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J. Biol. Chem., Vol. 276, Issue 33, 30786-30793, August 17, 2001

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Site-specific Protease Activity of the Carboxyl-terminal Domain of Semliki Forest Virus Replicase Protein nsP2^*

From the Program in Cellular Biotechnology, Institute of Biotechnology, Biocenter Viikki, P. O. Box 56, University of Helsinki, FIN-00014 Helsinki, Finland

Received for publication, May 25, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The virus-specific components (nsP1-nsP4) of Semliki Forest virus RNA polymerase are synthesized as a large polyprotein (P1234), which is cleaved by a virus-encoded protease. Based on mutagenesis studies, nsP2 has been implicated as the protease moiety of P1234. Here, we show that purified nsP2 (799 amino acids) and its C-terminal domain Pro39 (amino acids 459-799) specifically process P1234 and its cleavage intermediates. Analysis of cleavage products of in vitro synthesized P12, P23, and P34 revealed cleavages at sites 1/2, 2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expressed as thioredoxin fusion proteins (Trx12, Trx23, and Trx34), containing ~20 amino acids on each side of the cleavage sites. After exposure of these purified fusion proteins to nsP2 or Pro39, the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis, mass spectrometry, and amino-terminal sequencing. The expected amino termini of nsP2, nsP3, and nsP4 were detected. The cleavage at 3/4 site was most efficient, whereas cleavage at 1/2 site required 5000-fold more of Pro39, and 2/3 site was almost resistant to cleavage. The activity of Pro39 was inhibited by N-ethylmaleimide, Zn²⁺, and Cu²⁺, but not by EDTA, phenylmethylsulfonyl fluoride, or pepstatin, in accordance with the thiol proteinase nature of nsP2.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The genomes of many positive strand RNA viruses are expressed as polyproteins in order to achieve the expression of multiple proteins from a single message, unlike the mRNAs of their eukaryotic host cells, which mostly code for single proteins. Thus, proteolyses of the polyprotein precursors are essential events in the regulation of the replication and morphogenesis of these RNA viruses. In picornaviruses and flaviviruses, the entire RNA genome is translated as a single polyprotein, from which the structural and nonstructural proteins are processed by proteolysis. In picornavirus-infected cells, the processing is carried out by virus-encoded proteases within the polyprotein, whereas the processing of flavivirus polyprotein is assisted by host proteases (1, 2). The large RNA genomes of coronaviruses (approximately 30 kilobases) and arteriviruses (12.7-15.7 kilobases), together classified as Nidovirales, use in addition to the polyprotein strategy also a set of subgenomic mRNAs (3). Alphaviruses and rubella virus, members of the Togaviridae family, express two polyproteins. The nonstructural polyprotein is expressed directly from the RNA genome, whereas the structural polyprotein is synthesized from a subgenomic mRNA (4-6).

Semliki Forest virus (SFV)¹ is a typical alphavirus with a lipoprotein envelope surrounding the nucleocapsid. The 5' two-thirds of the 11.5-kilobase 42 S RNA genome codes for the nonstructural polyprotein (P1234) of 2432 aa, which is autocatalytically cleaved to finally yield the virus-specific components of the RNA polymerase complex, nsP1-nsP4 (4, 5, 7). The processing of the nonstructural polyproteins P1234 and P123 of Sindbis virus (SIN), another alphavirus, has been studied in detail (4, 8, 9). Using mostly in vitro translation and site-directed mutagenesis as tools, autocatalytic protease activity was detected in the polyprotein and its cleavage intermediates. The protease activity was localized to nsP2, and more precisely, to its carboxyl-terminal part (10, 11). Cysteine 481 and histidine 558 were identified as essential residues for the autoprotease activity (12), supporting the view that the protease is a thiol proteinase belonging to the papain superfamily. The same conclusion was reached by sequence comparisons (13). The respective mutation of Cys-478 in SFV nsP2 also inactivates the autocatalytic processing of P1234, P123, and P23 (14).

