key: cord-0688254-nqz4co2b
authors: Pastorino, Boris A.M.; Peyrefitte, Christophe N.; Almeras, Lionel; Grandadam, Marc; Rolland, Dominique; Tolou, Hugues J.; Bessaud, Maël
title: Expression and biochemical characterization of nsP2 cysteine protease of Chikungunya virus
date: 2007-10-25
journal: Virus Res
DOI: 10.1016/j.virusres.2007.09.009
sha: d127e9fa7bcad289c4435d88c3e779ec46dd6de0
doc_id: 688254
cord_uid: nqz4co2b

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemic fever, rash and polyarthralgia in Africa and Asia. Although it is known since the 1950s, new epidemiological and clinical features reported during the recent outbreak in the Indian Ocean can be regarded as the emergence of a new disease. Numerous severe forms of the infection have been described that put emphasis on the lack of efficient antiviral therapy. Among the virus-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. It is a multifunctional protein of approximately 90 kDa with a helicase motif in the N-terminal portion of the protein while the papain-like protease activity resides in the C-terminal portion. The nsP2 proteinase is an essential enzyme whose proteolytic activity is critical for virus replication. In this work, a recombinant CHIKV nsP2pro and a C-terminally truncated variant were expressed in Escherichia coli and purified by metal–chelate chromatography. The enzymatic properties of the proteinase were then determined using specific synthetic fluorogenic substrates. This study constitutes the first characterization of a recombinant CHIKV nsP2 cysteine protease, which may be useful for future drug screening.

Chikungunya virus (genus Alphavirus, family Togaviridae) is transmitted by Aedes mosquitoes (Porterfield, 1980; Strauss and Strauss, 1986) and is typically responsible in human of an acute illness, which includes fever, maculopapular rash, and incapacitating arthralgia (Robinson, 1955; Johnston and Peters, 1996; Jupp and Cornel, 1988) . Isolated for the first time during a Tanzanian outbreak in 1952 (Ross, 1956) , CHIKV is geographically distributed in Africa, India, and Southeast Asia (Lam et al., 2001; Pastorino et al., 2004; Saluzzo et al., 1983) . In Africa, until 2000, it was described as endemic through a sylvatic cycle involving wild primates, humans and mosquitoes of the genus Aedes (Powers et al., 2000) . However, during the last 6 years, large urban outbreaks have been also reported in central Africa (Pastorino et al., 2004) . Due to recent extension around the Indian Ocean, including Comoros, Mauritius, Reunion island, Madagascar and India (Bessaud et al., 2006b; Charrel et al., 2007; Schuffenecker et al., 2006; Ravi, 2006) , and now in Italia, CHIKV is regarded as a potential worldwide public health problem, with no preventive or therapeutic means available.

Both Sindbis virus (SINV) and Semliki Forest virus (SFV) have been studied extensively at the molecular level. They have served as models for determining alphavirus architecture and replication strategy. The genome of alphaviruses is a single RNA molecule of positive polarity, about 11.5 kb in length, encoding four non-structural proteins (nsP1-4) that are the essential components of the viral replicase and transcriptase. Functional proteins are first expressed as a polyprotein that is then cleaved by the viral proteases.

The nsP2 protein is multifunctional (Hardy and Strauss, 1989; Merits et al., 2001; Strauss et al., 1992; Vasiljeva et al., 2001) . Nucleoside triphosphatase, helicase, and RNA-dependent 5triphosphatase activities are located in the N-terminus of the protein while the proteolytic domain has been mapped to its C-terminal part. It forms a papain-like thiol protease with a catalytic dyad involving two conserved residues C and H. The nsP2 protease is responsible for cleavages in the non-structural polyprotein (Kim et al., 2004; Vasiljeva et al., 2003) that are crucial for the viral replication cycle. Therefore, this protease constitutes an attractive target for the development of antiviral compounds.

Previous functional mapping of SFV nsP2 had defined a soluble nsP2 region with protease activity and called Pro39 (SFV residues M 459 -C 799 ). The CHIKV nsP2pro sequence was determined by alignment of CHIKV (GenBank accession number AF369024), SINV (AF429428) and SFV (NC003215) amino acid sequences (residues E 417 -C 799 ) using the Clustal X 1.81 software (Thompson et al., 1997) . The overall nsP2pro sequence conservation was about 38% at the nucleotidic level and 63% at the amino-acidic level when CHIKV sequence was compared to SFV. The catalytic residues C 478 and H 548 were found invariant in all the three nsP2pro sequences, as well as the W 549 , which has been shown to be necessary for proteolytic activity (Golubtsov et al., 2006; Strauss et al., 1992) .

Based on this sequence analysis, we constructed two specific plasmids using the pET-DEST42 Vector, following specifications of Gateway Cloning Technology (Invitrogen, Cergy pontoise, France). The primers CHIK-D1 (genome position 2948-2965): 5 -AAAAAGCAGGCTCTGAAGGAGATAGA-ACCATGTGGAAAGACACTCTCCGGT and CHIK-C2 (genome position 4030-4048): 5 -AGAAAGCTGGGTAAC-ATCCTGCTCGGGTGGC were used for the first construction (nsP2pro) expressing the CHIKV nsP2pro from residue W 422 through residue C 799 . The primers CHIK-D1 and CHIK-C1 (genome position 3563-3581): 5 -AGAAAGCTG-GGTAGACCAGGAGCACGTGGTG were used for the second construct (nsP2proDel) (from residue W 422 through residue V 639 ). CHIKV nsP2pro and nsP2proDel were expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL (Stratagene) with 1 mM IPTG for 6 h at 22 • C in TB media (Fig. 1) . The two recombinant proteins were partially soluble. Different conditions were tested (addition of denaturing agents, detergents or glycylglycine (Ghosh et al., 2004) ) but failed to improve the protein solubility.

Purification was performed using metal-chelate chromatography (Ni-NTA, Qiagen, Courtaboeuf, France) loaded onto a XK16/20 column (Amersham Biosciences, Orsay, France) connected to anÄKTA FPLC system (Amersham Biosciences). After purification, the Coomassie-stained SDS-PAGE revealed additional bands accompanying the recombinant proteins. The use of Co 2+ matrix and the increase of the wash steps stringency, as well as size exclusion (Superdex 200 10/300 column, Amersham) and ion exchange (mono Q 5/50 column, Amersham) chromatographies did not improve the purity rate. The main contaminating proteins were identified by MALDI-TOF-MS analysis as the chaperone protein htpG of E. coli (molecular weight 71,378 Da, number Swiss-Prot/TrEMBL: HTPG ECO57), already described when recombinant proteins are overproduced in E. coli (Rikkonen et al., 1992; Liberek et al., 1991; Sherman and Goldberg, 1992) , and as the elongation factor EFCA of E. coli (molecular weight 42,422 Da); these proteins do not display any protease activity. Washing the Ni-NTA purified fractions with MgATP (Rial and Ceccarelli, 2002) for the purpose of eliminating the chaperone protein htpG resulted in a concomitant loss of a substantial proportion of the recombinant protein (data not shown).

The identity of nsP2pro and nsP2proDel were verified by immunoblotting using anti-His monoclonal antibodies (Fig. 1) . After the purification steps, nsP2pro and nsP2proDel protein concentration was determined to be ∼3 M with a purity close to 70% using a Lumi-Imager imaging system (Roche Applied Sciences, Meylan, France). Purified protein samples were then concentrated using Vivaspin concentrators (Vivascience, Hannover, Germany) .

To characterize the enzymatic activity of the purified protease nsP2pro and nsP2proDel, we investigated their ability to cleave peptidyl-7-amino-4-methylcoumarine-labelled substrates; this kind of substrate had been previously used to study the activ- ity of a wide range of proteases, such as eukaryotic and viral trypsin-like proteases (Jeohn et al., 1999; Kato et al., 1998; Bessaud et al., 2006a; Yusof et al., 2000) and cysteine proteases (Choe et al., 2006; Melo et al., 2001; Gosalia et al., 2005) . Such short substrates are useful to assay the catalytic activity of proteases and to map their specificity at non-prime sites. Residues in the P 4 -P 3 -P 2 -P 1 positions (according to Schechter & Berger's nomenclature, 1967 ) are thought to be important for nsP2 cleavage as the enzyme can only recognize substrates with small residues in these positions and specifically a G in the P2 site (Lulla et al., 2006) . Taking into account these data, we designed two synthetic peptides, Boc-AGA-MCA and Boc-AGG-MCA (JPT Peptide Technologies, Berlin, Germany), which represent the natural non-prime part of the CHIKV nsP1/nsP2 and nsP3/nsP4 cleavage sites, respectively.

Both the two substrates were found to be cleaved by nsP2pro. In contrast, no activity on Boc-AGA-MCA and Boc-AGG-MCA substrates was detected using the nsP2proDel protease even at an enzymatic concentration up to 5 M. This result confirmed the functional importance of the nsP2 C-terminal region for its protease activity and the absence of protease contaminating activity in the recombinant protein preparation. Previous studies had shown that SFV nsP2 protease activity was inhibited by deletion in the 200 carboxyl-terminal domain . Recently, the Venezuelan equine encephalitis virus (VEEV) nsP2 protease structure has been resolved (Russo and Watowich, 2006) and provided evidence that the C-terminal moiety may contribute to substrate recognition. Thus, the C-terminal region of alphavirus nsP2pro could fill a role similar to the NS2B cofactor of the flavivirus-encoded NS3 protease (Aleshin et al., 2007) .

The tri-peptide substrate Boc-AGG-MCA was used to determine the optimal conditions for enzymatic activity of nsP2pro. The pH dependence of proteolytic processing was studied at constant ionic strength and the optimal pH for enzyme catalysis was determined to be 9.5 ( Fig. 2A ). Enzymatic activity was higher than 80% between pH 8 and 10 and decreased rapidly out of this range. It was already the case for others purified viral recombinant proteases (Bessaud et al., 2006a; Li and Singh, 1999; Nall et al., 2004; Shiryaev et al., 2006) . The existence of such an alkaline optimal pH could indicate that proteolysis is a regulated process in vivo at physiological pH of the cytoplasm.

The CHIKV protease activity was slightly inhibited by NaCl (Fig. 2B) : the optimal activity values were obtained for NaCl concentration between 10 and 50 mM. Increasing salt concentration up to 250 mM resulted in a 50% decrease of the protease activity. This result can be explained by the importance of electrostatic interactions in cysteine protease/substrate binding.

Overproduced recombinant proteins had a tendency to aggregate and it had been shown that addition of glycerol or a neutral detergent could enhance the targeted activity by increasing accessibility of the enzyme site to substrate (Bessaud et al., 2005; Leung et al., 2001; Nall et al., 2004) . However, CHIKV nsP2pro processing activity was significantly inhibited by glycerol addition: at glycerol concentration 10%, the protease activity was already decreased about 50% and dropped to 20% with 20% glycerol (Fig. 2C) . pH; for each data point, relative activity was defined as the ratio (100 × V i )/(V i ) pH 9.5 . (B) Effect of ionic strength, observed in buffers containing Tris 50 mM pH 9.5 and varying concentration of NaCl; for each data point, relative activity was defined as the ratio (100 × V i )/(V i ) 10 mM . (C) Effect of glycerol; for each data point, relative activity was defined as the ratio

Therefore, the optimal experimental conditions were determined to be Tris 50 mM pH 9.5, NaCl 10 mM.

Under these optimal conditions, the purified CHIKV nsP2pro protease showed a 4.5-fold reduced activity for substrate Boc-AGA-MCA compared to Boc-AGG-MCA (Table 1) . For Boc-AGA-MCA substrate, the K m and k cat were, respectively 10 times and 2.5 times higher than the values obtained with the Boc-AGG-MCA substrate. The kinetic comparison showed that the CHIKV nsP2pro protease had a higher substrate affinity for the Boc-AGG-MCA substrate resulting in a more efficient cleavage. This result is consistent with the alphavirus replication cycle in which the speed of nsP1234 processing plays a crucial role in the control of RNA synthesis (Lemm and Rice, 1993; Lemm et al., 1994; Shirako and Strauss, 1994) . In alphavirus- Non-prime substrate sequences are indicated in bold and prime substrate sequences are shown in italics. Protease activity was assayed in Tris 50 mM pH 9.5, NaCl 10 mM using a substrate concentration ranging from 25 to 200 M. Standard reaction contained the CHIKV nsP2pro protease at a concentration of 0.5 M. The kinetic parameters for the degradation of Dabcyl-AGAGIIE-Edans have not been determined (ND) because of the too low activity of the protease towards this substrate.

infected cells, the non-structural polyprotein is cleaved at three sites in the specific order 3/4-1/2-2/3. The 3/4 site is rapidly cleaved during infection, and this cleavage is absolutely essential for virus replication and virus-specific RNA synthesis (Kim et al., 2004; Lemm et al., 1994) . Similar data have been previously reported with nsP2 proteases of SFV and SINV (Lulla et al., 2006; Vasiljeva et al., 2001) .

Our optimised assay was also performed using longer quenched fluorogenic hepta-and octa-peptides (Eurogentec S.A., Liège, Belgium) overlapping the natural cleavage sites 1/2 and 3/4 (Table 1 ), in order to identify the role of flanking sequences for proteolytic activity. The nsP2proDel protein was inactive on these substrates whereas enzymatic activity was detected using the nsP2pro recombinant protease. The highest activity was obtained using the fluorogenic peptide overlapping the nsP3/nsP4 natural cleavage site, confirming the previous result obtained with the MCA peptides. The CHIKV protease showed a higher affinity for this more authentic substrate than for the shorter Boc-AGG-MCA while the k cat /K m ratio was almost similar.

The hepta-peptide substrate corresponding to the natural nsP1/nsP2 cleavage site was very poorly cleaved; therefore, we were not able to determine the corresponding enzymatic constants for its degradation. This result could be the consequence of the poor substrate solubility due to a relatively high rate of hydrophobic residues (A, F, I, Y) in the hepta-peptide sequence (Petrassi et al., 2005) .

As an insight into the protease catalytic mechanism, the effect of different protease inhibitors on the enzymatic activity of CHIKV nsP2pro was also tested, using the cost-effective Boc-AGG-MCA peptide as substrate (Fig. 3) . As expected, the enzyme was completely resistant to the inhibitors of serine proteases (AEBSF, aprotinin), aspartic proteases (pepstatin), metalloproteases (EDTA). It was also resistant to one cysteine protease inhibitor (leupeptin). However, cleavage of the Boc-AGG-MCA substrate was completely inhibited by 25 mM N-ethylmaleimide (NEM) and by 2 mM of divalent cations Zn 2+ or Cu 2+ . These results are consistent with the classification of nsP2 as a thiol proteinase of the papain superfamily and with data already described for Pro39 , cathepsin B (Lockwood, 2004) or papain-like protease 2 (PLP2) from Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) (Han et al., 2005) . However, the mechanism of Zn 2+ , Cu 2+ and Fe 3+ inhibition is unclear due to the lack of molecular structures available, the diversity and the very low similarity inside the papain-like cysteine proteases family (Russo and Watowich, 2006) . It is interesting to note that zinc and zinc conjugates have been proven effective for inhibiting SARS-CoV replication in vivo through simultaneous inhibition of SARS-CoV PLP2 and 3CL proteases (Hsu et al., 2000) . As zinc salts (zinc acetate) are already used as therapeutics in humans, they could represent a promising drug for treatment of other viral infections.

The success of retroviral proteases inhibition and the early promise being shown by inhibitors of the NS3 hepatitis C virus (Lin et al., 2004) have demonstrated the feasibility and the interest of antiviral strategies based on protease inhibition. Functional assay such as the one we develop in this work will allow high-throughput screening of chemical libraries in search of inhibitors and should help for further characterization of the nsP2 alphaviruses' protease.

This work was partially funded by the French Armed Forces Medical Service and the French Délégation générale pour l'armement (DGA, contrat d'objectif 05CO008-05). The opinions and assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the French Armed Forces Medical Service or the French Army at large.

Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold

Identification and enzymatic characterization of NS2B-NS3 protease of Alkhurma virus, a class-4 flavivirus

Functional characterization of the NS2B/NS3 protease complex from seven viruses belonging to different groups inside the genus Flavivirus

Chikungunya virus strains, Reunion Island outbreak

Chikungunya outbreaks-the globalization of vectorborne diseases

Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities

Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli

Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays

Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition

Processing the nonstructural polyproteins of Sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans

Evaluation of metal-conjugated compounds as inhibitors of 3CL protease of SARS-coV

Purification and characterization of a detergent-requiring membrane-bound metalloendopeptidase from porcine brain

Alphaviruses

Vector competence tests with Rift Valley fever virus and five South African species of mosquito

Production of trypsins by human gastric cancer cells correlates with their malignant phenotype

Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts

Chikungunya infection-an emerging disease in Malaysia

Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription

Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus-and plus-strand RNA synthesis

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B cofactor, small peptide substrates, and inhibitors

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

The Escherichia coli DnaK chaperone, the 70 kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein

Combination of a hepatitis C virus NS3-NS4A protease inhibitor and alpha interferon synergistically inhibits viral RNA replication and facilitates viral RNA clearance in replicon cells

Cys-His proteases are among the wired proteins of the cell

Molecular determinants of substrate specificity for Semliki Forest virus nonstructural protease

Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides

Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease

Epidemic resurgence of Chikungunya virus in democratic Republic of the Congo: identification of a new central African strain

A strategy to profile prime and non-prime proteolytic substrate specificity

Arbovirus: structure et classification

Re-emergence of Chikungunya and O'nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships

Re-emergence of chikungunya virus in India

Removal of DnaK contamination during fusion protein purifications

Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2

An epidemic of virus disease in Southern Province, Tanganyika Territory

The Newala epidemic. III. The virus: isolation, pathogenic properties and relationship to the epidemic

Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

Outbreak of a Chikungunya virus epidemic in western Senegal in 1982

Genome microevolution of Chikungunya viruses causing the Indian Ocean outbreak

Involvement of the chaperonin DnaK in the rapid degradation of a mutant protein in Escherichia coli

Regulation of Sindbis virus RNA replication: uncleaved [123P] and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis

Cleavage targets and the d-argininebased inhibitors of the West Nile virus NS3 processing proteinase

Structure and Replication of the Alphavirus Genome

Identification of the active site residues in the nsP2 proteinase of Sindbis virus

The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools

Site-specific protease activity of the carboxyl-terminal domain of Semliki Forest virus replicase protein nsP2

Regulation of the sequential processing of Semliki Forest virus replicase polyprotein

Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro