Enterovirus 71 3C Protease Cleaves a Novel Target CstF-64 and Inhibits Cellular Polyadenylation

Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell–virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3C^pro) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3′ pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C^pro. CstF-64 was cleaved in vitro by 3C^pro but neither by mutant 3C^pro (in which the catalytic site was inactivated) nor by another EV71 protease 2A^pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C^pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3′-end pre-mRNA processing and polyadenylation in 3C^pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C^pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.     

The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3C^pro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3C^pro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3C^pro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3C^pro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.     

Conformational Plasticity of the 2A Proteinase from Enterovirus 71

The 2A proteinase (2A^pro) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A^pro from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A^pro was comprised of an N-terminal domain of a four-stranded antiparallel β sheet and a C-terminal domain of a six-stranded antiparallel β barrel with a tightly bound zinc atom. Unlike in other 2A^pro structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A^pro of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII β strands. This was supported by molecular dynamic simulation. The structural variation among different 2A^pro structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target.     

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2A^pro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID₅₀) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2A^pro region of the EV71 genome exerted antiviral effects in vitro.     

丙型肝炎病毒感染过程中其NS3/4A蛋白酶切割线粒体抗病毒信号蛋白的动态过程

已知丙型肝炎病毒(hepatitis C virus,HCV)可通过其蛋白酶NS3/4A切割线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)来逃逸天然免疫识别,但尚不清楚其切割动力学及切割在抑制干扰素中的作用。本研究旨在细胞模型中探讨HCV感染过程中病毒复制建立及病毒NS3/4A切割MAVS的动态过程,探究NS3/4A切割MAVS对病毒逃逸宿主天然免疫建立感染的贡献。首先构建基于绿色荧光蛋白(green fluorescent protein,GFP)的MAVS切割报告系统(GFP-NLS-MAVS-TM462),用 HCV Jc1-Gluc 感染Huh7.5/GFP-NLS-MAVS-TM462细胞。结果显示,病毒复制早期MAVS切割效率较低;NS3/4A高效切割MAVS发生于HCV复制晚期,且其切割效率与NS3蛋白水平相关。利用带有GFP ypet的HCV报告病毒Jc1-378-1感染Huh7.5/RFP-NLS-MAVS-TM462细胞,在单细胞水平观察HCV感染阳性细胞中MAVS被切割情况,发现HCV复制细胞中仅部分细胞MAVS被切割。进一步研究发现,不同基因型NS3/4A切割MAVS的效率仅与NS3表达水平相关。以上结果提示,HCV蛋白酶NS3/4A切割MAVS依赖NS3/4A蛋白在病毒复制过程中的累积,对在病毒复制早期逃逸宿主天然免疫建立感染可能无显著贡献。     

EV71 3C protease cleaves host anti-viral factor OAS3 and enhances virus replication

The global spread of enteroviruses (EVs) has become more frequent, severe and life-threatening. Intereron (IFN) I has been proved to control EVs by regulating IFN-stimulated genes (ISG) expression. 20-50-oligoadenylate synthetases 3 (OAS3) is an important ISG in the OAS/RNase L antiviral system. The relationship between OAS3 and EVs is still unclear. Here, we reveal that OAS3, superior to OAS1 and OAS2, significantly inhibited EV71 replication in vitro. However, EV71 utilized autologous 3C protease (3C^pro) to cleave intracellular OAS3 and enhance viral replication. Rupintrivir, a human rhinovirus 3C protease inhibitor, completely abolished the cleavage of EV71 3C^pro on OAS3. And the proteolytically deficient mutants H40G, E71A, and C147G of EV71 3C^pro also lost the ability of OAS3 cleavage. Mechanistically, the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3C^pro cutting site. Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3 (CVB3), Coxsackievirus A16 (CA16), Enterovirus D68 (EVD68), and Coxsackievirus A6 (CA6) subtypes. Notably, unlike other four subtypes, CA16 3C^pro could not cleave OAS3. Two key amino acids variation Ile36 and Val86 in CA16 3C^pro might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage. Our works elucidate the broad anti-EVs function of OAS3, and illuminate a novel mechanism by which EV71 use 3C^pro to escape the antiviral effect of OAS3. These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.     

Specific Cleavage of the Nuclear Pore Complex Protein Nup62 by a Viral Protease

Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A^pro) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A^pro is a major contributor to Nup62 processing. The ability of purified 2A^pro to cleave bacterially expressed and purified Nup62 demonstrated that 2A^pro directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A^pro in vitro. This analysis revealed that 2A^pro cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.     