The amino-terminal domain of SFV nsP2 (residues 1-470) has been shown to house several enzymatic activities including RNA triphosphatase (15), nucleoside triphosphatase (16), and RNA helicase (17). Interestingly, a significant amount of nsP2, synthesized during infection, is transported into the nucleus (18, 19). The core of the nuclear localization signal was mapped to a short sequence P⁶⁴⁷RRR (20). nsP2 mutant PRDR is not lethal for the virus in cell culture, but SFV carrying this mutation is apathogenic for mouse (21). In addition, the carboxyl-terminal domain of SFV nsP2 has been implicated in the regulation of the synthesis of the subgenomic mRNA (22-24).

Taken together, these data suggest that alphavirus nsP2 consists of two structurally independent domains, each possessing a distinct set of biological activities. However, direct proof that purified nsP2 or its carboxyl-terminal part has protease activity has been lacking. To this end, recombinant nsP2 and a set of its amino-terminally truncated variants were expressed in Escherichia coli, purified by metal-chelate chromatography, and assayed for the presence of protease activity. Both full-length nsP2 and its soluble carboxyl-terminal fragment Pro39 (aa 458-799) catalyzed site-specific proteolysis of SFV P1234 in vitro. Furthermore, both nsP2 and Pro39 could specifically cleave purified recombinant fusion proteins containing short (~40 aa) SFV-specific peptides, which span protease cleavage sites. Using this newly devised in vitro assay, we show that Pro39 was inactivated by N-ethylmaleimide, in accordance with the catalytic mechanism of cysteine proteases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

nsP2 Expression Plasmids-- Full-length nsP2 of SFV was produced and purified as described previously (15). To obtain a set of nsP2 mutants, containing progressive amino-terminal truncations, nine separate PCR amplifications were carried out using Pfu Turbo DNA polymerase (Stratagene), SFV infectious cDNA as a template and 3'-Mut (Table I) as common 3' primer. This oligonucleotide was designed to introduce a G  E mutation in the 2/3 cleavage site to prevent possible self-proteolysis of the expressed protein at the nsP2-His₆ tag boundary. Oligonucleotides N60, N120, N180, N240, N300, N350, N400, N458, and N470 (Table I) were used as 5' primers. Similarly, a set of bidirectional truncations of nsP2 was also prepared, using N300 as a common 5' primer and C40, C80, or C120 (Table I) as 3' primers. All PCR products were cleaved with NcoI-XhoI and cloned into pET21d vector (Novagen) to yield pP2N60, pP2N120, pP2N180, pP2N240, pP2N300, pP2N350, pP2N400, pP2N458, pP2N470, pP2N300C40, pP2N300C80, and pP2N300C120 expression plasmids, respectively. To obtain plasmid pP2N458^CA, encoding inactive protease, PCR amplification with N458 and 3'-Mut primers was carried out on the SFV^CA template (14), and the obtained PCR product was cloned as described above.

Site-directed Mutagenesis of the Cleavage Sites-- SFV cDNA fragments covering 1/2 and 3/4 cleavage sites (SFV genome regions 1444-1944 and 5306-6138, respectively) were subcloned into pBlueScript KS vector (Stratagene), and fragment covering 2/3 cleavage site (genome region 3791-5531) was subcloned into pUC18. The three resultant plasmids were used as templates for the PCR-based site-specific mutagenesis, using Pfu Turbo DNA polymerase (Stratagene) and the one of the following primer pairs: 1MutF and 1MutR, 2MutF and 2MutR, or 3MutF and 3MutR (Table I). The primers were designed to change the 1/2 processing site YHAGAGVVE to YHAEAGVVE, 2/3 site HTAGCAPSY to HTAECAPSY, and 3/4 site GRAGAYIFS to GRAEVYIFS, respectively (target residues underlined). PCR products were treated with DpnI (Stratagene) and T4 polynucleotide kinase (New England Biolabs) and self-ligated, using T4 DNA ligase (New England Biolabs). The plasmids obtained were verified by sequencing and designated pMut1^2, pMut2^3, and pMut3^4.

