Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication

Abstract

The subgenomic RNA 3 of IBV has been shown to be a tricistronic mRNA, encoding three products in IBV-infected cells. To explore if the least expressed ORF, ORF 3b, which encodes a nonstructural protein, is evolutionarily conserved and functionally indispensable for viral propagation in cultured cells, the Beaudette strain of IBV was propagated in chicken embryonated eggs for three passages and then adapted to a monkey kidney cell line, Vero. The 3b gene of passage 3 in embryonated eggs and passages 7, 15, 20, 25, 30, 35 50, and 65 in Vero cells were amplified by reverse transcription-polymerase chain reaction and sequenced. The results showed that viral RNA extracted from passages 35, 50, and 65 contained a single A insertion in a 6A stretch of the 3b gene between nucleotides 24075 and 24080, whereas the early passages carried the normal 3b gene. This insertion resulted in a frameshift event and therefore, if expressed, a C-terminally truncated protein. We showed that the frameshifting product, cloned in a plasmid, was expressed in vitro and in cells transfected with the mutant construct. The normal product of the 3b gene is 64 amino acids long, whereas the frameshifting product is 34 amino acids long with only 17 homogeneous amino acid residues at the N-terminal half. Immunofluorescent studies revealed that the normal 3b protein was localized to the nucleus and the truncated product showed a "free" distribution pattern, indicating that the C-terminal portion of 3b was responsible for its nuclear localization. Comparison of the complete genome sequences (27.6 kb) of isolates p20c22 and p36c12 (from passages 20 and 36, respectively) revealed that p36c12 contains three amino acid substitutions, two in the 195-kDa protein (encoded by gene 1) and one in the S protein, in addition to the frameshifting 3b product. Further characterization of the two isolates demonstrated that p36c12 showed growth advantage over p20c22 in both Vero cells and chicken embryos and was more virulent in chicken embryos than p20c22. These results suggest that the 3b gene product is not essential for the replication of IBV.

Fig. 1

Comparison of the nucleotide (a) and amino acid (b) sequences of the 3b gene between wt virus and the mutant. The site where an extra A was inserted in a 6A stretch and the predicted frameshift product with only 17 homogeneous amino acid residues are shown in bold. The resulting termination codons in the mutant 3B gene are underlined.

Fig. 2

The emergence of a 3′-end truncated 3b gene by insertion of a single nucleotide (A) during passage of IBV in Vero cells. The nucleotide sequences flanked the insertion site of both positive and complementary strands of passages 7, 20, 30, and 50 are shown. The percentage of the RNA population with the insertion in a given passage was estimated and indicated on the right, and the nucleotide and amino acid sequences of the normal and mutant 3b gene are indicated on the top and the bottom, respectively. The codons for the heterogeneous amino acid residues of the mutant 3B gene are indicated in red.

Fig. 3

Growth curves of p20c22 and p36c12 in Vero cells (a) and in chicken embryos (b). Vero cells were infected with p20c22 and p36c12, respectively, at a m.o.i. of 0.1 and embryonated eggs were inoculated with 4 × 10⁴ PFU of either virus in 0.2 ml medium. Samples were collected from the infected cells or allantoic fluids and titrated on Vero cells. The experiments were independently repeated twice and each titration contained three duplicates.

Fig. 4

Plaque sizes of p20c22 and p36c12. The viruses in passages 20 and 36 were plaque-purified three times, and the plaque sizes were compared after the genotypes were confirmed.

Fig. 5

Membrane fusion activity of the S gene. Plasmids containing the S protein derived from p20c22 (B) and p36c12 (C) were expressed in Vero cells using the T7/vaccinia expression system. The fusion activities were observed 2 days posttransfection.

Fig. 6

(a) Diagram of constructs used for in vitro expression of the normal 3b and mutant 3B gene. Plasmids pIBV3abc, pIBV3bc, and pIBV3c were constructed with PCR fragments derived from passage 20, and pIBV3aBc and pIBV3Bc were constructed with PCR fragments from passage 36. The nucleotide positions of ORF3a, 3b, and 3c; the six A stretch; and the single A insertion site are indicated. (b) in vitro translation of constructs containing the normal 3b and mutant 3B gene. Equal amounts of RNA derived from pIBV3abc and pIBV3aBc or pIBV3bc and pIBV3Bc were used for in vitro translation in wheat germ lysates. RNA derived from pIBV3c was included as a positive control and water was added as a negative control. Numbers on the left indicate molecular masses in kilodaltons. (c) Expression of constructs containing the normal 3b and mutant 3B gene in intact cells. Equal amounts of plasmid DNA from pIBV3abc and pIBV3aBc were used for in vivo translation in Vero cells using the vaccinia/T7 expression system. Plasmid pIBV3c was included as a positive control and water was added as a negative control. Anti-β-tubulin antibody was used in immunoprecipitation as loading controls. Numbers on the left indicate molecular masses in kilodaltons.

Fig. 7

(a) in vitro translation of constructs containing the T7-tagged, normal 3b, N-terminally truncated 3bcΔ1, and C-terminally truncated 3B gene. Equal amounts of RNA derived from pT7-3bc, pT7-3bcΔ1, and pT7-3Bc were used for in vitro translation in wheat germ lysates. Numbers on the left indicate molecular masses in kilodaltons. (b) Subcellular localization of the normal or truncated 3b proteins transiently expressed in Cos-7 cells. Cells were transfected with indicated plasmid DNA at 1 h postinfection. Cells in chamber slide were stained with anti-T7 monoclonal antibodies and the FITC-conjugated secondary antibodies at different time points. The fluorescence was viewed using a confocal scanning Zeiss microscopy.