Autoprocessing: an essential step for expression and purification of enterovirus 71 3Cpro in Escherichia coli

A gene encoding the 3BC of human enterovirus 71 (EV71) was cloned and inserted into a derivative of plasmid pET-32a(+) driven by T7 promoter. The expressed 3C protease (3C^pro) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3C^pro partitioned in the soluble fraction of bacterial lysate. The 13-amino-acid peptide substrates with the junction of 3B/3C were used to verify the proteolysis activity of the purified 3C^pro. The EV71 3C^pro had a K_m value of 63 μM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3C^pro was developed using HPLC to analyze the proteolytic products. The combination of two activity assays contributes to promote the identification of the specific inhibitors targeted to the EV71 3C^pro.     

Crystal structure of human enterovirus 71 3C protease

Human enterovirus 71 (EV71) is the major pathogen that causes hand, foot and mouth disease that particularly affects young children. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date. The genome of EV71 consists of a positive sense, single-stranded RNA of ∼ 7400 bp, encoding a large precursor polyprotein that requires proteolytic processing to generate mature viral proteins. The proteolytic processing mainly depends on EV71 3C protease (3C^pro) that possesses both proteolysis and RNA binding activities, which enable the protease to perform multiple tasks in viral replication and pathogen-host interactions. The central roles played by EV71 3C^pro make it an appealing target for antiviral drug development. We determined the first crystal structure of EV71 3C^pro and analyzed its enzymatic activity. The crystal structure shows that EV71 3C^pro has a typical chymotrypsin-like fold that is common in picornaviral 3C^pro. Strikingly, we found an important surface loop, also denoted as β-ribbon, which adopts a novel open conformation in EV71 3C^pro. We identified two important residues located at the base of the β-ribbon, Gly123 and His133, which form hinges that govern the intrinsic flexibility of the ribbon. Structure-guided mutagenesis studies revealed that the hinge residues are important to EV71 3C^pro proteolytic activities. In summary, our work provides the first structural insight into EV71 3C^pro, including a mobile β-ribbon, which is relevant to the proteolytic mechanism. Our data also provides a framework for structure-guided inhibitor design against EV71 3C^pro.     

Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2

Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A^pro or L^pro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2A^pro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) L^pro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2A^pro and L^pro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified L^pro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.     

Proteomic elucidation of the targets and primary functions of the picornavirus 2A protease

Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2A^pro and 3C^pro, which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2A^pro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2A^pro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2A^pro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions.     

Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus‐infected cell

Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A^pro). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET‐based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP²) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real‐time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A^pro catalytic activity, irrespective of other viral‐encoded protease, the activated caspases or general inhibition of protein synthesis in the EV‐infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease–substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus‐induced host translation inhibition. Biotechnol. Bioeng. 2009; 104: 1142–1152. © 2009 Wiley Periodicals, Inc.     

Differential processing of nuclear pore complex proteins by rhinovirus 2A proteases from different species and serotypes

Human rhinoviruses (HRVs) from the HRV-A, HRV-B, and HRV-C species use encoded proteases, 2A^pro and 3C^pro, to process their polyproteins and shut off host cell activities detrimental to virus replication. Reactions attributed to 2A^pro include cleavage of eIF4G-I and -II to inhibit cellular mRNA translation and cleavage of select nucleoporin proteins (Nups) within nuclear pore complexes (NPCs) to disrupt karyopherin-dependent nuclear-cytoplasmic transport and signaling. Sequence diversity among 2A^pro proteases from different HRV clades, even within species, suggested individual viruses might carry out these processes with unique mechanistic signatures. Six different recombinant 2A^pro proteases (A16, A89, B04, B14, Cw12, and Cw24) were compared for their relative substrate preferences and cleavage kinetics using eIF4G from cellular extracts and Nups presented in native (NPC) or recombinant formats. The enzyme panel attacked these substrates with different rates or processing profiles, mimicking the preferences observed during natural infection (A16 and B14). For eIF4G, all 2A^pro proteases cleaved at similar sites, but the comparative rates were species specific (HRV-A > HRV-C ≫ HRV-B). For Nup substrates, 5 of the 6 enzymes had unique product profiles (order of Nup selection) or reacted at different sites within Nup62, Nup98, and Nup153. Only A16 and A89 behaved similarly in most assays. Since each type of karyopherin receptor prefers particular Nups or uses a limited cohort of binding motifs within those Nups, the consequences of individual 2A^pro avidities could profoundly affect relative viral replication levels, intracellular signaling, or extracellular signaling, all of which are underlying triggers for different host immune responses.     