Thioredoxin Fusion Proteins as Substrates-- Fragments of the SFV nonstructural region, spanning 1/2, 2/3, or 3/4 junctions, in the context of 19-21 upstream and 18-21 downstream codons, were PCR-amplified, using one of the following primer pairs: S1/2up and S1/2down, S2/3up and S2/3down, or S3/4up and S3/4down, and using SFV infectious cDNA, pMut1^2, pMut2^3, or pMut3^4 as a templates. The PCR products treated with EcoRV-EcoRI were cloned into the MscI-EcoRI-cut vector pET32c(+) (Novagen) in frame with the thioredoxin gene. The obtained plasmids, designated as pTrx12, pTrx23, and pTrx34 (wild type cleavage sites) and pTrx1^2, pTrx2^3, and pTrx3^4 (their non-cleavable analogs), were used to produce respective thioredoxin fusion proteins.

Protein Expression and Purification-- Recombinant proteins were expressed in E. coli BL21(DE3) (Stratagene) and purified using Ni²⁺-affinity chromatography as described previously (16, 15). Protein purification was monitored by 10-17% SDS-PAGE (25), and protein concentration was determined with the Bradford assay (26) using Bio-Rad reagents. Purified proteins (1-5 mg/ml) were stored in a buffer, containing 50 mM Tris-HCl, pH 7.0, 200 mM NaCl, and 1 mM dithiothreitol at 4 °C.

In Vitro Synthesis of the Nonstructural Polyprotein Substrates-- Coupled transcription-translation of the constructs encoding SFV P12^CA, P2^CA3, P12^CA3, P12^CA34, and P34 was carried out in the T7 TNT rabbit reticulocyte lysate system (Promega) according to the manufacturer's instructions. Reaction mixtures (10 µl) supplemented with 10 µCi of [³⁵S]methionine (>1000 Ci/mmol; Amersham Pharmacia Biotech) and 1 µg of plasmid DNAs were incubated for 1 h at 30 °C, and the protein synthesis was stopped by adding 1 mM cycloheximide. For protease assay with ³⁵S-labeled nonstructural polyprotein substrates, typically 0.1-0.5 µg of the isolated protease was added to the cycloheximide-arrested translation mixtures (see above), and the mixture was incubated for 1 h at 30 °C. Reaction products were separated by SDS-PAGE. Gels were dried, and radioactive protein bands were visualized using phosphorimager (BAS-1500, Fuji).

Protease Assay with Purified Thioredoxin Fusion Protein Substrates-- Protease activity of nsP2 and Pro39 was assayed using thioredoxin (Trx) substrates (see above). Purified substrates and enzymes were mixed in the buffer containing 50 mM HEPES-NaOH, pH 7.2, 20-100 mM NaCl, and 1 mM dithiothreitol, and the mixtures were incubated for 1 h at 30 °C. Reaction products were analyzed by SDS-PAGE and/or mass spectrometry after HPLC purification. In the protease inhibition assays, enzyme was pre-incubated with the inhibitor for 10 min.

Reversed Phase Chromatography-- Substrates and the reaction products were separated on a 0.1 × 15-cm Vydac C8 column (300 Å, 5 µm, LC-Packings) using a SMART^TM system (Amersham Pharmacia Biotech, Uppsala, Sweden). Elution was performed using linear gradients of acetonitrile (0-60% in 100 min) in 0.1% trifluoroacetic acid. Chromatography was monitored for absorbancy at 214 nm, and the peptide-containing fractions were collected automatically.

Mass Spectrometry and NH₂-terminal Sequence Analysis-- MALDI-TOF mass spectrometry was performed on a Biflex^TM time-of-flight instrument (Bruker-Franzen Analytik, Bremen, Germany) equipped with a nitrogen laser operating at 337 nm. The reversed phase HPLC separated fractions were analyzed in the linear positive ion delayed extraction mode using saturated sinapic acid in a mixture of 0.1% trifluoroacetic acid and 50% acetonitrile (1:2) as a matrix. Samples were prepared by mixing 1 µl of reversed phase HPLC eluate with 1 µl of sinapic acid matrix on the target plate and dried under a gentle stream of warm air. All mass spectra were calibrated externally with either cytochrome C or myoglobin as standards. Electrospray ionization mass spectra were obtained using a Micromass Q-TOF quadrupole/time-of-flight hybrid mass spectrometer (Micromass, Manchester, United Kingdom). Pro39 was dissolved in a mixture of 0.1% trifluoroacetic acid and 50% acetonitrile (1:2) and directly injected into the electrospray ionization mass spectrometer with a syringe pump at a flow rate of 30 µl/h. The mass spectrometer was calibrated using sodium trifluoroacetate as described (27). Protein masses were calculated by deconvulation in MassLynx 3.4 (Micromass). NH₂-terminal sequence analyses were performed by Edman degradation using a Procise 494A Sequencer (PerkinElmer Applied Biosystems Division).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Purification of Recombinant Proteins-- Full-length SFV nsP2 was expressed in E. coli and purified using metal-chelate chromatography as described (15). The same expression strategy was utilized to prepare a set of amino-terminally truncated variant proteins of nsP2: P2N60, P2N120, P2N180, P2N240, P2N300, P2N350, P2N400, P2N458, and P2N470; as well as three bidirectional deletion mutants P2N300C40, P2N300C80, and P2N300C120. All amino-terminally truncated mutants were at least partially soluble. On the other hand, deletions from the carboxyl terminus rendered nsP2 fragments insoluble, except for P2N300C120, which was partly soluble but had no protease activity (Fig. 1). Interestingly, the P2N458 mutant showed solubility comparable to, or even higher than, that of full-length nsP2. Since this 341-aa-long fragment coincides with the putative nsP2 protease domain, it will be hereafter referred to as Pro39 (Fig. 2A). This soluble carboxyl-terminal fragment was purified to near homogeneity from cleared bacterial lysates using metal-chelate chromatography, as shown in Fig. 2B. Pro39 migrated in SDS-PAGE as a 39-kDa protein. Its molecular mass was determined by electrospray mass spectrometry to be 39.3 kDa (Fig. 2C).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Schematic presentation and properties of nsP2 deletion mutants. Filled boxes represent the deleted regions. C478 represents catalytic residue of the active site; +, protease activity; , no protease activity; nd, activity could not be determined.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Protease domain Pro39 of nsP2. A, a schematic presentation of nsP2. The carboxyl-terminal protease domain of 341 aa (Pro39) with the proposed active site residue Cys-478 is indicated. B, purification of Pro39 by metal affinity chromatography monitored by SDS-PAGE in 10% gel. Lane 1, total extract from the isopropyl-1-thio--D-galactopyranoside-induced bacterial cells; lane 2, supernatant after centrifugation at 100,000 × g; lane 3, purified Pro39 eluted from the nickel-chelate column. Arrow indicates the position of Pro39. Molecular mass markers in kDa are shown in lane M. C, determination of molecular mass of Pro39 by electrospray mass spectrometry. The peak of 39.3 kDa is the calculated molecular mass of Pro39.

Both nsP2 and Pro39 Are Proteolytically Active in Vitro-- To assay protease activities of the isolated proteins, several SFV nonstructural polyprotein substrates containing [³⁵S]methionine were synthesized in a cell-free transcription-translation system. These were P12^CA, P2^CA3, P12^CA3, P12^CA34 (with a protease-inactivating mutation of C478A in the nsP2 moiety), as well as P34. Both nsP2 and its amino-terminally truncated fragments were active as proteases; however, Pro39 showed the highest specific activity (data not shown). From the polyprotein pairs, both preparations cleaved readily at sites 1/2 (Fig. 3A, lanes 2-5) and 3/4 (Fig. 3A, lanes 12-15), whereas cleavage at site 2/3 was much less efficient, but detectable (Fig. 3A, lanes 9 and 10). The two larger polyproteins were cleaved also at sites 1/2 and 3/4 and to some extent at site 2/3, as revealed by immunoprecipitation of the cleavage products (Fig. 3B, lanes 4-6 and 10-13). The cleavage at site 3/4 was more efficient than that at site 1/2, as seen after reduction of enzyme concentration or time of incubation (data not shown). As expected, the C478A mutation inactivated both nsP2 and Pro39 completely (data not shown). Overall, these data demonstrate site-specific protease activity in vitro of purified nsP2 and Pro39. The high solubility and specific activity of Pro39 suggest that this fragment represents a structurally compact protease domain of nsP2.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Protease activity of purified nsP2 and Pro39 assayed on in vitro synthesized SFV polyprotein substrates with active site mutation C478A. A, polyproteins P12CA, P2CA3, and P34 as substrates. Identification of the reaction products of Pro39 with pertinent antiserum is shown in lanes 4, 5, 9, 10, 14, and 15. In vitro synthesized nsP1 and polyproteins containing nsP1 show regularly a truncated form P1, due to internal initiation of translation. B, polyproteins 12CA3 and 12CA34 as substrates. Immunoprecipitation analyses are shown in lanes 4-6 and 10-13. The proteins were synthesized using a cell-free in vitro system in the presence of [35S]methionine for 1 h at 30 °C. The synthesis was stopped by adding cycloheximide. The enzyme preparations (100 ng) were added to the reaction mixture followed by incubation for 1 h at 30 °C. The reaction products were analyzed by SDS-PAGE in 10% gels before and after immunoprecipitation, followed by visualization by autoradiography. Positions of the polyproteins and their cleavage products are indicated with the arrows.

Protease Activity of nsP2 and Pro39 Assayed with Purified Substrates-- In the following experiments, protease activity of nsP2 and Pro39 was assayed with a set of purified recombinant Trx fusion proteins as substrates (Fig. 4A). These fusion proteins (~18 kDa) contained approximately 40-aa sequences spanning the SFV P1234 polyprotein cleavage sites (Trx12, Trx23, and Trx34). As controls we used non-cleavable analogs with point mutations (glycine to glutamic acid) in the penultimate amino acid of the predicted cleavage site (Trx1^2, Trx2^3) as well as an additional mutation (Ala  Val) for 3/4 cleavage site yielding Trx3^4 (Fig. 4). Both nsP2 and Pro39 cleaved Trx34 with the highest efficiency. As expected, two proteolytic products were formed, one larger (L; ~14 kDa) and one smaller (S; ~4 kDa). The L-fragment (thioredoxin moiety plus 19 carboxyl-terminal residues from nsP3) could be detected in standard SDS-PAGE gels (Fig. 4B). The S-fragment migrated in this gel system in the front (data not shown). However, Tricine SDS-PAGE method (28) resolved this fragment as a defined band (Fig. 4C). Pro39 also cleaved Trx12, as judged by the appearance of the L-fragment (Fig. 4D). Under the same conditions, the cleavage products obtained with nsP2 as protease and Trx12 as substrate were barely detectable (data not shown). No cleavage of Trx23 could be detected with either nsP2 or Pro39 when analyzed by SDS-PAGE (Fig. 4E). Neither nsP2 nor Pro39 could cleave Trx1^2, Trx2^3, or Trx3^4, which served as controls (Fig. 4, B, D, and E). The relative cleavage efficiency by Pro39 of sites 1/2 and 3/4 was further analyzed using serial dilutions of the enzyme (Fig. 5). From these results it could be estimated that complete cleavage of Trx12 requires ~5000-fold more enzyme than cleavage of Trx34.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Thioredoxin fusion protein substrates containing cleavage sites 1/2, 2/3, and 3/4 of SFV P1234, and their cleavage by nsP2 and Pro39. A, schematic presentation of Trx12, Trx23, and Trx34 substrates. The amino-terminal sequence of thioredoxin is followed by 42-37 aa from the cleavage regions of the 1/2, 2/3, and 3/4 sites; 13 aa from pET32C; and a His6 tag. Six residues on both sides of the expected cleavage sites are shown. The uncleavable fusion proteins Trx1^2 and Trx2^3 differ only in replacements of the penultimate glycine (bold) to glutamic acid at the cleavage site. In Trx3^4, an additional mutation of alanine (bold) to valine was engineered. Arrowheads indicate the predicted cleavage positions. B, proteolysis of Trx34 by nsP2 and Pro39 (lanes 5 and 6); D, proteolysis of Trx12 (lane 4); E, proteolysis of Trx23. The products were analyzed by SDS-PAGE in 17% gels, which were stained by Coomassie Blue 250. The position of Pro39, uncleaved substrates, and the large cleavage product (L) are indicated by arrows. Molecular mass markers in B, D, and E are given in lanes 1. C, separation of large (L; 14 kDa) and small (S; 4 kDa) proteolytic products of Trx34 by Pro39 using Tricine-SDS electrophoresis (28).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Dependence of Trx12 and Trx34 proteolysis on Pro39 concentration. A, Trx34 (1 µg) was treated by different nanogram amounts of Pro39 as indicated for lanes 1-5. B, Trx12 (1 µg) was treated with microgram amounts of Pro39 as indicated for lanes 1-5. The reactions were carried out for 1 h at 30 °C. The proteolysis products were separated by SDS-PAGE, and the gel was stained with Coomassie Blue R-250. Positions of Pro39, Trx12, Trx34, and large (L; ~14 kDa) products are indicated with the arrows.

