This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martinez, M. A.
Right arrow Articles by Clotet, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martinez, M. A.
Right arrow Articles by Clotet, B.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, May 2003, p. 1760-1765, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1760-1765.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genetic Screen for Monitoring Hepatitis C Virus NS3 Serine Protease Activity

Miguel Angel Martinez* and Bonaventura Clotet

Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Barcelona, Spain

Received 17 October 2002/ Returned for modification 17 January 2003/ Accepted 11 February 2003


arrow
ABSTRACT
 
We have developed a genetic system to monitor the activity of the hepatitis C virus (HCV) NS3 serine protease. This genetic system is based on the bacteriophage lambda regulatory circuit where the viral repressor cI is specifically cleaved to initiate the switch from lysogeny to lytic infection. An HCV protease-specific target, NS5A-5B, was inserted into the lambda phage cI repressor. The target specificity of the HCV NS5A-5B repressor was evaluated by coexpression of this repressor with a ß-galactosidase (ßgal)-HCV NS32-181/421-34 protease construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI.HCV5AB-cro and the ßgal-HCV NS32-181/421-34 protease constructs, lambda phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS32-181/421-34 protease. This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine protease, and it has the potential to be used for screening specific inhibitors.


arrow
TEXT
 
The activity of specific proteases is essential in many fundamental cellular and viral processes. Viral polyprotein processing is indispensable in the replication and maturation of many viruses (6). Consequently, site-specific proteolysis has been an attractive target for the development of antiviral therapies based on potent and selective viral inhibitors. The generation of such therapies based on the inhibition of site-specific proteolysis has been clearly illustrated in the development of effective inhibitors of human immunodeficiency virus type 1 (HIV-1) (3, 8, 9).

The hepatitis C virus (HCV) is a positive-stranded RNA virus which is the causal agent for a chronic liver infection afflicting more than 170 million people worldwide. The infection is usually persistent, and after an asymptomatic period often lasting years, many patients develop chronic liver disease, including cirrhosis and hepatocellular carcinoma (1, 4). The HCV genome is approximately 9.6 kb long and encodes a polyprotein of around 3,000 amino acid residues. This polyprotein is processed into structural and nonstructural proteins by host signal peptidases and by two viral proteases, NS2/3 and NS3 (reference 23 and references therein). The role of the NS2/3 protease appears to be limited to the autoproteolytic cleavage of the NS2-NS3 junction in cis (22). The amino-terminal 180-amino-acid sequence of the NS3 protein encodes a serine protease which cleaves at the NS3/4A junction in cis, which is followed by cleavage at the NS4/4B, NS4B/5A, and NS5A/B sites in trans (10, 29). Of note, the NS3 serine protease requires an accessory viral protein, NS4A, for optimal cleavage activity. The contribution of NS4A to NS3 protease activity can be mimicked by a synthetic peptide encompassing amino acid residues 21 to 34 of NS4 (30). The three-dimensional structure of the NS3 protease domain (residues 1 to 181) complexed with a synthetic NS4A cofactor (residues 21 to 34) has shown that the NS4A peptide is an integral component of the NS3 protease structure (12).

It has previously been demonstrated that a bacteriophage lambda-based genetic screen can be used to monitor the activity and phenotype of the HIV-1 protease (2, 17, 26, 27). This genetic screen system is based on the bacteriophage lambda cI-cro regulatory circuit where viral repressor cI is specifically cleaved to initiate the switch from lysogeny to lytic infection (21). The inherent difficulties in the purification and characterization of the HCV NS3 protease by in vitro classical methodologies prompted us to explore this genetic system as a simple alternative approach for the characterization of HCV NS3 protease activity. Moreover, the different biological properties of the HCV NS3 protease and the HIV-1 protease (serine protease and aspartic protease, respectively) offered us the opportunity to explore whether this system can be used to characterize proteases with different mechanisms of action.

