Structural and dynamics characterization of norovirus protease

Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X‐ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C‐like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹⁵N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C‐terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹⁵N spin relaxation and Carr–Purcell–Meiboom–Gill (CPMG)‐based relaxation dispersion analyses reveal the dynamic properties of residues in the C‐terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123–G133 show fast motion (ps‐ns), and the residues in the bII–cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (μs–ms), suggesting their important role in substrate recognition.     

Recent development of 3C and 3CL protease inhibitors for anti-coronavirus and anti-picornavirus drug discovery

SARS-CoV (severe acute respiratory syndrome-associated coronavirus) caused infection of ~8000 people and death of ~800 patients around the world during the 2003 outbreak. In addition, picornaviruses such as enterovirus, coxsackievirus and rhinovirus also can cause life-threatening diseases. Replication of picornaviruses and coronaviruses requires 3Cpro (3C protease) and 3CLpro (3C-like protease) respectively, which are structurally analogous with chymotrypsin-fold, but the former is a monomer and the latter is dimeric due to an extra third domain for dimerization. Subtle structural differences in the S2 and S3 pockets of these proteases make inhibitors selective, but some dual inhibitors have been discovered. Our findings as summarized in the present review provide new potential anti-coronavirus and anti-picornavirus therapeutic agents and a clue to convert 3CLpro inhibitors into 3Cpro inhibitors and vice versa.     

Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.     

Putative structural rearrangements associated with the interaction of macrocyclic inhibitors with norovirus 3CL protease

Human noroviruses are the primary cause of outbreaks of acute gastroenteritis worldwide. The problem is further compounded by the current lack of norovirus-specific antivirals or vaccines. Noroviruses have a single-stranded, positive sense 7 to 8 kb RNA genome which encodes a polyprotein precursor that is processed by a virus-encoded 3C-like cysteine protease (NV 3CLpro) to generate at least six mature nonstructural proteins. Processing of the polyprotein is essential for virus replication, consequently, NV 3CLpro has emerged as an attractive target for the discovery of norovirus therapeutics and prophylactics. We have recently described the structure-based design of macrocyclic transition state inhibitors of NV 3CLpro. In order to gain insight and understanding into the interaction of macrocyclic inhibitors with the enzyme, as well as probe the effect of ring size on pharmacological activity and cellular permeability, additional macrocyclic inhibitors were synthesized and high resolution cocrystal structures determined. The results of our studies tentatively suggest that the macrocyclic scaffold may hamper optimal binding to the active site by impeding concerted cross-talk between the S₂ and S₄ subsites.     

Inhibition of SARS-CoV 3CL protease by flavonoids

Abstract

There were severe panics caused by Severe Acute Respiratory Syndrome (SARS) and Middle-East Respiratory Syndrome-Coronavirus. Therefore, researches targeting these viruses have been required. Coronaviruses (CoVs) have been rising targets of some flavonoids. The antiviral activity of some flavonoids against CoVs is presumed directly caused by inhibiting 3C-like protease (3CLpro). Here, we applied a flavonoid library to systematically probe inhibitory compounds against SARS-CoV 3CLpro. Herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of SARS-CoV 3CLpro. The interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. An induced-fit docking analysis indicated that S1, S2 and S3′ sites are involved in binding with flavonoids. The comparison with previous studies showed that Triton X-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. With the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors.     

Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.     

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.     

MERS冠状病毒3C样蛋白酶是一种新的细胞自噬诱导蛋白

冠状病毒（Coronavirus, CoV）3C样蛋白酶（3CLpro）在冠状病毒复制过程中起重要作用,是一种重要的潜在抗病毒药物候选靶标。细胞自噬是宿主重要抗病毒防御机制之一,但目前冠状病毒诱导细胞自噬及其机制还不很清楚。本研究以人类新发高致病性冠状病毒 --中东呼吸综合征冠状病毒（MERS CoV）为研究对象,探讨人类冠状病毒感染与细胞自噬的关系。通过免疫荧光法检测发现,MERS 3CLpro引起细胞内eGFP-LC3B绿色荧光点状聚集,同时MERS 3CLpro诱导自噬标志蛋白微管相关蛋白1-轻链3基 (LC3-II)表达增多,表明MERS 3CLpro可激活细胞自噬。进一步研究发现,MERS 3CLpro诱导细胞自噬体形成而阻断或抑制自噬溶酶体形成,即MERS 3CLpro诱导不完全细胞自噬效应,而且MERS 3CLpro诱导细胞自噬具有时间依赖性且不依赖于其蛋白酶催化活性。此外发现SARS CoV和NL63 CoV等其它人类冠状病毒3CLpro也具有诱导细胞自噬效应,表明3CLpro诱导细胞自噬可能是人类冠状病毒所具有的一种普遍生物学特性。本研究首次发现冠状病毒蛋白酶3CLpro能诱导宿主细胞自噬,是一种新型冠状病毒来源的宿主细胞自噬诱导蛋白,这一发现拓展了对人类冠状病毒蛋白酶功能的新认识,为研究冠状病毒与宿主抗病毒天然免疫以及以病毒蛋白酶为靶标的抗病毒药物研究提供了理论基础。     

Novel P1 chain-extended HIV protease inhibitors possessing potent anti-HIV activity and remarkable inverse antiviral resistance profiles

A novel series of tyrosine-derived HIV protease inhibitors was synthesized and evaluated for in vitro antiviral activity against wild-type virus and two protease inhibitor-resistant viruses. All of the compounds had wild-type antiviral activities that were similar to or greater than several currently marketed HIV protease inhibitors. In addition, a number of compounds in this series were more potent against the drug-resistant mutant viruses than they were against wild-type virus.     

