Date Published: June 29, 2018
Publisher: Public Library of Science
Author(s): Miguel Rodríguez Pulido, María Teresa Sánchez-Aparicio, Encarnación Martínez-Salas, Adolfo García-Sastre, Francisco Sobrino, Margarita Sáiz, Bert L. Semler.
The RNA helicase LGP2 (Laboratory of Genetics and Physiology 2) is a non-signaling member of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), whose pivotal role on innate immune responses against RNA viruses is being increasingly uncovered. LGP2 is known to work in synergy with melanoma differentiation-associated gene 5 (MDA5) to promote the antiviral response induced by picornavirus infection. Here, we describe the activity of the foot-and-mouth disease virus (FMDV) Leader protease (Lpro) targeting LGP2 for cleavage. When LGP2 and Lpro were co-expressed, cleavage products were observed in an Lpro dose-dependent manner while co-expression with a catalytically inactive Lpro mutant had no effect on LGP2 levels or pattern. We further show that Lpro localizes and immunoprecipitates with LGP2 in transfected cells supporting their interaction within the cytoplasm. Evidence of LGP2 proteolysis was also detected during FMDV infection. Moreover, the inhibitory effect of LGP2 overexpression on FMDV growth observed was reverted when Lpro was co-expressed, concomitant with lower levels of IFN-β mRNA and antiviral activity in those cells. The Lpro target site in LGP2 was identified as an RGRAR sequence in a conserved helicase motif whose replacement to EGEAE abrogated LGP2 cleavage by Lpro. Taken together, these data suggest that LGP2 cleavage by the Leader protease of aphthoviruses may represent a novel antagonistic mechanism for immune evasion.
Antiviral response against RNA viruses greatly relies on detection of infection by cytoplasmic sensors. Among the different pattern-recognition receptors (PRRs) involved in antiviral immunity, the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), recognize non-self RNA species derived from viral infection triggering the downstream signaling cascade leading to type-I interferon (IFN) secretion and host antiviral response [1,2]. RLRs are ubiquitous cytosolic RNA helicases including RIG-I, melanoma differentiation-associated gene 5 (MDA5) and LGP2 (Laboratory of Genetics and Physiology 2). All three RLRs share a DExD/H box RNA helicase domain and a C-terminal domain (CTD). The helicase domain and the CTD bind to viral RNA, CTD being essential for the specific recognition of RNA substrate features. The helicase domain generally functions to coordinate RNA binding, ATP hydrolysis, and conformational rearrangements upon RNA recognition [2,3]. The RLRs share the ability to detect molecular signatures of virus infection, but differ in both their RNA recognition specificity and signaling properties. RIG-I senses primarily 5´-triphosphate blunt-end dsRNA, while MDA5 is activated by long dsRNA, consequently responding to different but overlapping sets of viruses [4,5]. LGP2 has the highest RNA binding affinity of the RLRs, and has the ability to recognize diverse dsRNAs, regardless of the presence of 5´-triphosphate or RNA length . RIG-I and MDA5 contain N-terminal tandem caspase activation and recruitment domains (CARDs) which upon recognition of viral RNA, interact with the CARD of the mitochondrial activator of virus signaling (MAVS) protein, the essential adaptor molecule for RLR signaling. LGP2 lacks the N-terminal CARDs and then independent signaling activity. However, LGP2 is known to be widely involved in viral RNA recognition and regulation during innate immune responses, remaining the most enigmatic member of the RLR family . Both negative and positive regulatory roles have been reported for LGP2 in antiviral immunity. An enhancing effect on MDA5-mediated signaling was found when LGP2 was present at low cellular concentrations. According to a model based on a concentration dependent biphasic switch, at early stages of infection low levels of LGP2 would enhance MDA5-mediated antiviral signaling, but as infection progresses and LGP2 production is induced by IFN, LGP2 would act as a negative feedback regulator inhibiting MDA5 signaling [7–9]. Single molecule RNA binding experiments and biochemical analysis revealed that ATP hydrolysis activity is required to enable LGP2 to efficiently engage diverse dsRNA species, and for enhancement of MDA5 signaling . An RNA- and virus-independent inhibitory role for LGP2 in antiviral signaling has also been reported, likely involving CARD-independent interaction with MAVS by competition with an essential kinase for binding and interfering with downstream signaling .
In this study, we identified the innate immune sensor LGP2 as a target for the FMDV Leader protease. The IFN system is a powerful component of the antiviral response and viruses have evolved sophisticated strategies to evade the host innate immune response by interfering with the different events involved in PRR activation and signaling [15,16]. FMDV is no exception, and viral proteases have been found to counteract the innate responses induced in cells during the course of infection [17,18]. Lpro is known to prevent the host antiviral response by several mechanisms including cleavage of initiation factor eIF4G – and then prevention of the synthesis of IFN and other cytokines immediately after infection-, degradation of NF-κB, and deubiquitination of immune signaling molecules [18,24]. Though the contribution of LGP2 to innate immune activation is still not fully understood, recent work unveils a relevant regulatory role on RLR signaling through CARD-independent interactions. The dsRNA generated during picornavirus replication is sensed by MDA5, and LGP2 is believed to promote the viral RNA-MDA5 interaction leading to efficient antiviral signaling . Indeed, RNA derived from EMCV infection with strong MDA5-stimulatory activity was immunoprecipitated with LGP2 . It is also known that either LGP2 or MDA5 deficiency results in higher susceptibility to picornavirus infections [38,39]. A recent report shows that, in the absence of infection or viral proteins, LGP2 functioned as a biphasic master activator of numerous innate immunity genes, sequentially induced in a cascade fashion leading to production of IFN. In turn, LGP2 was subject to negative control by cellular translation regulators .