Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7622-7626, August 1995 Biochemistry

The hepatitis C virus NS3 serine proteinase and NS4A cofactor: Establishment of a cell-free trans-processing assay (flaviviridae/proteinase inhibitor/antiviral assay) CHAo LIN AND CHARLES M. RICE* Department of Molecular Microbiology, Washington University School of Medicine, Box 8230, 660 South Euclid Avenue, St. Louis, MO 63110-1093 Communicated by Robert H. Purcell, National Institutes ofHealth, Bethesda, MD, March 31, 1995

ABSTRACT The hepatitis C virus RNA genome encodes a polyprotein of "3000 amino acids (reviewed in refs. 5 and 6). long polyprotein that is proteolytically processed into at least This polyprotein is cleaved at multiple sites by cellular and viral 10 products. The order of these cleavage products in the proteinases. The order and nomenclature of these cleavage polyprotein is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A- products for the HCV-H strain are NH2-C-El-E2-p7-NS2-NS3- NS5B-COOH. A serine proteinase domain located in the NS4A-NS4B-NS5A-NSSB-COOH. C, El, and E2 are putative N-terminal one-third of nonstructural protein NS3 mediates structural proteins and the remaining proteins are presumed cleavage at four downstream sites (the 3/4A, 4A/4B, 4B/SA, nonstructural (NS) proteins involved in RNA replication. The and 5A/5B sites). In addition to the proteinase catalytic N-terminal domain of NS3 functions as a serine proteinase domain, the NS4A protein is required for processing at the which cleaves at four sites in the NS region (at the 3/4A, 4B/5A site but not at the 5A/5B site. These cleavage events 4A/4B, 4B/5A, and 5A/5B sites) (7-12). Substitutions for are likely to be essential for virus replication, making the residues in the putative catalytic triad (such as Ser-1165) serine proteinase an attractive antiviral target. Here we abolish processing at all four sites. While the NS3 serine describe an in vitro assay where the NS3-4A polyprotein, NS3, proteinase domain alone is sufficient for cleavage at the 5A/5B the serine proteinase domain (the N-terminal 181 residues of site, coexpression of the 54-residue NS4A protein is required NS3), and the NS4A cofactor were produced by cell-free for processing at the 3/4A and 4B/SA sites, and perhaps also translation and tested for trans-processing of radiolabeled at the 4A/4B site (13-15). It has been suggested that the NS3 substrates. Polyprotein substrates, NS4A-4B or truncated and NS4A proteins associate as a complex which is important NS5A-5B, were cleaved in trans by all forms of the proteinase, not only for modulating substrate specificity of the serine whereas NS4A was also required for NS4B-5A processing. proteinase (13-15) but also for membrane association of the Proteolysis was abolished by substitution mutations previ- HCV RNA replicase complex (16). ously shown to inactivate the proteinase or block cleavage at Thus far, to our knowledge, in vitro assays useful for specific sites in vivo. Furthermore, N-terminal sequence anal- purification and further characterization of the NS3 serine ysis established that cleavage in vitro occurred at the authentic proteinase and NS4A cofactor have not been described. In this 4A/4B site. Translation in the presence of microsomal mem- report, we have established an in vitro trans-cleavage assay for branes enhanced processing for some, but not all, proteinase- these activities and have studied the effects of microsomal substrate combinations. Trans-processing was both time and membranes, incubation temperature, detergents, and several temperature dependent and was eliminated by treatment with common proteinase inhibitors. a variety of detergents above their critical micelle concentra- tions. Among many common proteinase inhibitors tested, only high (millimolar) concentrations of serine proteinase inhib- MATERIALS AND METHODS itors tosyllysyl chloromethyl ketone and 4-(2-aminoethyl)ben- Cell-Free Transcription and Translation. HCV cDNA- zenesulfonyl fluoride inactivated the NS3 proteinase. This in containing expression constructs used in this study have been vitro assay should facilitate purification and further charac- described (15). These plasmids (with the encoded proteins terization of the viral serine