The Beaudette strain of IBV, grown in Vero cells at 37 °C, was plaque-purified and was initially adapted to grow at 35 °C. After 4 passages, the virus was subsequently adapted to grow at progressively lower temperatures by culturing at 34 °C for 5 passages, 33 °C for 7 passages, 32 °C for 44 passages, 30 °C for 12 passages, 29 °C for 21 passages, and 28 °C for 29 passages. When the virus was initially adapted to a lower temperature, no CPE was observed within 72–96 h postinfection. Apparent CPE appeared usually after 2 to 5 passages. The virus was continuously passaged at the same temperature until CPE became visible within 24–48 h postinfection, and then shifted to a lower temperature.

Plaque assays of different passages were performed at both 32 and 40 °C. It was found that viruses from early passages grown at 32 °C could form plaques at both temperatures at similar levels. But passages grown at 29 °C produced much fewer plaques at 40 °C than at 32 °C, suggesting the emergence of ts mutants during cold-adaptation. These ts mutants dominated quickly at 28 °C.

Two ts mutants, designated ts291602 and ts282902, were plaque-purified from passage 16 grown at 29 °C and passage 29 grown at 28 °C, respectively. Plaque assays showed that both ts291602 and ts282902 formed plaques only at the permissive temperature, but not at all at the nonpermissive temperature. By growing the ts mutant at 37 °C overnight and then at 40 °C for 2 days, a revertant, rev-1, was obtained which produced plaques at both 32 and 40 °C at a similar level (Table 1) . The titers of rev-1 were much higher than that of the wild-type virus wt6501 (isolated from passage 65 at 37 °C) at 32 °C, as it was derived from the cold-adapted virus (Table 1).Table 1

Titers of wild type, mutant and revertant viruses at 32 °C and 40 °C (PFU/ml)

To map the defects responsible for the ts phenotype described above, the complete nucleotide sequences of wt6501, ts291602, ts282902, and rev-1 were determined using RT-PCR products and cDNA clones from each virus. Comparison of the sequence of wt6501 with ts291602 revealed 22 point mutations, a 5-base-deletion, a 1-base-, 3-base- and a 9-base-insertion (Table 2) . These alterations caused 15 amino acid substitutions in eight mature viral proteins, a single amino acid insertion in the 3a protein, a truncated 3b (compared with early passages of wt virus) caused by a single base insertion in the 3b of ts291602, and a 3-amino acid insertion in the N protein. In ts282902, similar point mutations, deletion and insertion were found (Table 2). The most different feature of the two ts mutant is the insertion in the 3b gene. In ts291602, a single A insertion was found at a poly-A stretch at positions 24,075 to 24,080, causing frameshift of the 3b gene and resulting in a C-terminally truncated 3b protein (Shen and Liu, 2003). However, a three-A-insertion at the same poly-A stretch was found in ts282902, which does not affect the open reading frame of the 3b gene. Comparison of the sequence of rev-1 with the two ts mutants showed only one amino acid change (Q²⁹⁴-to-L²⁹⁴) in the S protein and one amino acid deletion in the nonstructural protein 3a (I²⁸), which exists in both ts291602 and ts 282902 but not in rev-1 and wt virus. The genotype of the rev-1 at these two positions (Q²⁹⁴ and I²⁸) are therefore the same as that of wt6501, but different from the two ts mutants. As both wt virus and the revertant could form plaques at 32 and 40 °C, these two mutations might be responsible for the ts phenotype. A Blast search of GenBank clearly showed that the amino acid at position 294 of the S protein was a conserved glutamine. The Leu²⁹⁴ residue in the two ts mutants is located immediately downstream of a hypervariable region and the upstream of a conserved region of the S1 subunit. One additional mutation, D⁷⁰⁹–N⁷⁰⁹, was found in ts291602 only, and an I⁷⁶⁹–M⁷⁶⁹ mutation in the two ts mutants and rev-1.Table 2

Summary of nucleotide (nt) and amino acid (aa) changes in the wild type, mutant and revertant viruses

After mapping the potential determinants responsible for the ts phenotype, we would like to explore the mechanisms by which these mutations affect the propagation of the ts mutants at the nonpermissive temperature. Synthesis of the S protein in cells infected with wt virus, ts mutants, and revertant was analyzed. Two sets of experiments were carried out. First, Vero cells were infected with wt6501, ts291602, ts282902, and rev-1 at 32 and 40 °C, respectively, and lysates were immunoprecipitated with anti-IBV antibodies. It was obvious that synthesis of the ts mutant S protein was detectable at 32 °C but not at 40 °C (Fig. 1a , lanes 3, 4, 5, and 6) under these conditions. In contrast, expression of the S protein was observed in cells infected with wt6501 and rev-1 at both temperatures (Fig. 1a, lanes 1, 2, 7, and 8), though the level of the proteins detected in cells infected with wt6501 was much lower at 32 °C. This was consistent with the lower titers of wt virus at 32 °C.Fig. 1

Analysis of the expression of the S protein from wt (wt6501), ts mutants (ts291602 and 282902) and revertant (rev-1) viruses. (a) Cells were infected with each virus at 32 °C and 40 °C as indicated on the top for 1 h and were maintained at the same temperatures. (b) Two dishes of cells were infected with each virus at 32 °C for 1 h. One of the duplicates was maintained at 32 °C (lanes 1, 3, 5, and 7) and the other one was shifted to 40 °C (lanes 2, 4, 6, and 8). Radiolabeled cell lysates were immunoprecipitated with anti-IBV antibodies. The proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. Numbers on the left indicate molecular mass in kilodalton and the position of the S protein is indicated on the right.

Comparison of the nucleotide sequences and the S protein synthesis of wt, ts mutants and revertant indicate that the single amino acid mutation (Q²⁹⁴–L²⁹⁴) in the S protein may be responsible for the temperature sensitivity of the mutant virus. This possibility was studied by biochemical and functional characterization of the S protein from the revertant and a ts mutant.

The S gene of rev-1 and ts291602 was cloned under the control of a T7 promoter to investigate whether membrane-fusion activity was affected by the mutations in the S protein. Cells were infected with recombinant vaccinia/T7 virus and were transfected with plasmids containing the S gene from either ts291602 or rev-1. The expression of the S proteins was analyzed by Western blotting using anti-IBV antibodies. As shown in Fig. 2a , the S protein of both revertant and ts mutant was expressed at 32 and 40 °C (lanes 3, 4, 5, and 6), though the S protein of ts291602 was expressed at a relatively lower level than the rev-1 at 40 °C (compare lanes 4 and 6).Fig. 2

Membrane fusion activity of the S protein expressed from wt and ts291602. (a) Vero cells were transfected with plasmids without insert (lanes 1 and 2) or with the S gene from the wt (lanes 3 and 4) and ts291602 (lanes 5 and 6), and were cultured at 32 °C (lanes 1, 3, and 5) or 40 °C (lanes 2, 4, and 6). The expression of the S protein was examined in Western blot using anti-IBV antibodies, and β-tubulin was immunostained as loading controls. (b) The membrane-fusion activity of the S protein from the wt (panels B, E, and H) and ts mutant (panels C, F, and I) at 32 °C (panels B and C) and 40 °C (panels C and F) 2 days post-transfection was compared. Panels H and I show induction of membrane fusion on cells transfected with the wt and ts291602 by shifting cells shown in panels E and F to 32 °C for 1 day. Panels A, D, and G show cells transfected with empty plasmids.

The recombinant S genes of the ts mutant and revertant were transfected into cells in 35 mm dishes at 32 °C and one of the duplicates was shifted to 40 °C at 4 h posttransfection. Radiolabeled proteins were immunoprecipitated and treated with Endo-H at 37 °C. As shown in Fig. 3a , the ts mutant S protein, synthesized at 40 °C, was sensitive to Endo-H digestion, as no 180-kDa form was observed (lane 12). In contrast, a portion of the ts mutant S protein, synthesized at 32 °C (lanes 10), was resistant to Endo-H digestion. The revertant S protein, synthesized at both 32 and 40 °C, was also resistant to Endo-H digestion (lanes 6 and 8). These results indicate that the ts mutant S protein synthesized at 40 °C was not a mature form of the glycoprotein.Fig. 3