HPLC Purification and MALDI-TOF Mass Spectrometry of the Proteolytic Products-- In addition to the SDS-PAGE analysis, the proteolytic products of Trx12, Trx23, and Trx34 were also separated by reversed phase HPLC (see "Experimental Procedures" for details). Fractions containing S- and L-fragments, as well as non-cleaved substrates, were analyzed by MALDI-TOF mass spectrometry as shown in detail for Trx12 (Fig. 6). Amino-terminal sequences of the S-fragments were also determined. The results of these experiments confirmed that Pro39 cleaves Trx-fusion protein substrates exactly at the predicted positions of P1234, and were supported by previous NH₂-terminal radiosequence analyses of in vivo labeled nsP2, nsP3, and nsP4 (29, 30) (Table II). Importantly, this approach was sensitive enough to detect the cleavage of the Trx23 substrate. Thus, 2/3 site can be hydrolyzed correctly in vitro by Pro39 but with a very poor efficiency (Table II).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   HPLC separation and MALDI-TOF mass spectra of Trx12 and its cleavage products by Pro39. A, reversed phase HPLC purification of Trx12 and MALDI-TOF mass spectrum of the indicated peak in the inset. B, HPLC separation of the reaction products and MALDI-TOF mass spectrometric analyses of S- and L-fragment-containing fractions (filled arrows) in the insets. The peaks corresponding to Trx12 are shown with empty arrows in A and B.

Characterization of the Pro39-catalyzed Reaction-- Effects of several reaction parameters on the protease activity of Pro39 were studied systematically. The enzyme was active over a broad range of pH values (pH 6.8-9.5) and different ionic strengths (0-500 mM NaCl), and the optimal reaction temperature was 30 °C (data not shown). Omission of reducing reagents from the reaction mixture had no detectable effect on Pro39 activity. The time course of the proteolysis under optimized conditions was also studied. In this experiment, Pro39 demonstrated a very high specific activity, hydrolyzing 50% of 400-fold molar excess of Trx34 substrate in 5 min (Fig. 7).

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Kinetics of the proteolytic cleavage of Trx34 catalyzed by Pro39. A 50-µl reaction mixture contained 20 µg of Trx34 and 100 ng of the enzyme. Aliquots of 5 µl were taken during 120 min of incubation at 30 °C, and then reaction was stopped by addition of sample buffer and analyzed by SDS-PAGE in 17% gel. The gel was stained with Coomassie Blue R-250. Arrows show the positions of Pro39, Trx34, and 14-kDa large proteolytic product (L).

The effect of different protease inhibitors on the enzymatic activity of Pro39 was also tested. The enzyme was completely resistant to the inhibitors of serine proteases (PMSF), metalloproteases (EDTA), aspartic proteases (pepstatin), and some cysteine protease inhibitors (leupeptin and E-64) (Fig. 8A). Cleavage of both Trx12 and Trx34 was completely inhibited by 2.5 mM N-ethylmaleimide (NEM), a typical cysteine protease inhibitor. Surprisingly, the protease was also sensitive to some divalent cations in the reaction mixture. Addition of 2 mM Zn²⁺ or Cu²⁺ resulted in total inactivation of the protease activity, and Co²⁺ and Ni²⁺ caused partial inhibition (Fig. 8B). On the other hand, the same concentrations of Ca²⁺, Mg²⁺, and Mn²⁺ had no effect on Pro39 activity.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Effects of inhibitors (A) and divalent cations (B) on proteolytic activity of Pro39. Pro39 (10 ng) was incubated with the following inhibitors: 10 mM EDTA; 2.5 mM NEM; 100 µM E-64; and 2 mM Zn2+, Co2+, Mg2+, Mn2+, Ca2+, and Ni2+ for 20 min prior to addition of 5 µg of Trx34, followed by 60-min incubation at 30 °C. Lanes 2 contain Trx34 incubated alone. Lanes 3 show the result of pre-incubation of Pro39 with the reaction buffer (Tris-HCl, pH 7.5, 25 mM NaCl) prior to substrate addition. Molecular mass markers (kDa) are indicated in lanes 1. Arrows show the positions of Pro39, Trx34, and the large proteolytic product of 14 kDa (L).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous work on Semliki Forest virus showed that early in infection the synthesis of the negative strand RNA was strictly dependent on protein synthesis and ceased in about 15 min after addition of cycloheximide (31), whereas late in infection the synthesis of positive strand RNAs could continue for several hours in the absence of protein synthesis. Solution to this dilemma came from findings with Sindbis virus, another alphavirus, where the processing intermediate P123 of the nonstructural polyprotein together with nsP4 was shown to be responsible for the synthesis of the negative strand RNA (32-34). Cleavage of P123 is essential for the synthesis of the positive RNA strands. Thus, the regulated processing of the nonstructural polyprotein controls the early events of virus infection. An overactive processing mutant of Sindbis virus nsP2 (N614D) cannot replicate, as the P123 intermediate is too short-lived to enable the necessary synthesis of the complementary RNA (35).

Our knowledge of the processing of alphavirus nonstructural polyprotein(s) is based mostly on experiments of in vitro translation of Sindbis virus RNA. Ingenious constructions by which the cleavage sites were mutated alone and in different combinations, together with constructs coding for enzymatically inactive polyprotein as substrates, have been used to analyze this complex process (4, 8, 9). These experiments showed that the polyprotein P1234 itself and all its cleavage intermediates containing nsP2 to be active proteases. The cleavability of the different sites varied and was dependent on the order of removal of different nsPs from the polyprotein substrate. Particularly interesting was the finding where the cleavage of site 2/3 in P1234 or P123 was only possible after the cleavage of nsP1 (36). Evidently, conformational changes in P1234 and its cleavage products affect the interactions between the cleavage sites and the protease domain of nsP2 in a complex manner. To understand the processes better, we have characterized purified SFV nsP2 and its carboxyl-terminal fragment Pro39 as proteolytic enzymes. As substrates we used in vitro synthesized polyproteins P1234, P123, P23 and P34, as well as recombinant thioredoxin fusion proteins, which contain short SFV-specific fragments, spanning the polyprotein processing sites 1/2, 2/3, and 3/4.

We show for the first time that purified nsP2 has proteolytic activity, which cleaves readily the 3/4 site of P1234 and P34. Deletion series of nsP2 resulted in a soluble, active carboxyl-terminal fragment consisting of amino acid residues 459-799, which was designated as Pro39. It was purified to near homogeneity by metal-affinity chromatography. The specificities of Pro39 and nsP2 were identical, indicating that the amino-terminal half of nsP2 does not affect the fidelity of the protease. According to sequence alignments with the thiol protease superfamily, Pro39 contains a conserved protease domain (459), but also almost 200 carboxyl-terminal extra amino acids (9, 13). Our attempts to delete 40-120 amino acids from the carboxyl terminus of nsP2 resulted in insoluble or inactive proteins (Fig. 1). Experiments with temperature-sensitive mutants of SIN and SFV nsP2 have shown that amino acid replacements N700K in SIN ts133, K736S in SIN ts24, and M781T in SFV ts4 result in inhibition of protease activity at 39 °C, suggesting that the extreme carboxyl terminus of nsP2 participates somehow in the protease function (24).

Establishment of a biochemical assay system consisting of isolated Pro39 and thioredoxin attached cleavage regions of the SFV nonstructural polyprotein allowed characterization of the viral protease under defined experimental conditions. Pro39 was inactivated by N-ethylmaleimide but not with pepstatin, EDTA, or PMSF. These are properties, which are in accordance with its classification as a thiol proteinase of the papain superfamily. However, Pro39 is not inhibited by E-64, which is a typical inhibitor of cysteine proteases (37). Sensitivity for NEM and resistance for E-64 have been previously reported for poliovirus 3C thiol proteinase (38, 39). Another interesting feature of Pro39 is the inhibition by zinc ions (Fig. 8).