proteinase and identification of given in parentheses) include pTM3/HCV1027-1207 (NS3181), molecules which selectively inhibit its activity. pTM3/HCV1027-1657 (NS3), pBRTM/HCV1027-1711 (NS3- 4A), pTM3/HCV1658-1711 (NS4A), pTM3/HCV1658-1972 Hepatitis C viruses (HCVs) have been recently identified as (NS4A-4B), pTM3/HCV1712-2420 (NS4B-5A), and pTM3/ agents of the parenterally transmitted form of non-A non-B HCV2269-2508 (NS5A297-5B88) (Fig. 1). Upstream (5') of the hepatitis (1, 2). Blood screening has virtually eliminated HCV cDNA sequences, all of these constructs contain a T7 HCV-contaminated samples and greatly reduced the incidence promoter followed by the internal ribosome entry element of of post-transfusion hepatitis. However, HCV remains respon- encephalomyocarditis virus (17). 5'-Uncapped RNA tran- sible for a significant proportion of community-acquired hep- scripts were synthesized from linearized cDNA templates using atitis (3). Chronic infections are often established and can lead T7 RNA polymerase (Epicenter Technologies, Madison, WI) to chronic hepatitis and cirrhosis, which is strongly associated (18). Cell-free translations using rabbit reticulocyte lysates with development of hepatocellular carcinoma (reviewed in (Promega) were performed according to the supplier's instruc- ref. 4). There is considerable interest in developing additional tions. For 35S-labeled substrates, reaction mixtures were incu- HCV-specific antiviral agents to complement currently avail- bated for 60 min at 30°C in the presence of [35S]methinine able a-interferon therapy, which effectively controls disease in (Amersham) at 300 tLCi/ml (1 ,uCi = 37 kBq). To produce only a minority of HCV-infected patients. proteinase components, reaction mixtures were incubated for The positive-strand HCV genome RNA is approximately 9.4 90 min at 30°C with a mixture containing all 20 unlabeled kb in length and consists of a highly conserved 5' noncoding amino acids (Promega). In some translation reactions, micro- region followed by a long open reading frame that encodes a Abbreviations: HCV, hepatitis C virus; NS, nonstructural; TLCK, The publication costs of this article were defrayed in part by page charge tosyllysyl chloromethyl ketone; AEBSF, 4-(2-aminoethyl)benzenesul- payment. This article must therefore be hereby marked "advertisement" in fonyl fluoride. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7622 Downloaded by guest on September 28, 2021 Biochemistry: Lin and Rice Proc. Natl. Acad. Sci. USA 92 (1995) 7623 - + *-membrane - * * OR... I .. <- A ...... z + -- proteinase ElE2 p7 2" "34SA4BSB - _ _ It ; I a .. II + at at o _ T . I _ _ _ +1$ 97.4- -4B-5A 34A 69- 3.5A 3 A 46- 3 4A 30- 4A-4B ....._I,... ._ -4B 4B-SA 21.5- 5A~SB I 46- I I I I I I I FIG. 1. HCV polyprotein, cleavage products, and expression con- -5A297-5BB structs. The diagram of the HCV-H strain polyprotein indicates the positions and identities of the cleavage products including C, El, E2, B 30- p7, NS2, NS3, NS4A, NS4B, NS5A, and NSSB. Sites of proteolytic processing are shown at the top, including those for host signal ...... _om 0 -5A297448 peptidase (*), the HCV NS2-3 autoproteinase (h), and the HCV NS3 21.5- X1 24t- serine proteinase ( I ). The serine proteinase domain in the N-terminal I I I I I I I I one-third of NS3 is shaded. Shown below are the seven expression constructs used in this study. HCV polypeptide sequences present in 30- -4A4B each plasmid are indicated by black lines, which are drawn to scale and C -4B N- oriented with respect to the diagram of the HCV-H polyprotein. 21.5- and C-terminal boundaries of truncated proteins are indicated by I subscript numbers except for 3181, which denotes the N-terminal 181 residues of NS3. For simplicity, the NS prefix is not used here or in ra subsequent figures. SC 3181 + 4A