Maturation defects of the S protein from the ts mutant. (a) Endo-H treatment of the S protein from rev-1 and ts291602. The S protein derived from rev-1 (lanes 5, 6, 7, and 8) and ts291602 (lanes 9, 10, 11, and 12) were expressed in Vero cells at 32 °C (lanes 5, 6, 9, and 10) and 40 °C (lanes 7, 8, 11, and 12), using a T7-vaccinia expression system. The radiolabeled proteins were immunoprecipitated with anti-IBV antibodies, the eluted proteins were Endo-H- (lanes 6, 8, 10, and 12) or mock-treated (lanes 5, 7, 9, and 11) and analyzed by SDS-PAGE. (b) Detection of the less matured S protein from ts291602 at 40 °C and the defect in cleavage of the mutant S protein. Cells were infected with ts291602 and rev-1 for 5 h at 32 °C, one of the duplicates was maintained at 32 °C (lanes 1, 3, 5, and 7), and the other one was shifted to 40 °C (lanes 2, 4, 6, and 8). Cells were radiolabeled and viral proteins were immunoprecipitated with anti-IBV antibodies and analyzed by SDS-PAGE. Lanes 1, 2, 3, and 4 refer to viral products detected from cell lysates and lanes 5, 6, 7, and 8 refer to viral proteins detected from virus particles released to the cultured media.

To detect whether the ts mutant S protein would be transported to the plasma membrane at the nonpermissive temperature, the S protein was transiently expressed in Vero cells using the vaccinia virus-T7 expression system. The transfected cells were treated with cycloheximide at 3.5 h posttransfection for 30 min to inhibit further protein synthesis. The cells were then incubated at 32 and 40 °C, respectively. The subcellular localization of the S protein was analyzed by indirect immunofluorescent staining at 4, 8, 16, and 24 h postinfection, respectively. The confocal microscopy images of transfected cells are shown in Fig. 4 . Plasma membrane staining of the ts mutant S protein was not observed at any time points posttransfection at 40 °C, but was clearly seen at 32 °C at 16 and 24 h postinfection (Fig. 4). The revertant S protein was expressed on the cell surface at both temperatures at 16 and 24 h posttransfection. These results may explain the lack of membrane fusion activity of the ts mutant S protein in both infected and transfected cells.Fig. 4

Subcellular localization and translocation of the wt and ts mutant S protein in Vero cells at the permissive- and nonpermissive temperatures. Vero cells transiently expressing the wt and ts mutant S protein (as indicated on the top) were treated with cycloheximide at 3.5 h posttransfection for 30 min and were cultured at either 32 °C or 40 °C as indicated on the top. Cells were incubated with anti-IBV antibodies and then with the FITC-conjugated secondary antibodies at different time points as indicated on the left. The fluorescence was viewed using a confocal scanning Zeiss microscope.

To investigate whether the S protein of ts mutant was assembled into virion or not, ts291602 and rev-1-infected cells were radiolabeled and the virus particles were purified through sucrose gradients twice. Immunoprecipitation of the purified virions using anti-IBV antibodies showed that the S protein of ts291602 was not detected at 40 °C (Fig. 6 , lane 2), but was observed at 32 °C (Fig. 6, lane 1). The S protein was detected at both temperatures for rev-1 (Fig. 6, lanes 3 and 4). These results render support that the spike protein of the ts mutant may not be assembled into virus particles at 40 °C.Fig. 6

Analysis of the structural proteins on purified ts291602 and rev-1 virions. Immunoprecipitations were performed using virions, purified from ts291602- (lanes 1 and 2) and rev-1-infected (lanes 3 and 4) Vero cells at 32 °C (lanes 1 and 3) and 40 °C (lanes 2 and 4). The viral proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. Numbers on the left indicate molecular masses in kilodalton and the positions of the three structural proteins are indicated on the right.

Vero cells were maintained in complete DMEM medium (GIBCO BRL), supplemented with newborn calf serum (10%), streptomycin (1000 μg) and penicillin (1000 units/ml).

