A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase

Abstract

Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3C pro cleavage sites at the 5= and 3= ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-␤ expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event.IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order Picornavirales) and torovirus (order Nidovirales). This naturally occurring recombination event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that was inserted into the EVG genome encodes a deubiquitinase/ deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights into how a gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity.KEYWORDS enterovirus G, torovirus, genetic recombination, papain-like protease, deubiquitinase

First detection of novel enterovirus G recombining a torovirus papain‐like protease gene associated with diarrhoea in swine in South Korea

“…The viral 3C pro protease trims the EV‐G precursor polyprotein posttranslationally into the 2C and 3A proteins at a well‐defined cleavage sequence (ALFQ↓GPPT) (Blom, Hansen, Brunak, & Blaas, ). Furthermore, a previous study using reverse genetics revealed that the US EV‐G17‐PLCP strain functionally produces the exogenous PLCP gene in virus‐infected cells, demonstrating its own ALFQ↓GPPV and AEFQ↓GPPT sequences as the putative cleavage sites (Shang et al., ). Considering the sequence similarity of the putative cleavage sites including GPPT−ALFQ, GPPA−ALFQ, and GPPE−ALPQ among global strains (Conceição‐Neto et al., ; Knutson et al., ; Tsuchiaka et al., ), therefore, the cleavage of the inserted PLCP gene appears to be guaranteed using each corresponding cleavage sequence.…”

“…The length of the inserted PLCP sequence varied among strains from different geographical locations (194/223‐aa, 212‐aa, and 211/217‐aa for the US, Belgium, and Japanese strains respectively) (Figure b). The PLCP sequence of Korean EV‐G only shared 75.0%−89.3% aa sequence identity with other recombinant EV‐G1, ‐G2, and ‐G17 strains, exhibiting the highest identity with the US EV‐G17 strain 08/NC_USA/2015 (Shang et al., ). The viral 3C pro protease trims the EV‐G precursor polyprotein posttranslationally into the 2C and 3A proteins at a well‐defined cleavage sequence (ALFQ↓GPPT) (Blom, Hansen, Brunak, & Blaas, ).…”

A novel defective recombinant porcine enterovirus G virus carrying a porcine torovirus papain-like cysteine protease gene and a putative anti-apoptosis gene in place of viral structural protein genes

“…In addition to type 1 recombinant EV-G, second type of recombinant EV-G (we call type 2 recombinant EV-G in this study), which carried the PLCP gene in place of viral structural genes, has been identified in a Chinese pig farm (Wang et al, 2018). The PLCP gene is encoded in the ORF1 of the genome of torovirus, a member of family Coronaviridae, and the order Nidovirales (Conceição-Neto et al, 2017;Knutson et al, 2017;Shang et al, 2017;Tsuchiaka et al, 2017;Wang et al, 2018). The PLCP of nidoviruses serves as a protease to cleave viral gene 1 polyproteins to mature proteins (Mielech et al, 2014).…”

“…The PLCP of nidoviruses serves as a protease to cleave viral gene 1 polyproteins to mature proteins (Mielech et al, 2014). It also has functions of deubiquitinating and deISGylating (interferonstimulated gene15-removing) enzymes, which plays important roles in viral pathogenesis by acting as an innate immunity antagonist (Shang et al, 2017).…”

“…1016/j.meegid.2019.103975 Received 18 April 2019; Received in revised form 17 July 2019; Accepted 19 July 2019 prevalence of type 1 recombinant EV-G in the EV-G population (Tsuchiaka et al, 2018). Type 1 recombinant EV-Gs have been discovered from feces of neonatal pigs showing clinical symptoms, such as diarrhea, in the US, Belgium, Germany and South Korea (Bunke et al, 2018;Conceição-Lee and Lee, 2019;Neto et al, 2017;Knutson et al, 2017;Shang et al, 2017), while type 1 recombinant EV-Gs identified in Japan are detected from normal as well as diarrhea pig feces (Tsuchiaka Fig. 1.…”