- + + membrane (3181) somal membranes (Promega) were added to a final concen- 0-+ + memnbranem (4A) tration of 1.8 per 25-,ul reaction volume unless otherwise 97.4- r.C. Al -4B-5A indicated. Translation reactions were terminated by the addi- 69- 25A tion of RNase A (Boehringer Mannheim) to 10 ,tg/ml and 46- cycloheximide to 0.3 mg/ml, followed by incubation for 10 min D at 30°C. Unlabeled methionine was also added to the 35S- 30- labeling reaction mixtures to a final concentration of 1 mM. -4B Control experiments showed no further incorporation of 1 2 3 4 5 6 7 [35S]methionine after the addition of these reagents. In Vitro Activity Assay for the NS3 Serine Proteinase. After FIG. 2. In vitro activity of various forms of the NS3 serine pro- termination of the translation reactions, standard trans- teinase. Cell-free transcription and translation were used to synthesize volumes the indicated HCV proteins. Proteinase components were synthesized cleavage reactions were initiated by mixing equal of in the absence or presence of microsomal membranes as indicated at the translation reaction mixtures for proteinase components the top of each lane forA-C. 35S-labeled HCV substrates NS4B-5A (A) and substrates followed by incubation under the conditions and NS5A297-5B88 (B) were synthesized in the absence of microsomal specified in the figure legends. In some experiments, deter- membranes, whereas NS4A-4B (C) was produced in the presence of gents (Boehringer Mannheim) or proteinase inhibitors microsomal membranes. Trans-cleavage reaction mixtures were incu- (Boehringer Mannheim, Calbiochem, or Sigma) were added to bated for 3 h at 30°C and analyzed by SDS/PAGE. (D) Effects of the trans-cleavage reaction mixtures. Reaction mixtures con- microsomal membranes on activity of NS318g and NS4A synthesized taining proteinase inhibitors or appropriate solvent controls separately. 35S-labeled NS4B-5A substrate was produced in the ab- were preincubated for 10 min on ice and then incubated for 1 sence of microsomal membranes (lanes 1-7). Unlabeled proteinase h were ana- translations: lane 1, no RNA; lanes 2 and 3, NS318g and NS4A RNAs at 37°C. Following incubation, reaction products cotranslated in the same reaction in the absence (-; lane 2) or lyzed by SDS/10% PAGE (15, 19). presence (+; lane 3) of microsomal membranes. In lanes 4-7, NS3181 and NS4A RNAs were translated in separate reactions in the absence RESULTS (-) or presence (+) of microsomal membranes and then mixed together with the NS4B-5A substrates. In this and subsequent figures, An in Vitro Assay for HCV NS3 Serine Proteinase Activity. HCV-specific proteins are indicated on the right and the sizes (in kDa) Various forms of the proteinase, with or without the NS4A of 14C-labeled protein markers are on the left. The identities of cofactor, were produced by cell-free translation and tested for HCV-specific in vitro cleavage products were verified by immunopre- their ability to process radiolabeled substrates containing the cipitation with region-specific antisera and shown to comigrate with 4A/4B, 4B/5A, or 5A/5B cleavage sites (see Fig. 1). We first the corresponding HCV-specific proteins produced in vivo (data not tested the NS4B-5A polyprotein substrate, whose cleavage shown). should be dependent upon expression of both the serine and proteinase and the NS4A cofactor. The full-length NS4B-5A indicator of virus-specific proteolytic activity. When NS3 translation product (predicted molecular mass 76.5 kDa) was NS4A were synthesized as a single polyprotein NS3-4A, sig- not processed after incubation with either the NS3 serine nificantly higher proteolytic activity was observed (Fig. 2A, proteinase domain, NS3181 (Fig. 2A, lane 3), or the full-length lane 7). Due to the limited quantities of the cleavage products, NS3 protein (lane 5). When either form of the proteinase was we have not attempted N-terminal sequence analysis of NS5A. produced by cotranslation with NS4A, proteolytic activity was However, the sizes of the cleavage products, the requirement observed as evidenced by the appearance of the NS4B and for both the NS3 serine proteinase and NS4A, and the NS5A cleavage products (Fig. 2A, lanes 4 and 6). The putative immunoreactivity of the products (data not shown) suggest NS5A cleavage product was difficult to observe in this assay, that authentic cleavage occurred in our in vitro assay. Further, since it often comigrated with truncated translation products. substitution of an Ala residue for Ser-1165, the putative NS4B, however, was readily observed and used as the major nucleophile of the NS3 serine proteinase (20), abolished Downloaded by guest on September 28, 2021 7624 Biochemistry: Lin and Rice Proc. Natl. Acad. Sci. USA 92 (1995) processing in the in vitro assay, and processing was not ob- membrane association of NS4A-4B allows formation of an served for substrates containing mutations at the 4B/5A accessible 4A/4B cleavage site as well as a functional NS4A cleavage site that blocked cleavage in vivo (Pl, Cys -* Arg or cofactor. N-terminal sequence analysis of the [35S]methionine- Asp; ref. 21) (data not shown). labeled NS4B cleavage product yielded methionine residues at As mentioned earlier, the 5A/5B site can be cleaved by the positions 11 and 12 (data not shown), verifying site-specific NS3 proteinase in vivo in the absence of NS4A. Besides the cleavage at the authentic 4A/4B site (9) in vitro. full-length NS5A-5B polyprotein substrate, a truncated 240- Further Characterization of the in Vitro Assay. A time residue polypeptide containing the 5A/5B cleavage site course of NS3-4A-mediated processing of the NS4B-5A sub- (NS5A297-5B88) was also efficiently processed in trans by strate is shown in Fig. 14. At 30°C, a gradual increase in the NS3181 in vivo (15). As shown in Fig. 2B, this substrate was amount of the NS4B and NS5A cleavage products was ob- processed inefficiently in the in vitro assay. Upon the addition served throughout the 3-h incubation period (Fig. 14). Inter- of the various proteinases, we observed a slight but significant estingly, after 3 h of incubation, the NS5A band shifted to a increase in the level of a 26-kDa species, tentatively identified slower-migrating form, which is due to post-translational phos- as the truncated NS5A cleavage product (NS5A297-U8). It phorylation (K. Reed, A. Grakoui, J. Xu, C.L., and C.M.R., should be noted that this protein comigrated with a faint unpublished data). Compared with the rate at 30°C, NS4B-5A background band present in the original NS5A297-5B88 trans- cleavage was slower at 23°C and faster at 37°C. No processing lation reaction (lane 2). The truncated NS5B cleavage product was observed at 0°C even after a 3-h incubation. Processing of (NS5B1 88), which is presumably unstable, has not been de- NS4A-4B by the NS3181 proteinase (Fig. 3B) occurred rapidly tected either in the in vitro assay or in vivo when the vaccinia even at 23°C, reaching a plateau after 15-30 min. Similar virus-17 system has been used (15). In contrast to the results were obtained at 30°C or 37°C (data not shown). Even NS4B-5A substrate, the appearance of the NS5A297448 cleav- at 0°C, a weak band corresponding to the NS4B cleavage age product was not markedly influenced by the presence of product was observed after 2 or 3 h of incubation (Fig. 3B). NS4A. Translation and processing of the HCV polyprotein are A temperature time (min) believed to occur in the rough endoplasmic reticulum in 97.4- association with microsomal membranes (6). Through their 69- -4B-5A interaction with segments of the NS polyprotein rich in hy- -5A drophobic amino acids (either components of the proteinase 46- or the substrates), microsomal membranes may also influence serine proteinase-dependent processing. We examined this 30-j possibility by studying the effects of microsomal membranes -4B added to proteinase or substrate translation reactions. When the NS4B-5A substrate was synthesized in the presence of 37°C temperature microsomes, the degree of processing by the various protein- '0 15 30 60 180' time (min) 97.4- -iLA,,ik ases was unchanged (data not shown). Production of the NS4B 69- A -4B-5A and NS5A cleavage products was enhanced when NS4A was 25A cotranslated with NS3181 or NS3 in the presence of microsomal 46- membranes (Fig. 2A, lane 9 or 11). No stimulation was observed when microsomal membranes were added post- 30- translationally (data not shown). Interestingly, the proteolytic -4B activity of NS3-4A was not increased by inclusion of microso- mal membranes during translation (lane 12) (see Discussion). B 0°C 230C temperature Microsomal membranes might exert their influence on '0O 15 30 60 120 180 0 15 30 60 120 180' time (nin) interaction with or both of proteinase activity by NS3, NS4A, 30- -4A-4B these components. To examine this, transcripts encoding the -4B serine proteinase domain, NS3181, and NS4A were translated in separate reactions in the absence or presence of microsomal 21.5- membranes and then combined and incubated with the NS4B-5A substrate. As shown in Fig. 2D, stimulation of C proteinase activity correlated with the presence of microsomal ,318, membranes in the translation reaction mixtures ofNS4A (lanes -4A-4B 4 and 6) but not NS3181 (lanes 6 and 7). These data suggest that nascent NS4A may cotranslationally interact with microsomal 30- -4A-4B membranes in a way which stimulates serine proteinase activ- -4B ity, possibly by facilitating formation of an active proteinase 21.5- complex between NS4A and the catalytic domain of NS3 (see Discussion). In contrast to our observations with the NS4B-5A FIG. 3. Processing kinetics of NS4A-4B and NS4B-5A substrates. substrate, microsomal membranes present during translation (A) Trans-cleavage reaction mixtures containing unlabeled NS3-4A of the proteinase components did not enhance processing of proteinase and 35S-labeled NS4B-5A substrate (both synthesized in the NS5A297-5B88 (Fig. 2B). This is perhaps not surprising, given absence ofmicrosomal membranes) were incubated at 0°C, 23°C, 30°C, that this cleavage is not NS4A-dependent. or 37°C. At the indicated time points, an equal portion of each The most striking effect of microsomal membranes was seen trans-cleavage reaction mixture was analyzed by SDS/PAGE. (B) for the NS4A-4B substrate. NS4A-4B synthesized in the ab- Trans-cleavage reaction mixtures containing unlabeled NS3181 pro- sence of microsomal membranes was not processed by any of teinase (synthesized in the absence of microsomal membranes) and the proteinases (data not also see In contrast, 35S-labeled NS4A-4B substrate (synthesized in the presence of micro- shown; Fig. 4). somal membranes). Samples were processed and analyzed as inA. (C) NS4A-4B synthesized in the presence of microsomal mem- Aliquots of NS3181 proteinase and 35S-labeled NS4A-4B substrate branes was efficiently processed by all sources of active serine were preincubated for 30 min at the indicated temperatures and proteinase, including NS3-4A, NS3, or NS3181 (Fig. 2C), but assayed for trans-processing by mixing and incubation for 1 h at 37°C. not the inactive proteinase containing the Ser-1165 -- Ala Samples not preincubated were used as positive controls (+). Products substitution (data not shown). These results suggest that were resolved by SDS/PAGE. Downloaded by guest on September 28, 2021 Biochemistry: Lin and Rice Proc. Natl. Acad. Sci. USA 92 (1995) 7625 To determine whether active proteinase or properly folded substrate was limiting in these trans-cleavage reactions, samples detergent ofNS3181 or NS4A-4B were preincubated atvarious temperatures (%) prior to the in vitro trans-cleavage assay. As shown in Fig. 3C, 97.4- preincubation at 37°C for 30 min resulted in only slight inactiva- 69- -4B-5A tion of the proteinase (lane 4) or the substrate (lane 8). Both 2.5A appeared to be stable at 0°C (lane 2) or 30°C (lanes 3 and 7). 46- Higher temperatures resulted in increased inactivation of the proteinase (lanes 5 and 6) and, to a lesser extent, the substrate 30- (lanes 9 and 10). NS3181 activity was not detected after preincu- > -4B bation for 30 mi at 50°C (lane 6). FIG. 5. Effects of detergents on trans-processing by NS3-4A. Since the proteinase was reasonably stable at 30°C, these NS3-4A proteinase and 35S-labeled NS4B-5A substrate were produced experiments suggest that the rate and extent of NS4A-4B by translation in the absence of microsomal membranes. After trans- cleavage may be limited by the amount of substrate in the lation, reaction mixtures were mixed in a 1:1 ratio and detergents were correct conformation for efficient processing. Since cleavable added to the final concentrations shown above each lane. MEGA-8, octanoyl-N-methylglucamide; n-OG, n-octyl glucoside; CHAPS, 3-[(3- NS4A-4B was produced only when microsomal membranes cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPSO, were present during translation, we examined the effects of 2-hydroxy-CHAPS. Trans-cleavage reaction mixtures were incubated varying the concentration of microsomal membranes or for 3 h at 30°C and analyzed by SDS/PAGE. NS4A-4B RNA transcript on cleavage efficiency of the sub- strate. Increasing the concentration of microsomal membranes led to increased accumulation of primary translation product like serine proteinase superfamily (reviewed in ref. 20), and and enhanced processing efficiency at the 4A/4B site (Fig. 4). the importance of predicted catalytic residues was subse- At the highest concentration of microsomal membranes tested quently verified by site-directed mutagenesis (see ref. 6 and (1.8 ,lI per 25-,ul reaction mixture), decreasing the input RNA citations therein). The in vitro assay allowed us to test the transcript lowered the level of the NS4A-4B translation prod- effects of common proteinase inhibitors on trans-cleavage by uct but did not significantly alter its cleavage efficiency (data the HCV serine proteinase. The serine proteinase inhibitors not shown). tosyllysyl chloromethyl ketone (TLCK) and 4-(2-aminoethyl)- We also examined the effects of several detergents. All of benzenesulfonyl fluoride (AEBSF) at millimolar concentra- the detergents tested showed significant inhibition of trans- tions inhibited both the NS3181 and NS3-4A proteinases in the cleavage of NS4B-5A by the NS3-4A proteinase (Fig. 5). Least trans-cleavage assays utilizing either the NS4A-4B or NS4B-5A severe inhibition was observed for MEGA-8 [critical micelle substrates (Fig. 6). Slight inhibition was observed with phe- concentration (cmc) of 58 mM], which at 0.5% (15.6 mM) only nylmethylsulfonyl fluoride (PMSF; 2 mM), tosylphenylalanyl slightly decreased the proteolytic activity of NS3-4A. Proteo- chloromethyl ketone (TPCK; 0.2 mM), and EDTA (10 mM) lysis was diminished in the presence of 0.1% (3.4 mM) n-octyl (data not shown). Other proteinase inhibitors, even at the glucoside (cmc of 20-25 mM) and was abolished by the highest concentrations tested, had no detectable effect on addition of n-octyl glucoside to 0.5% (17.1 mM). For CHAPS activity. These included serine proteinase in- and CHAPSO (cmc of 8 mM), 0.1% (1.6 mM) but not 0.5% trans-cleavage (8.0 mM) allowed detectable processing of NS4B-5A. No hibitors a1-antichymotrypsin (2 ,uM), aprotinin (2 mg/ml), cleavage was observed in the presence of 0.1% of any of the benzamidine (20 mM), chymostatin (2 mM), 3,4-dichloroiso- following detergents: n-dodecyl f3-D-maltoside, Triton X-100, coumarin (2 mM), leupeptin (2 mM; also a cysteine proteinase Triton X-114, Thesit, isotridecylpoly(ethylene glycol ether), or inhibitor), soybean trypsin inhibitor (0.2 mM), and the cysteine N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate proteinase inhibitor E-64 (0.4 mM) (data not shown). (also called Zwittergent 3-12) (data not shown). It should be noted that this concentration (0.1%) is above or close to the cmc for each of these six detergents. Similar results were obtained for NS3181-mediated trans-processing of NS4A-4B (data not shown). These results suggest an inverse correlation A 30a -4A-4B between formation of detergent micelles and proteinase ac- -4B tivity of NS3181 or NS3-4A. 21.5- The serine proteinase domain ofHCV NS3 was identified on the basis of sequence homology with members of the trypsin- 30- '-4A4B B -4B 0 0.3 0.6 0.9 1.2 1.5 1.8*(>XV25-RI)membrane) 215- 97A4- 30- 4A_-44B -4B-5A -CA 21.5- 30- - 30- _ -4A4B -k W.O !-4B -4B 21.5- I S.';...... , . 21.5- FIG. 6. Effects ofproteinase inhibitors on the activity ofthe NS3181 and NS3-4A proteinases. Unlabeled proteinases included NS3181 (A) FIG. 4. Concentration effect of microsomal membranes on pro- or NS3-4A (B and C). 35S-labeled substrates included NS4A-4B (A and cessing of NS4A-4B by NS3181. 35S-labeled NS4A-4B was produced by B) or NS4B-5A (C). The NS4A-4B substrate was translated in the translation (transcript RNA at 50 jug/ml) in the absence or presence presence of microsomal membranes. The first lane in each set (-) is of increasing amounts of microsomal membranes (indicated above a solvent control containing no inhibitor (2 mM sodium acetate, pH each lane in ,ul per 25-,l reaction mixture). NS4A-4B substrates were 5.2, for TLCK; 4 mM sodium phosphate, pH 7.0, for AEBSF). Inhibitor incubated for 1 h at 37°C in the absence (Upper) or presence (Lower) concentrations (increasing from left to right) were: TLCK (2 ,uM, 20 of unlabeled NS3181 (produced by translation in the absence of ,uM, 200 ,uM, or 2 mM); AEBSF (20 ,uM, 200 ,uM, 2 mM, or 20 mM). microsomes). Samples were analyzed by SDS/PAGE. Trans-cleavage reaction products were analyzed by SDS/PAGE. Downloaded by guest on September 28, 2021 7626 Biochemistry: Lin and Rice Proc. Natl. Acad. Sci. USA 92 (1995) DISCUSSION (7-12) are all consistent with the assignment of the HCV proteinase to the trypsin/chymotrypsin-like superfamily. This In this report, cell-free translation was used to develop a hypothesis is further supported by our finding that only serine trans-cleavage assay for the HCV serine proteinase. This proteinase inhibitors, in particular TLCK and AEBSF, mark- approach has proven valuable for studying proteinases en- edly inhibited the HCV proteinase trans-cleavage activity. coded by many animal and plant viruses. For HCV, proteolytic Millimolar inhibitor concentrations were needed for complete activity was obtained by expression of an NS3-4A polyprotein, inhibition, suggesting that these common trypsin (TLCK) or full-length NS3, or the serine proteinase domain (NS3181). general (AEBSF) serine proteinase inhibitors are not partic- Active NS4A cofactor could be expressed as a cis-cleavage ularly effective for the HCV enzyme. Only slight inhibition was product of the NS3-4A polyprotein, as an individual polypep- observed with other serine proteinase inhibitors, phenylmeth- tide, or as part of the NS4A-4B substrate. Thus far, the ylsulfonyl fluoride and tosylphenylalanyl chloromethyl ketone, requirements for trans-cleavage in vitro generally mimic those or the metal ion chelator EDTA. The latter observation previously observed in transient expression experiments in suggests that metal ions may somehow be involved in HCV mammalian cell cultures (13-15). Both the NS3 serine pro- serine proteinase activity. In any case, the establishment of an teinase and the NS4A cofactor were required for trans- cleavage at the 4B/5A site, whereas the serine proteinase in vitro trans-cleavage assay and the preliminary results with these inhibitors are for future development of domain alone was sufficient for cleavage of NS4A-4B or a promising truncated substrate containing the 5A/5B site. specific inhibitors of the HCV serine proteinase. The role of NS4A in facilitating cleavage at certain sites in We thank Karen Reed and Drs. Stephen Chambers, James Landro, the NS polyprotein is still unclear. Of many possibilities, NS4A David Livingston, Vicki Sato, and John A. Thomson of Vertex might help in the formation of an active proteinase by acting Pharmaceuticals for discussion and critical reading of the manuscript. as a chaperone to facilitate proper folding, by interacting with This work was supported by a grant from the U.S. Public Health the catalytic subunit in a way which facilitates substrate Service (CA57973). C.L. was a doctoral candidate and was supported recognition and cleavage at NS4A-dependent sites, or by in part by the Division of Biology and Biomedical Sciences at localizing NS3 to cellular membranes (16) where proteolysis of Washington University. membrane-associated substrates might occur more efficiently (13-15). Our results (this report; ref. 22) argue against an 1. Choo, Q.-L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & obligate chaperone-like role, since similar trans-cleavage ac- Houghton, M. (1989) Science 244, 359-362. H. G. A. at was 2. Kuo, G., Choo, Q.-L., Alter, J., Gitnick, L., Redeker, G., tivity the 5A/5B site observed for all forms of the Purcell, R. H., Miyamura, T., Dienstag, J. L., Alter, M. J., Stevens, proteinase, regardless of NS4A coexpression. Coimmunopre- C. E., Tegtmeier, G. E., Bonino, F., Colombo, M., Lee, W.-S., Kuo, C., cipitation studies have provided evidence for an NS3-NS4A Berger, K., Shuster, J. R., Overby, L. R., Bradley, D. W. & Houghton, complex which may be responsible for cleavage at NS4A- M. (1989) Science 244, 362-364. dependent cleavage sites (22). Interestingly, we found that 3. Alter, M. J., Hadler, S. C., Judson, F. N., Mares, A., Alexander, W. J., translation of NS4A in the presence of microsomal membranes Hu, P. Y., Miller, J. K., Moyer, L. A., Fields, H. A., Bradley, D. W. & enhanced activity. Similarly, microsomal mem- Margolis, H. S. (1990) J. Am. Med. Assoc. 264, 2231-2235. proteolytic 4. Shimotohno, K. (1993) Semin. Virol. 