The Beaudette stain of IBV was purchased from ATCC and propagated in chicken embryonated eggs for three passages. The virus was then adapted to grow and passage on Vero cells for 65 times at 37 °C. In this study, the viruses were further passaged on Vero cells at progressively lower temperatures for 121 passages (at 35, 34, 33, 32, 30, 29, and 28 °C for 4, 5, 7, 44, 12, 21, and 29 passages). Virus stocks were used for plaque assays, plaque-to-plaque purification and viral RNA extraction as described previously (Shen et al., 1994). The wild-type virus, wt6501, was plaque-purified from passage 65 grown at 37 °C, while ts mutants, ts291602 and ts282902, were purified from passage 16 grown at 29 °C and passage 29 grown at 28 °C, respectively. The revertant, rev-1, was plaque-purified from cells infected with ts 291602 for 12 h at 37 °C and then shifted to 40 °C for 2 days.

Recombinant vaccinia/T7 (V/T3) virus was propagated and was tittered on Vero cells. Virus stocks were kept at −80 °C until use.

Confluent monolayers of Vero cells were infected with viruses at a multiplicity of infection (MOI) of 0.1. After 2-h absorption, the viruses were radiolabeled by replacing medium with methionine-free DMEM supplemented with 30 μci/ml [³⁵S]-methionine. After 16-h incubation at temperatures appropriate for each virus, cells were harvested and virus stock was prepared by freezing and thawing three times. Cell debris was removed by centrifugation at 5000 rpm for 15 min (Beckmen, 25.50). The supernatant was centrifuged through a 20% sucrose cushion and the resulting pellet was resuspended in TNE buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA). The viruses were further purified by centrifugation through a 20–55% sucrose gradient in TNE buffer at 45,000 rpm (Beckmen, SW50) twice. Fractions containing the virus were pooled together and used for protein analysis.

Viral structural proteins were labeled with [³⁵S]-methionine, immunoprecipitated and separated on 12.5% polyacrylamide gels as described previously (Liu et al., 1998). The anti-IBV serum was raised in rabbit against purified IBV virions and was used in immunoprecipitation and Western blot as previously described (Liu and Inglis, 1991).

A portion of cell lysates was immunoprecipitated with anti-IBV serum. The pellets were washed three times with standard RIPA buffer, dissolved in 20 μl of digestion buffer (50 mM Tris, pH 6.8, 0.25% SDS) and incubated at 95 °C for 5 min. Ten microliters of the supernatants were mixed with 10 μl of digestion buffer with or without Endo-H (0.2 mU, Boehringer Mannheim) and incubated for 4 h at 37 °C.

Confluent monolayers of Vero cells were infected with recombinant vaccinia/T7 viruses at a MOI of 0.1. After 1-h absorption, cells (2 × 10⁷) were trypsinized, centrifuged at 250 g and resuspended in 2 ml of PBS. The cells (0.3 ml) were mixed with 5 μg plasmid and electroporated (Easyject, EquiBio) at 600 V in a 0.4-cm cuvette (BioRad). The cells were then plated in a 35-mm dish containing 3 ml of DMEM with 2% of newborn calf serum and incubated at temperatures indicated.

Viral RNA was extracted from the purified viruses using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Reverse transcription and polymerase chain reaction (RT-PCR) were performed using the Expand Reverse Transcription and High Fidelity PCR Kits (Boehringer Mannheim). Annealing and extension times of PCR were optimized for amplification of PCR products with different sizes using different primers. More than 100 specific primers were used for amplification, sequencing and cloning. Automated sequencing was carried out using PCR products or cDNA clones and specific primers as previously described (Shen et al., 1999). Sequence analysis was carried out using the GCG and Blast suite of programs as described previously (Shen and Liu, 2000).

HeLa cells were infected with vaccinia/T7 virus, transfected with constructs encoding wt and ts mutant S protein using QIAGEN effectene transfection reagent, and incubated at the permissive and nonpermissive temperatures, respectively, for 18 h. Cells were harvested, washed once with PBS, resuspended in blocking buffer (20% FBS and 1% BSA in PBS), incubated on ice for 30 min. A half of the cells were permeabilized with 0.1% saponin in FACS washing buffer (2.5% FBS and 0.05% sodium azide in PBS) incubated for 10 min at room temperature, and stained with 1:100 diluted primary antiserum, the rabbit anti-IBV S protein. The other half was stained directly with the same primary antibody. Cells were then washed two times with the FACS washing buffer, stained with 1:20 diluted FITC conjugated swine anti-rabbit antibody (DAKO). After washing two times with the FACS washing buffer, cells were then fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry.