Functional and genetic studies of the substrate specificity of coronavirus infectious bronchitis virus 3C-like proteinase

Abstract

Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.

FIG. 1.

IBV genome organization and proteolytic processing of the replicase polyproteins. Cleavage sites and the processed products of IBV replicase polyproteins pp1a (nsp2 to nsp10) and pp1ab (nsp2 to nsp10 and nsp12 to nsp16) are shown. The cleavage sites of 3CLpro are indicated with black arrows, and the conserved glutamine (Q) residue at the P1 position of each cleavage site is indicated. The cleavage sites of papain-like proteinase (PLpro) are indicated with block arrows. UTR, untranslated region; S, spike protein; E, envelope protein; M, membrane protein; N, nucleocapsid protein; a, 3a or 5a protein; b, 3b or 5b protein.

FIG. 2.

Proteolytic processing of IBV polyproteins by IBV and SARS-CoV 3CLpros. (A) Diagram showing the constructs containing sequences flanking IBV cleavage sites and the 3CLpro-mediated processing in the present study. Uncleaved (U) and cleaved (C) products are represented with straight lines. The predicted sizes of the Flag-tagged (black box) uncleaved and cleaved products are indicated. Western blot analyses of the 3CLpro-mediated processing of wild-type and mutant substrates flanking nsp4-5/Q2779 (B), nsp5-6/Q3086 (B), nsp6-7/Q3379 (B), nsp7-8/Q3462 (C), nsp8-9/Q3672 (C), nsp9-10/Q3783 (C), and nsp12-13/Q4868 (C) and nsp13-14/Q5468 (D), nsp14-15/Q5989 (D), and nsp15-16/Q6327 (D) in IBV 1a and 1ab polyproteins, respectively. The Flag-tagged wild-type and P1-mutant substrates were coexpressed with IBV (I-3C) or SARS-CoV (S-3C) 3CLpro in H1299 cells using the vaccinia virus-T7 system. The transfected cell lysates were resolved on an SDS-15 to 20% polyacrylamide gel and subjected to Western blotting with anti-Flag antibodies. Q, E, H, N, and P represent constructs containing the corresponding amino acid residue at the P1 position. As a control, the amount of expressed 3CLpro was probed with IBV or SARS-CoV anti-3CLpro antibodies. The uncleaved (U) and cleaved (C) products and the relative cleavage efficiency (RCE) are indicated.

FIG. 3.

Characterization of the growth properties and genetic stability of mutant viruses. (A) Growth properties of rIBV and mutant viruses. Vero cells were infected with wild-type and mutant viruses at an MOI of 1 PFU/cell and harvested at 0, 4, 8, 12, 16, 24, and 36 h postinoculation. Viral stocks were prepared by freeze-thawing the cells three times, and the TCID₅₀ of each viral stock was determined by infecting five wells of Vero cells on 96-well plates in triplicate with a 10-fold serial dilution of each viral stock. Error bar shows standard error of the mean. (B) Northern blot analysis of the genomic and subgenomic RNAs in cells infected with wild-type and mutant viruses. Vero cells infected with wild-type and mutant viruses at an MOI of 1 PFU/cell. Total RNA (10 μg) extracted from the infected cells at 12 h postinfection was separated on 1% agarose gel and transferred to a Hybond N⁺ membrane. Viral RNAs were probed with a Dig-labeled DNA probe corresponding to the 3′-end 400 nucleotides of the IBV genome. Numbers on the right indicate the genomic and subgenomic RNA species of IBV. (C) Partial reversion of Asn to Gln at amino acid position 3462 during passage of the Q3462N mutant virus. The plaque-purified Q3462N mutant virus was successively passaged on Vero cells using supernatants from the cells infected with this mutant virus at an MOI of 1 PFU/cell for 16 h. Total RNA was extracted from the infected cells, and RT-PCR was carried out. Sequencing the gel-purified RT-PCR products displayed an increasing trend reverting AAC to CAA, causing partial reversion of Asn to wild-type Gln from passage 2 to 5.