4, 305-312. branes were required to produce an NS4A-4B substrate which 5. Houghton, M. (1995) in Fields Virology, eds. Fields, B. N., Knipe, could be processed in trans. These results suggest that the D. M. & Howley, P. M. (Raven, New York), 3rd Ed., in press. nascent NS4A region, perhaps by means of its N-terminal 6. Rice, C. M. (1995) in Fields Virology, eds. Fields, B. N., Knipe, D. M. hydrophobic domain, interacts with membranes to allow for- & Howley, P. M. (Raven, New York), 3rd Ed., in press. mation of an active proteinase complex, attain proper sub- 7. Bartenschlager, R., Ahlborn-Laake, L., Mous, J. & Jacobsen, H. strate conformation (as for NS4A-4B), or both. Microsomal (1993) J. Virol. 67, 3835-3844. 8. Eckart, M. R., Selby, M., Masiarz, F., Lee, C., Berger, K., Crawford, membranes were not absolutely required to generate active K., Kuo, C., Kuo, G., Houghton, M. & Choo, Q.-L. (1993) Biochem. NS4A cofactor and did not enhance the activity of the NS3-4A Biophys. Res. Commun. 192, 399-406. proteinase. Thus, while membrane association of the NS3- 9. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M. & NS4A complex may be important for replicase function, it is Rice, C. M. (1993) J. Virol. 67, 2832-2843. not absolutely required for cleavage at NS4A-dependent sites. 10. Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., For a variety of detergents, we found that trans-cleavage Tanaka, T., Kimura, K & Shimotohno, K (1993)J. Virol. 67,4665-4675. 11. Manabe, S., Fuke, I., Tanishita, O., Kaji, C., Gomi, Y., Yoshida, S., activity was inhibited or abolished as the detergent concen- Mori, C., Takamizawa, A., Yoshida, I. & Okayama, H. (1994) Virology trations approached or exceeded their critical micelle concen- 198, 636-644. trations. This observation contrasts with the flavivirus NS2B- 12. Tomei, L., Failla, C., Santolini, E., De Francesco, R. & LaMonica, N. NS3 serine proteinase complex, where activity is recovered (1993) J. Virol. 67, 4017-4026. only after solubilization of cellular membrane fractions with 13. Bartenschlager, R., Ahlborn-Laake, L., Mous, J. & Jacobsen, H. nonionic detergents (23). Computer modeling (24) and the (1994) J. Virol. 68, 5045-5055. that the HCV 14. Failla, C., Tomei, L. & De Francesco, R. (1994) J. Vrol. 68, 3753-3760. results of mutagenesis studies (21) indicate NS3 15. Lin, C., Pragai, B., Grakoui, A., Xu, J. & Rice, C. M. (1994) J. Virol. proteinase has a rather hydrophobic substrate binding pocket 68, 8147-8157. which might be capable of binding detergents or detergent 16. Hijikata, M., Mizushima, H., Tanji, Y., Komoda, Y., Hirowatari, Y., micelles, leading to inhibition of proteinase activity. Alterna- Akagi, T., Kato, N., Kimura, K. & Shimotohno, K. (1993) Proc. Natl. tively, detergent micelles could exert their inhibitory effect by Acad. Sci. USA 90, 10773-10777. binding to NS4A or its cognate binding site on the NS3 17. Jang, S. K, Krausslich, H.-G., Nicklin, M. J. H., Duke, G. M., Palimen- proteinase domain. The NS3-NS4A complex, although not berg, A. C. & Wimmer, E. (1988) JVruol. 62, 2636-2643. 18. Rice, C. M., Levis, R., Strauss, J. H. & Huang, H. V. (1987) J. Virol. disrupted by treatment with nonionic detergents, appears to be 61, 3809-3819. stabilized by multiple hydrophobic interactions involving res- 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. idues in the N-terminal and central regions of NS4A (22). 20. Bazan, J. F. & Fletterick, R. J. (1990) Semin. Virol. 1, 311-322. Inhibition could be mediated either by changing proteinase 21. Kolykhalov, A. A., Agapov, E. V. & Rice, C. M. (1994) J. Virol. 68, conformation and specificity or by sterically hindering sub- 7525-7533. strate access. Further studies are needed to distinguish among 22. Lin, C., Thomson, J. & Rice, C. M. (1995) J. Virol. 69, 4373-4380. 23. Amberg, S. M., Nestorowicz, A., McCourt, D. W. & Rice, C. M. these possibilities. (1994) J. Virol. 68, 3794-3802. Sequence alignments with known proteinases (see ref. 20 for 24. Pizzi, E., Tramontano, A., Tomei, L., LaMonica, N., Failla, C., a review), structural modeling of the active site (24), and Sardana, M., Wood, T. & De Francesco, R. (1994) Proc. Natl. Acad. mutagenesis studies of the proposed catalytic triad residues Sci. USA 91, 888-892. Downloaded by guest on September 28, 2021