Proteolytic processing of polyproteins 1a and 1ab between non-structural proteins 10 and 11/12 of Coronavirus infectious bronchitis virus is dispensable for viral replication in cultured cells

Abstract

Coronavirus 3C-like proteinase (3CLpro) plays important roles in viral life cycle through extensive processing of the polyproteins 1a and 1ab into 12 mature, non-structural proteins (nsp5-nsp16). Structural and biochemical studies have revealed that all confirmed 3CLpro cleavage sites have a conserved Gln residue at the P1 position, which is thought to be absolutely required for efficient cleavage. Recent studies on murine hepatitis virus (MHV) showed that processing of the 1a polyprotein at the position between nsp10-nsp11 is essential for viral replication. In this report, we investigated the requirement of processing at the equivalent position for replication of avian coronavirus infectious bronchitis virus (IBV), using an infectious cloning system. The results showed that mutation of the P1 Gln to Pro or deletion of the Gln residue in the nsp10-nsp11/12 site completely abolished the 3CLpro-mediated processing, but allowed production of infectious recombinant viruses with variable degrees of growth defect, suggesting that cleavage at the nsp10-nsp11/12 site of IBV is dispensable for viral replication in cultured cells. This study would pave a way for potential vaccine development by generation of attenuated IBV from field isolates through manipulation of the nsp10-nsp11/12 cleavage site. Similar approaches would be also applicable to other human and animal coronaviruses.

Fig. 1

(a) IBV genome organization and proteolytic processing of IBV replicase polyproteins. Shown are the replicase polyproteins 1a and 1ab of IBV. The processing end-products of 1a are designated non-structural proteins (nsp) 2 to nsp11, and those of polyprotein 1ab are designated nsp2 to nsp10 and nsp12 to nsp16. The cleavage sites of IBV 3CLpro are indicated with black arrow. The cleavage sites of papain-like proteinase (PLP) are indicated with block arrow. (b) Effect of deletion and mutation of the P1 residue at the cleavage site between nsp10–nsp11/12. Plasmids carrying wild type and mutant IBV sequences from nucleotides 12100–12729 with insertion of an extra T at position between nucleotides 12347–12348 were co-expressed with the IBV 3CLpro in Vero cells using the vaccinia virus-T7 system. Cells were harvested at 16 h post-transfection, lysates were prepared and separated on 15% SDS-PAGE. Western blot was performed using anti-FLAG antibody. The cleaved product (9.1 kDa) and uncleaved substrate (24.8 kDa) were indicated.

Fig. 2

(a) Analysis of the growth properties of wild type and mutant viruses. Vero cells were infected with wild type (rIBV) and three mutant viruses Q3928N, Q3928P and ΔQ3928 at a multiplicity of infection of approximately 1. Cells were harvested at 0, 4, 8, 12, 16, 24 and 36 h post-infection, respectively. Viral stocks were prepared by freezing/thawing of the cells three times, and TCID₅₀ of each viral preparation was determined by infection of five wells of Vero cells on 96-well plates in triplicate with 10-fold serial dilution of each viral stock. (b) Processing of nsp10–nsp12 in wild type and mutant viruses. Vero cells were infected with rIBV, Q3928N, Q3928P and ΔQ3928, respectively, at a multiplicity of infection of approximately 1 for 4 h and labeled with ³⁵S-Met-containing medium for 16 h. Cells were harvested, lysed and proteins were immunoprecipitated with polyclonal antiserum against IBV nsp12. After resolved by 6% SDS-PAGE, proteins were detected by fluorography. Bands corresponding to nsp12 (100 kDa, nsp12) and the nsp10–nsp12 fusion protein (120 kDa, nsp10–12) are indicated.

Fig. 3

(a) Northern blot analysis of RNA synthesis in cells infected with wild type IBV and passage 20 of the mutant viruses Q3928N, Q3928P and ΔQ3928, respectively. Vero cells were infected with wild type and the mutant viruses at a multiplicity of infection of approximately 1, and harvested at 16 h post-infection. Total RNA was extracted from the infected cells and separated on a 0.8% denatured agarose gel. After transferring to nylon membrane, viral RNAs were detected by hybridization with a DIG-labeled DNA probe specific for the 3′-UTR of IBV. (b) Genetic stability of the mutant viruses. Total RNA was prepared from Vero cells infected with passage 20 of the mutant virus Q3928N, Q3928P, ΔQ3928 and ΔQ3928–S3929, and the region covering mutation or deletion was amplified by RT-PCR and sequenced. The changed or deleted nucleotides were indicated.