FIG. 4.

Analysis of IBV genomic RNA replication and subgenomic RNA transcription in cells electroporated with full-length mutant transcripts. (A) Detection of positive- and negative-strand genomic RNA (gRNA) replication in cells electroporated with wild-type and mutant full-length transcripts. Total RNA was prepared from Vero cells electroporated with in vitro-synthesized full-length transcripts at 48 h postelectroporation. The region corresponding to nucleotides 14931 to 15600 of the positive (+)- and negative (−)-sense IBV genomic RNA was amplified by RT-PCR and analyzed on 1.2% agarose gel. Lane 1 shows DNA markers, and lanes C show negative control. (B) Detection of the negative- and positive-strand subgenomic RNA synthesis in cells electroporated with wild-type and mutant transcripts. Total RNA was prepared from Vero cells electroporated with in vitro synthesized full-length transcripts at 48 h postelectroporation. Regions corresponding to the 5′-terminal 415 and 1010 nucleotides of subgenomic RNA 4 and 3, respectively, were amplified by RT-PCR and analyzed on 1.2% agarose gel. Lane 1 shows DNA markers. The sizes of the molecular weight markers are indicated on the left.

FIG. 5.

Effects of amino acid substitutions at Q6327 on IBV replication. (A) Growth properties of the Q6327H mutant virus. Vero cells were infected with wild-type and mutant viruses at an MOI of 1 PFU/cell and harvested at 0, 4, 8, 12, 16, 24, and 36 h postinoculation. Viral stocks were prepared by freeze-thawing the cells three times, and the TCID₅₀ of each viral stock was determined by infecting five wells of Vero cells on 96-well plates in triplicate with a 10-fold serial dilution of each viral stock. Error bar shows standard error of the mean. (B) Detection of genomic and subgenomic RNA synthesis in cells electroporated with full-length Q6327P mutant transcripts. Total RNA was prepared from Vero cells electroporated with in vitro-synthesized full-length transcripts at 48 h postelectroporation. Regions corresponding to nucleotides 14931 to 15600 of the positive (+)- and negative (−)-sense IBV genomic RNA, and the 5′-terminal 415 and 1010 nucleotides of subgenomic RNA 4 and 3, respectively, were amplified by RT-PCR and analyzed on 1.2% agarose gel. Lanes 1 and 9 show DNA markers. The sizes of the molecular weight markers are indicated on the left.

FIG. 6.

Effects of a single amino acid mutation (P166S or P166L) in 3CLpro on the cleavage efficiency for the nsp15-16/Q6327N mutant substrate and IBV replication. (A) Existence of mixed nucleotides at nucleotide positions 9361 and 9362 in passage 1, 3, and 9 of the recovered double mutant viruses. Wild-type sequence in passage 6 of the recovered Q6327N mutant virus, which did not show syncytium formation, is shown on top. Viral RNA was extracted, and RT-PCR was performed. The purified RT-PCR products were directly sequenced. (B) Growth properties of mutant viruses. Vero cells were infected with the indicated viruses at an MOI of 1 PFU/cell and harvested at 0, 4, 8, 12, 16, and 24 h postinoculation. Viral stocks were prepared by freeze-thawing the cells three times, and the TCID₅₀ of each viral stock was determined by infecting five wells of Vero cells on 96-well plates in triplicate with a 10-fold serial dilution of each viral stock. Error bar shows standard error of the mean. (C) Comparison of the cleavage efficiencies of wild-type (wt) and mutant 3CLpro at the nsp15-16 site containing the Q6327N mutation. Wild-type and mutant substrates were coexpressed with wild-type and mutant 3CLpro in H1299 cells using the vaccinia virus-T7 system. The transfected cell lysates were resolved on a 15% SDS-polyacrylamide gel and subjected to Western blotting with anti-Flag antibody. As a control, the amount of expressed 3CLpro was probed with anti-3CLpro antibody. The relative cleavage efficiency (RCE) is shown.