When Pro39 (or nsP2) was added to the reaction mixture, after in vitro translation of P12 or P123, almost a quantitative release of nsP1 was observed. Similarly, when P34 or P1234 were used as substrates, quantitative release of nsP4 was seen, whereas only a small amount of nsP3 was released from P23, P123, or P1234 (Fig. 3). These results suggested that sites 1/2 and 3/4 were exposed to the added protease, whereas site 2/3 was not. To study this phenomenon under controlled conditions, in which the large protein domains would not interfere sterically with the proteolysis, we constructed fusion proteins with a different number of amino acid residues around the cleavage sites.

Constructs with less than 10 amino acids on both sides of the cleavage site were not digested by Pro39 or nsP2 (data not shown). We ended up using thioredoxin fusion proteins with about 40 residues of each cleavage region (Trx12, Trx23, and Trx34). Isolation of the cleavage products and their mass spectrometric analysis, as well as amino-terminal sequencing showed that Pro39 cleavage products were derived exactly from the predicted cleavage sites, determined previously by radiosequence analysis (29, 30) (Table II). As controls we used thioredoxin fusion proteins with mutations close to the cleavage site, which were not digested by Pro39 or nsP2 (Fig. 4). Thus, we conclude that both proteases recognize specifically the three cleavage sites of the SFV nonstructural polyprotein. However, there were large differences in the sensitivity of the different cleavage sites, the 3/4 junction being most sensitive. Roughly 5000-fold more Pro39 was needed for complete cleavage of site 1/2 (Fig. 5). Under the same conditions, only a small amount of Trx23 was cleaved.

The different sensitivities of the three cleavage sites may well reflect the different specificities of the protease associated with the polyproteins, in which the cleavage at site 2/3 of P123 or P1234 does not take place unless preceded by cleavage of nsP1. The fact that Pro39 can catalyze cleavage at 1/2 site of in vitro translated SFV P12, P123, and P1234, which normally undergoes cleavage in cis (14), is better understood when realized that the estimated molar enzyme to substrate ratio represents an excess of 50 to 1, which is difficult to imagine to take place during virus infection. Thus, we cannot exclude the possibility that cleavages at sites 1/2 and 2/3 require cofactor(s), which might be derived from the other nsPs. Such a situation has been characterized thoroughly for the NS3 protease of hepatitis C virus. The site-specific proteolytic activity of NS3 protease was greatly increased by a short amino acid sequence of NS4 protein adjacently located in the polyprotein (40, 41, 43, 44).

The processing intermediate P123, together with nsP4, enables the synthesis of the complementary RNA for a short time period, whereafter P123 is autocatalytically cleaved to yield the components of the stable RNA polymerase among them nsP2. The released nsP2 exercises its role in two different forms. As a part of the RNA polymerase complex (45), the amino-terminal domain provides RNA triphosphatase and RNA helicase activities (15, 17). As "soluble nsP2," the carboxyl-terminal domain acts as a regulator of 26 S RNA synthesis (24) and as a trans-acting protease, which catalyzes the rapid cleavage of P1234 and P123, thus preventing the negative strand RNA synthesis late in infection.

	ACKNOWLEDGEMENTS

We thank Airi Sinkko for excellent technical assistance. We are grateful to Dr. Nisse Kalkkinen for valuable advice and discussions. We thank Dr. Marja Makarow and Dr. Tero Ahola for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by Academy of Finland Grant 8397 and by grants from the Technology Development Center and Center for International Mobility.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Biocentrum Helsinki fellow. To whom correspondence should be addressed. Tel.: 358-9-191-59400; Fax: 358-9-191-59560; E-mail: leevi.kaariainen@helsinki.fi.

Published, JBC Papers in Press, June 15, 2001, DOI 10.1074/jbc.M104786200

	ABBREVIATIONS

The abbreviations used are: SFV, Semliki Forest virus; nsP, nonstructural protein; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; NEM, N-ethylmaleimide; E-64, L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane; PMSF, phenylmethylsulfonyl fluoride; aa, amino acid(s); PCR, polymerase chain reaction; S, short; L, long; Trx, thioredoxin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SIN, Sindbis virus.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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