 and 6), or BeauR-M41-S-BSM P₃-CK (lanes 7 and 8). Cells were lysed at 24 h postinfection, and spike glycoproteins were analyzed by Western blotting with anti-S2 antibody. Actin was detected with anti-beta-actin antibody. Two replicates of BeauR-S-MM P₃-CK and BeauR-M41-S-BSM P₃-CK were used. Bands corresponding to uncleaved spike, the S2 subunit, and the S2′ cleavage product are labeled. The S2′ cleavage product was detected in lanes 2, 7, and 8 only.