Modulation of Innate Immune Signalling by Lipid-Mediated MAVS Transmembrane Domain Oligomerization

RIG-I-like receptors detect viral RNA in infected cells and promote oligomerization of the outer mitochondrial membrane protein MAVS to induce innate immunity to viral infection through type I interferon production. Mitochondrial reactive oxygen species (mROS) have been shown to enhance anti-viral MAVS signalling, but the mechanisms have remained obscure. Using a biochemical oligomerization-reporter fused to the transmembrane domain of MAVS, we found that mROS inducers promoted lipid-dependent MAVS transmembrane domain oligomerization in the plane of the outer mitochondrial membrane. These events were mirrored by Sendai virus infection, which similarly induced lipid peroxidation and promoted lipid-dependent MAVS transmembrane domain oligomerization. Our observations point to a role for mROS-induced changes in lipid bilayer properties in modulating antiviral innate signalling by favouring the oligomerization of MAVS transmembrane domain in the outer-mitochondrial membrane.     

Differential Regulation of TLR Signaling on the Induction of Antiviral Interferons in Human Intestinal Epithelial Cells Infected with Enterovirus 71

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease, which can lead to fatal neurological complications in young children and infants. Few gastrointestinal symptoms are observed clinically, suggesting the presence of a unique immunity to EV71 in the gut. We reported a robust induction of interferons (IFNs) in human intestinal epithelial cells (HT-29), which was suppressed in other types such as RD and HeLa cells. The underlying mechanism for the apparent difference remains obscure. In this study we report that in EV71-infected HT-29 cells, TLR/TRIF signaling was essential to IFN induction; viral replication increased and the induction of IFN-α, -β, -ω, -κ, and -ε decreased markedly in TRIF-silenced HT-29 cells. Importantly, TRIF was degraded by viral 3C^pro in RD cells, but resisted cleavage, and IRF3 was activated and translocated into the nucleus in HT-29 cells. Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection.     

Mitochondrial anti-viral immunity

In the cytosol, the sensing of RNA viruses by the RIG-I-like receptors (RLRs) triggers a complex signaling cascade where the mitochondrial antiviral signaling protein (MAVS) plays a crucial role in orchestrating the innate host response through the induction of antiviral and inflammatory responses. Hence, in addition to their known roles in the metabolic processes and the control of programmed cell death, mitochondria are now emerging as a fundamental hub for innate anti-viral immunity. This review summarizes the findings related to the MAVS adapter and mitochondria in the innate immune response to RNA viruses.     

The mitochondrial antiviral signaling protein, MAVS, is cleaved during apoptosis

Apoptosis of virus-infected cells is one important host strategy used to limit viral infection. Recently a member of the innate immune signaling pathway, MAVS, was localized to mitochondria, an organelle important for apoptosis regulation. Here we investigate what role MAVS may play in apoptosis. Induction of cell death led to the rapid cleavage of MAVS, resulting in its release from the outer mitochondrial membrane. This cleavage is blocked in cells incubated with proteasome or caspase inhibitors. Transfection of synthetic viral dsRNA and dsDNA also led to cleavage of MAVS, indicating that this process may be important during infection. Preventing apoptosis by over-expression of anti-apoptotic Bcl-xL blocks MAVS cleavage, placing this process downstream of caspase activation in the apoptotic program.     

Virus-Infection or 5′ppp-RNA Activates Antiviral Signal through Redistribution of IPS-1 Mediated by MFN1

In virus-infected cells, RIG-I-like receptor (RLR) recognizes cytoplasmic viral RNA and triggers innate immune responses including production of type I and III interferon (IFN) and the subsequent expression of IFN-inducible genes. Interferon-β promoter stimulator 1 (IPS-1, also known as MAVS, VISA and Cardif) is a downstream molecule of RLR and is expressed on the outer membrane of mitochondria. While it is known that the location of IPS-1 is essential to its function, its underlying mechanism is unknown. Our aim in this study was to delineate the function of mitochondria so as to identify more precisely its role in innate immunity. In doing so we discovered that viral infection as well as transfection with 5′ppp-RNA resulted in the redistribution of IPS-1 to form speckle-like aggregates in cells. We further found that Mitofusin 1 (MFN1), a key regulator of mitochondrial fusion and a protein associated with IPS-1 on the outer membrane of mitochondria, positively regulates RLR-mediated innate antiviral responses. Conversely, specific knockdown of MFN1 abrogates both the virus-induced redistribution of IPS-1 and IFN production. Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes.     

Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL^pro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL^pro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL^pro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL^pro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL^pro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL^pro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL^pro domain was found to suppress IFN-β promoter activation, PL^pro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL^pro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PL^pro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL^pro as a viral DUB during MERS-CoV infection.