X-ray crystallographic structure of the Norwalk virus protease at 1.5-A resolution

Norwalk virus (NV), a member of the Caliciviridae family, is the major cause of acute, epidemic, viral gastroenteritis. The NV genome is a positive sense, single-stranded RNA that encodes three open reading frames (ORFs). The first ORF produces a polyprotein that is processed by the viral cysteine protease into six nonstructural proteins. We have determined the structure of the NV protease to 1.5 and 2.2 A from crystals grown in the absence or presence, respectively, of the protease inhibitor AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride]. The protease, which crystallizes as a stable dimer, exhibits a two-domain structure similar to those of other viral cysteine proteases with a catalytic triad composed of His 30, Glu 54, and Cys 139. The native structure of the protease reveals strong hydrogen bond interactions between His 30 and Glu 54, in the favorable syn configuration, indicating a role of Glu 54 during proteolysis. Mutation of this residue to Ala abolished the protease activity, in a fluorogenic peptide substrate assay, further substantiating the role of Glu 54 during proteolysis. These observations contrast with the suggestion, from a previous study of another norovirus protease, that this residue may not have a prominent role in proteolysis (K. Nakamura, Y. Someya, T. Kumasaka, G. Ueno, M. Yamamoto, T. Sato, N. Takeda, T. Miyamura, and N. Tanaka, J. Virol. 79:13685-13693, 2005). In the structure from crystals grown in the presence of AEBSF, Glu 54 undergoes a conformational change leading to disruption of the hydrogen bond interactions with His 30. Since AEBSF was not apparent in the electron density map, it is possible that these conformational changes are due to subtle changes in pH caused by its addition during crystallization.     

Chemical Derivatives of a Small Molecule Deubiquitinase Inhibitor Have Antiviral Activity against Several RNA Viruses

Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale). Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility.     

N-Terminal Finger Stabilizes the S1 Pocket for the Reversible Feline Drug GC376 in the SARS-CoV-2 Mpro Dimer

The main protease (M^pro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug’s properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar K_i values with the M^pro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 M^pro. The comparison of a new X-ray crystal structure of M^pro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 M^pro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the M^pro, which facilitates coordination of the drug’s P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.     

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 μM) and La Crosse virus (EC(50)=0.27 μM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).     

Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs

Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-(P1) in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs.     

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.     

Synthesis, metabolic stability and antiviral evaluation of various alkoxyalkyl esters of cidofovir and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine

Alkoxyalkyl esters of cidofovir (CDV) are orally active agents which inhibit the replication of a variety of double stranded DNA (dsDNA) viruses including variola, vaccinia, ectromelia, herpes simplex virus, cytomegalovirus, adenovirus and others. One of these compounds, hexadecyloxypropyl-CDV (HDP-CDV, CMX001) is in clinical development for prevention and treatment of poxvirus infection, vaccination complications, and for infections caused by cytomegalovirus, adenovirus, herpesviruses and other dsDNA viruses. This class of lipid analogs is potentially prone to undergo omega oxidation of the alkyl moiety which can lead to a short chain carboxylic acid lacking antiviral activity. To address this issue, we synthesized a series of alkoxyalkyl or alkyl glycerol esters of CDV and (S)-HPMPA having modifications in the structure of the alkyl residue. Antiviral activity was assessed in cells infected with vaccinia, cowpox or ectromelia viruses. Metabolic stability was determined in S9 membrane fractions from rat, guinea pig, monkey and human liver. All compounds had substantial antiviral activity in cells infected with vaccinia, cowpox or ectromelia. Metabolic stability was lowest in monkey liver S9 incubations where rapid disappearance of HDP-CDV and HDP-(S)-HPMPA was noted. Metabolic stability in monkey preparations increased substantially when a ω-1 methyl group (15-methyl-HDP-CDV) or a terminal cyclopropyl residue (14-cyclopropyl-tetradecyloxypropyl-CDV) was present in the alkyl chain. The most stable compound was 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV) which was not metabolized extensively by monkey liver S9. In rat, guinea pig or human liver S9 incubations, most of the modified antiviral compounds were considerably more stable.     

High Content Image-Based Screening of a Protease Inhibitor Library Reveals Compounds Broadly Active against Rift Valley Fever Virus and Other Highly Pathogenic RNA Viruses

High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and any possible deleterious effects on host cellular biology.     

Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.