The ADRP domain from a virulent strain of infectious bronchitis virus is not sufficient to confer a pathogenic phenotype to the attenuated Beaudette strain

Abstract

The replicase gene of the coronavirus infectious bronchitis virus (IBV) encodes 15 non-structural proteins (nsps). Nsp 3 is a multi-functional protein containing a conserved ADP-ribose-1″-phosphatase (ADRP) domain. The crystal structures of the domain from two strains of IBV, M41 (virulent) and Beaudette (avirulent), identified a key difference; M41 contains a conserved triple-glycine motif, whilst Beaudette contains a glycine-to-serine mutation that is predicted to abolish ADRP activity. Although ADRP activity has not been formally demonstrated for IBV nsp 3, Beaudette fails to bind ADP-ribose. The role of ADRP in virulence was investigated by generating rIBVs, based on Beaudette, containing either a restored triple-glycine motif or the complete M41 ADRP domain. Replication in vitro was unaffected by the ADRP modifications and the in vivo phenotype of the rIBVs was found to be apathogenic, indicating that restoration of the triple-glycine motif is not sufficient to restore virulence to the apathogenic Beaudette strain.

Keywords: ADRP; coronavirus; infectious bronchitis virus; macrodomain; nsp3; pathogenicity.

Fig. 1.

The ADRP domain. (a) Schematic of the IBV genome. The ADRP domain is located within non-structural protein (nsp) 3, encoded within the replicase gene. Nsp 3 is the largest of the nsps, and consists of several domains, including transmembrane domains, an acidic hypervariable region (Ac), papain-like cysteine proteinases (PL1 and PL2) and the Y region. (b) Schematic representation of the rIBVs analysed in this study. The sequence derived from M41 is shown in red and the sequence derived from Beau-R is shown in blue. (c) Sequence alignment of the ADRP domain from M41 and Beaudette, amino acid residues 1003–1171 of the IBV replicase protein. The ADRP domain is largely conserved. An asterisk (*) highlights the difference within the binding cleft.

Fig. 2.

Activation of ADRP has no effect on viral replication in vitro. Primary chicken kidney (CK) cells and DF-1 cells, a continuous cell line derived from chicken embryo fibroblasts, were inoculated with 5×10⁴ plaque-forming units (p.f.u.) of (a) rIBV BeauR-G-ADRP, Beau-R or BeauR-M41-ADRP, or (b) rIBV BeauR-M41(S), BeauR-M41(S)-G-ADRP or BeauR-M41(S)-M41-ADRP. Supernatant, containing viral progeny, was harvested at 1, 24, 48, 72 and 96 h post-infection (h.p.i.), and was titrated in triplicate on CK cells. The means of three independent experiments are plotted, with the error bars representing the standard error of the mean. The data were analysed using a one-way analysis of variance (ANOVA) with a Friedman test and Dunn’s multiple comparison test; the replication of the rIBVs was not statistically different to that of the parent viruses, rIBV Beau-R and rIBV BeauR-M41(S).

Fig. 3.

An active ADRP domain is insufficient to confer pathogenicity to rIBV Beau-R. Eight-day-old chickens were inoculated with 10⁴ p.f.u. of either M41-CK, Beau-R, BeauR-M41(S), BeauR-G-ADRP, BeauR-M41-ADRP, BeauR-M41(S)-G-ADRP or BeauR-M41(S)-M41-ADRP. Clinical signs, such as (a) snicking and (b) rales, were assessed on days 3 to 7 post-infection. Birds infected with BeauR-G-ADRP displayed no clinical signs, similarly to those infected with Beau-R. In contrast, snicking and rales were observed in chickens infected with M41-CK from day 3 post-inoculation. (c) Four and 6 days post-infection, three birds per group were culled and the ciliary activity in 10×1 mm trachea sections was assessed by light microscopy and the percentage ciliary activity was calculated. The retention of less than 50 % tracheal ciliary activity 4 days post-infection is indicative of the presence of a pathogenic isolate of IBV. The error bars represent the standard error of the mean. Ciliary activity was analysed using a one-way ANOVA with a Friedman test and Dunn’s multiple comparison test. The ciliary activity was statistically comparable between Beau-R- and BeauR-G-ADRP-infected chickens, confirming that rIBV BeauR-G-ADRP is avirulent in chickens.