1. Introduction
Pattern recognition receptors (PRRs) are responsible for sensing the presence of pathogens, including viruses, since they recognise conserved features of microbes known as pathogen-associated molecular patterns (PAMPs) (Akira et al., 2006). Four different classes of PRRs have been identified: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and NOD-like receptors (NLRs) (Takeuchi and Akira, 2010).
RLRs are PRRs expressed both in professional and in various non-professional immune cells, including epithelial cells (Takeuchi and Akira, 2010). RLRs play a major role in triggering and modulating antiviral immunity by detecting exogenous viral RNAs (Loo and Gale, 2011; Yoneyama et al., 2015). The RLR family is composed of retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). RLRs localise to the cytosol, and their expression is maintained at low levels in resting cells but is greatly increased after virus infection (Loo and Gale, 2011; Chow et al., 2018). The activation of RIG-I and MDA5 is regulated by multiple ubiquitin E3 ligases of the tripartite motif (TRIM) proteins such as TRIM containing 25 (TRIM25) and Riplet (Oshiumi et al., 2013). RIG-I and MDA5 sense viral RNAs through the mitochondrial antiviral signalling (MAVS) protein (Seth et al., 2005). Although the majority of MAVS is present on the outer mitochondrial membrane (OMM), a small proportion is located in the mitochondria-associated membranes (MAMs) as well as in the peroxisomes (Goubau et al., 2013). RIG-I and MDA5 harbour caspase activation and recruitment domains (CARDs), and they bind to and activate MAVS via CARD-CARD interactions, triggering polymerisation of MAVS into prion-like structures required for antiviral signalling (Hou et al., 2011; Banoth and Cassel, 2018). Activation of MAVS on mitochondria and MAMs results in stimulation of the kinases TBK1 and IKK and, consequently, of the transcription factors IRF3, IRF7, and NF-κB for the induction of genes encoding type I and type III interferon and pro-inflammatory cytokines (Onoguchi et al., 2007; Chow et al., 2018; Ablasser and Hur, 2020). LGP2 lacks antiviral signalling activity. LGP2 has been proposed to be an accessory protein important for regulating RIG-I and MDA5 signalling (Chow et al., 2018). Indeed, LGP2 interacts with MAVS in microsomes, blocking RIG-I/MAVS. After virus infection, LGP2 is rapidly released from MAVS and redistributed to mitochondria, which correlates with IRF3 activation (Esser-Nobis et al., 2020).
Besides RNA ligands from RNA viruses, RLRs recognise DNA ligands from DNA viruses as well as those derived from bacteria (Chow et al., 2018). RLRs are known to detect herpesviruses, adenoviruses, and poxviruses (Goubau et al., 2013; Chow et al., 2018; Huo et al., 2019). Recently, it has been shown that high-risk human papillomaviruses (HPVs) can downregulate RLR expression, thus creating a cellular milieu suitable for their persistence (Albertini et al., 2018).
Bovine papillomaviruses (BPVs), a heterogeneous group of species-specific viruses distributed worldwide (IARC, 2007), comprise 29 types assigned to five genera (https://pave.niaid.nih.gov/; Yamashita-Kawanishi et al. 2020). Bovine δPVs are known to infect epithelial and mesenchymal cells and are unique among BPVs to show cross-species transmission and infection (IARC, 2007). Besides skin tumours, bovine δPVs play a very important role in the bladder carcinogenesis of large ruminants, such as cattle and buffaloes (Campo et al., 1992; Roperto et al., 2013). Bovine δPVs show their transforming activity through the E5 protein, a highly conserved oncoprotein, believed to be the major δPV oncoprotein (DiMaio and Petti, 2013). E5 forms dimers and displays pathogen activity via numerous pathways in the absence of other viral genes (DiMaio and Petti, 2013). E5 can bind to the activated form of platelet-derived growth factor ß receptor (PDGFßR) (Borzacchiello et al., 2006; Roperto et al., 2013). E5 can also act via the calpain3 pathway and/or it binds to the subunit D of the V1-ATPase proton pump (Roperto et al., 2010b; Roperto et al., 2014). E6 and E7 are less studied δPV oncoproteins.
We aimed to investigate the interaction between E5 oncoprotein and E3 ubiquitin ligase TRIM25 and the downregulation of multiple downstream effectors of the host antiviral response pathway mediated by the RLRs in a spontaneous model of bovine papillomavirus disease.