Using a unique restriction site (BssH2) located in the linker domain of cI (26), the HCV NS5A-5B cleavage site was inserted into the {lambda} cI repressor (cI.HCV5A5B) (Fig. 1). Oligonucleotides encoding the HCV proteolytic NS5A-5B cleavage site (EDVVCCSMSYTWTG, NS5AB sense [5'-CGCGCGCTAGTGAGGACGTCGTCTGCTGCTCAATGTCCTATACGTGGACAGGCGCCG-3'], and NS5AB antisense [5'-CGCGCGGCGCCTGTCCACGTATAGGACATTGAGCAGCAGACGACGTCCTCACTAGCG-3']) were inserted in frame in the bacteriophage {lambda} cI repressor, pcI.Bss-cro, to generate pcI.HCV-cro. Similarly, a plasmid containing a mutant proteolytic NS5A-5B cleavage site (EDVVGGSMSYTWTG), pcI.HCVmt-cro, was also constructed. As shown in Fig. 2A, these two repressors efficiently repressed the infecting phage.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 1. Lambda-based genetic screen to monitor the activity of HCV NS3/4 protease. This genetic screen system is based on the bacteriophage lambda cI-cro regulatory circuit where the viral repressor cI is specifically cleaved to initiate the switch from lysogeny to lytic infection. When phages infect E. coli cells that express recombinant cI.HCV5A5B repressor and ßgal-HCV NS32-181/421-34 protease, infection results in lytic replication. In contrast, phage replication is repressed in cells that do not express the specific ßgal-HCV NS32-181/421-34 protease (lysogeny). The cI.HCV5A5B repressor contains the HCV NS5A-NS5B cleavage site sequence shown here.







View larger version (125K):
[in this window]
[in a new window]
  		FIG. 2. Selective growth of {lambda} in E. coli cells coexpressing the ßgal-HCV NS32-181/421-34 protease construct and the cI.HCV5A5B repressor. Expression of the protease was induced by IPTG treatment for 1 h, and the cells were infected with {lambda} for an additional 3 h. The graph illustrates the resulting phage titer (in PFU per microliter). Plasmids pBluescript SK- and pAlterEX-2 were used as controls of the ßgal-HCV NS32-181/421-34 protease construct and the cI.HCV5A5B repressor, respectively. pcI.HCV5A5Bmt-cro was also used as a negative control of the cI.HCV5A5B repressor. As shown, selection in cells coexpressing the ßgal-HCV NS32-181/421-34 protease construct and the cI.HCV5A5B repressor resulted in {lambda} replication, whereas the replication of {lambda} was severely compromised in cells expressing the mutant cI.HCV5A5B repressor (pcI.HCV5A5Bmt-cro) or in cells that do not express active proteases. Absence of protease induction with IPTG also compromised {lambda} replication. Values are the means ± standard deviations (error bars) of at least four experiments.

Next we tested the target specificity of the HCV repressors by coexpressing these repressors with a ß-galactosidase (ßgal)-HCV NS32-181/421-34 protease construct (Fig. 1, 2A, and 3). The ßgal-HCV NS32-181/421-34 construct contained NS4 residues 21 to 34 fused in frame via a short linker to the amino terminus of the NS3 protease domain (residues 2 to 181) (Fig. 3). After viral RNA was isolated from an individual infected with HCV genotype 1b (patient 1), 10 µl of resuspended RNA was reverse transcribed at 42°C by using the avian myeloblastosis virus reverse transcriptase (Promega) and the oligonucleotide HCVproR1 (antisense) (5'-GGATGAGTTGTCTGTGAAGAC-3'; residues 3966 to 3984 of the BK strain). An aliquot of the reverse transcriptase product was amplified by PCR with AmpliTaq Gold DNA polymerase (Applied Biosystems) using the buffers and conditions specified by the manufacturer. The oligonucleotides used for the amplification were HCVproL1 (sense) (5'-GCAAGGGTGGCGACTCCTTGC-3'; residues 3401 to 3421 of the BK strain) and HCVproR1. Nested PCR was then performed with a 5' oligonucleotide (HCVproL2) encoding an EcoRI site, residues 21 to 34 of NS4, and a dipeptide linker, Gly-Gly, along with residues 2 to 8 of NS3 (residues 3433 to 3446 of the BK strain) (5'-GGGTTGAATTCTATGGCTCCTATTGGATCTGTTGTTATTGTTGGAAGAATTATTTTGTCTGGAAGAGGAGGACCTATCACGGCCTACTCCCAA-3'). The 3' oligonucleotide (HCVproR2) was complementary to residues 175 to 181 of NS3 (residues 3942 to 3962 of the BK strain) and encoded an in-frame stop codon flanked by an XhoI site (5'-GGGAGGGGCTCGAGTCAAGACCGCATAGTAGTTTCCAT-3'). The 621-bp fragment was digested with EcoRI and XhoI and inserted into pBSK- (Stratagene) to generate a ßgal-HCV NS32-181/421-34 protease fusion protein (pHCVNS32-181/421-34 protease).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3. The ßgal-HCV NS32-181/421-34 protease construct contains NS4 residues 21 to 34 fused in frame via a short linker to the amino terminus of the NS3 protease domain (residues 2 to 181). It has been previously demonstrated that the kinetic parameters of a single-chain protein containing the NS4A cofactor and the HCV NS3 protease were identical to those of the bona fide NS31-631/NS4A1-54 protein complex generated in eucaryotic cells (5, 28). The HCV NS32-181/mutNS421-34 and HCV NS32-181delNS4 protease constructs are also shown.

Escherichia coli JM109 cells containing plasmid pcI.HCV-cro or pcI.HCVmt-cro were then transformed with plasmid pHCVNS32-181/421-34 protease (Fig. 1). The resulting cells were grown overnight at 30°C in the presence of 0.2% maltose, harvested by centrifugation, and resuspended to an optical density at 600 nm of 2.0 per ml in 10 mM MgSO4. To induce the expression of HCV NS32-181/421-34 protease, cells (200 µl) were incubated in 1 ml of Luria-Bertani (LB) medium containing 12.5 µg of tetracycline, 20 µg of ampicillin, 0.2% maltose, 10 mM MgSO4, and 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 1 h. Thereafter, cell cultures were infected with 107 PFU of {lambda} phage. After 3 h at 37°C, the titer of the resulting phage was determined by coplating the cultures with 200 µl of E. coli XL-1 Blue cells (adjusted to an optical density at 600 nm of 2.0 per ml in 10 mM MgSO4) on LB plates using 3 ml of top agar containing 12.5 µg of tetracycline per ml, 0.2% maltose, and 0.1 mM IPTG. After incubation at 37°C for 6 h, the resulting phage plaques were counted in order to score growth. In the experiments in which E. coli cells express the HCV NS32-181/421-34 protease (Fig. 2A), {lambda} phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS32-181/421-34 construct.

The target specificity of the HCV repressor was further analyzed by coexpression of a control mutant cI.HCV5Abmt-cro target site (EDVVGGSMSYTWTG) with the ßgal-HCV NS32-181/421-34 protease construct. As shown in Fig. 2A, the mutant cI.HCV5Abmt-cro target site prevented phage replication, arguing that the cI.HCV5AB-cro degradation was specifically mediated by the HCV NS32-181/421-34 protease. Finally, when the expression of ßgal-HCV NS32-181/421-34 protease was not induced with IPTG, phage replication was significantly reduced (Fig. 2A).

The specificity of this trans-cleavage reaction was further demonstrated by the lack of phage replication in cells expressing mutated forms of the HCV NS32-181/421-34 protease that included substitutions within the catalytic triad, that is, H57A, D81A, and S139A (Fig. 2B). NS3 protease single mutants were constructed by site-directed mutagenesis using the overlap extension protocol (24). Any of the former substitutions within the catalytic triad completely abolished the proteolytic activity detected with the genetic system presented here. Interestingly, the substitution S138A, which is not part of the catalytic triad, reduced phage growth up to five times, showing the lower catalytic efficiency of this mutant protease (Fig. 2B). In contrast, the Q80L substitution, a very frequent polymorphism found between HCV NS3 proteases of genotype 1, did not affect the catalytic efficiency of the enzyme (Fig. 2B). These findings demonstrate that quantitation of the protease activity with this lambda-based genetic system makes it possible to monitor HCV NS3 proteases with different catalytic efficiencies.

Next, we investigated the influence of the central region of NS4A (residues 21 to 34) in this genetic system. We constructed a deletion form of the HCV NS32-181/421-34 protease construct in which the NS4 residues were absent; we also constructed a mutated form in which the I residues at positions 25 and 29 of NS4A were replaced by A residues (Fig. 3). NS4 mutants were generated by PCR by directly amplifying the pHCVNS32-181/421-34 construct with the appropriate mutated HCVproL2 oligonucleotide. Although the NS3 protease has proteolytic activity of its own, complex formation with the viral NS4A polypeptide is essential for efficient processing of the NS3-NS4A and NS4B-NS5A sites and improved cleavage at the NS4A-NS4B and NS5A-NS5B junctions (reviewed in reference 31). Moreover, NS4 amino acids I25 and I29 seemed to be critical for NS4 activation of NS3 protease (25). The former two constructs HCV NS32-181delNS4 and HCV NS32-181/mutNS421-34, tested here using the lambda-based genetic system showed very little proteolytic activity (Fig. 2C). Although the HCV NS32-181/mutNS421-34 construct showed 10 times more activity than the negative control, its activity represented only 0.2% of the wild-type HCV NS32-181/421-34 construct activity (Fig. 2C). These results indicate that the NS4A peptide is essential in our system for the NS3 cleavage of the NS5A-NS5B junction. In short, we have developed a simple, rapid, and highly specific assay that can be used to monitor the activity of the HCV NS3 serine protease.

Because the NS3 protease has been an attractive target in developing effective therapeutic agents against HCV, several assay systems have been developed to identify specific inhibitors. These assay systems have included a system in mammalian cells in which NS3 protease cleavage is required for transcription of a reporter gene (11), the generation of stable chimeric viruses (e.g., Sindbis virus-HCV) whose propagation depends on the activity of the NS3 protease (7), and the reconstitution of the NS3 activity in yeast (13, 16). Another reason for the development of these systems has been the lack of a robust ex vivo cellular system for the propagation of live HCV. Recently, subgenomic replicons have been developed as the first cellular system to study the dynamics of HCV replication (15). However, these replicons do not produce infectious virus, and hence, they cannot be used to study viral infection and cell-to-cell viral transmission. In the absence of a cell culture system to propagate HCV, the HCV proteases have been studied mainly in in vitro cell-free enzymatic experiments (reviewed in references 14, 20, and 31).

The simplicity of our system can be seen as a complement to the classical biochemical approach for monitoring NS3 proteolytic activity. An interesting result of the present study has been the finding that our system allows the characterization of enzymes with different proteolytic activities. The activity of the protease carrying the S138A substitution is reduced fivefold compared to wild-type protease (Fig. 2B). Since this mutant shows a proteolytic activity 1,000 times higher than the inactive mutants carrying substitutions within the catalytic triad, the genetic system used here is sensitive enough to allow the quantification of a wide range of different proteolytic activities. The reproducibility showed by this assay (Fig. 2) and previous work that we have performed with {lambda}-based HIV-1 expression vectors (2, 17) strongly suggest that the different proteolytic activities observed here with the S138A variant (Fig. 2A) were not due to different expression levels of HCV protease variants but resulted from the lower catalytic efficiency of the S138A mutant. Coupling mutant sequence libraries with this positive genetic selection system will allow the study of a huge number of functional mutants (2, 18, 19, 27). Finally, as we have previously demonstrated for the HIV-1 protease (2, 17), this system may be used to search for new protease inhibitors and to predict the protease inhibitor resistance profile in the course of viral infection and antiviral therapy.

Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. The system described here in which proteases with different mechanisms of action can be monitored should be useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities.

Nucleotide sequence accession number. The NS3 nucleotide sequence from the HCV-infected individual (patient 1) reported in this paper has been deposited in the GenBank database under accession number AF510039.


arrow
ACKNOWLEDGMENTS
 
We thank H. J. Sices and T. M. Kristie for cI.HIV-cro constructs and for helping us set up the lambda-based genetic system.

This work was supported in part by Fundació irsiCaixa and by grants from the Spanish Fondo de Investigación Sanitaria (FIS 01/0067-02) and the Fundación para la Investigación y la Prevención del SIDA en España (FIPSE 36293/02 and 36207/01).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, 08916 Barcelona, Spain. Phone: 34-934656374. Fax: 34-934653968. E-mail: mamartz{at}ns.hugtip.scs.es. Back


arrow
REFERENCES
 
    1
  1. Alter, H. J. 1995. To C or not to C: these are the questions. Blood 85:1681-1695.[Free Full Text]
    2. 2
  2. Cabana, M., G. Fernandez, M. Parera, B. Clotet, and M. A. Martinez. 2002. Catalytic efficiency and phenotype of HIV-1 proteases encoding single critical resistance substitutions. Virology 300:71-78.[CrossRef][Medline]
    3. 3
  3. Carpenter, C. C., D. A. Cooper, M. A. Fischl, J. M. Gatell, B. G. Gazzard, S. M. Hammer, M. S. Hirsch, D. M. Jacobsen, D. A. Katzenstein, J. S. Montaner, D. D. Richman, M. S. Saag, M. Schechter, R. T. Schooley, M. A. Thompson, S. Vella, P. G. Yeni, and P. A. Volberding. 2000. Antiretroviral therapy in adults: updated recommendations of the International AIDS Society-USA Panel. JAMA 283:381-390.[Abstract/Free Full Text]
    4. 4
  4. Choo, Q. L., K. H. Richman, J. H. Han, K. Berger, C. Lee, C. Dong, C. Gallegos, D. Coit, R. Medina-Selby, P. J. Barr, et al. 1991. Genetic organization and diversity of the hepatitis C virus. Proc. Natl. Acad. Sci. USA 88:2451-2455.[Abstract/Free Full Text]
    5. 5
  5. Dimasi, N., A. Pasquo, F. Martin, S. Di Marco, C. Steinkuhler, R. Cortese, and M. Sollazzo. 1998. Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein. Protein Eng. 11:1257-1265.[Abstract/Free Full Text]
    6. 6
  6. Dunn, B. M. (ed.) 1999. Proteases of infectious agents. Academic Press, San Diego, Calif.
    7. 7
  7. Filocamo, G., L. Pacini, and G. Migliaccio. 1997. Chimeric Sindbis viruses dependent on the NS3 protease of hepatitis C virus. J. Virol. 71:1417-1427.[Abstract]
    8. 8
  8. Gulick, R. M., J. W. Mellors, D. Havlir, J. J. Eron, C. Gonzalez, D. McMahon, D. D. Richman, F. T. Valentine, L. Jonas, A. Meibohm, E. A. Emini, and J. A. Chodakewitz. 1997. Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. N. Engl. J. Med. 337:734-739.[Abstract/Free Full Text]
    9. 9
  9. Hammer, S. M., K. E. Squires, M. D. Hughes, J. M. Grimes, L. M. Demeter, J. S. Currier, J. J. Eron, Jr., J. E. Feinberg, H. H. Balfour, Jr., L. R. Deyton, J. A. Chodakewitz, M. A. Fischl, et al. 1997. A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. N. Engl. J. Med. 337:725-733.[Abstract/Free Full Text]
    10. 10
  10. Hijikata, M., H. Mizushima, T. Akagi, S. Mori, N. Kakiuchi, N. Kato, T. Tanaka, K. Kimura, and K. Shimotohno. 1993. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J. Virol. 67:4665-4675.[Abstract/Free Full Text]
    11. 11
  11. Hirowatari, Y., M. Hijikata, and K. Shimotohno. 1995. A novel method for analysis of viral proteinase activity encoded by hepatitis C virus in cultured cells. Anal. Biochem. 225:113-120.[CrossRef][Medline]
    12. 12
  12. Kim, J. L., K. A. Morgenstern, C. Lin, T. Fox, M. D. Dwyer, J. A. Landro, S. P. Chambers, W. Markland, C. A. Lepre, E. T. O'Malley, S. L. Harbeson, C. M. Rice, M. A. Murcko, P. R. Caron, and J. A. Thomson. 1996. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87:343-355.[CrossRef][Medline]
    13. 13
  13. Kim, S. Y., K. W. Park, Y. J. Lee, S. H. Back, J. H. Goo, O. K. Park, S. K. Jang, and W. J. Park. 2000. In vivo determination of substrate specificity of hepatitis C virus NS3 protease: genetic assay for site-specific proteolysis. Anal. Biochem. 284:42-48.[CrossRef][Medline]
    14. 14
  14. Kwong, A. D., J. L. Kim, G. Rao, D. Lipovsek, and S. A. Raybuck. 1998. Hepatitis C virus NS3/4A protease. Antivir. Res. 40:1-18.[CrossRef][Medline]
    15. 15
  15. Lohmann, V., F. Korner, J. Koch, U. Herian, L. Theilmann, and R. Bartenschlager. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110-113.[Abstract/Free Full Text]
    16. 16
  16. Mak, P., O. Palant, P. Labonte, and S. Plotch. 2001. Reconstitution of hepatitis C virus protease activities in yeast. FEBS Lett. 503:13-18.[CrossRef][Medline]
    17. 17
  17. Martinez, M. A., M. Cabana, M. Parera, A. Gutierrez, J. A. Este, and B. Clotet. 2000. A bacteriophage lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease. Antimicrob. Agents Chemother. 44:1132-1139.[Abstract/Free Full Text]
    18. 18
  18. Martinez, M. A., V. Pezo, P. Marliere, and S. Wain-Hobson. 1996. Exploring the functional robustness of an enzyme by in vitro evolution. EMBO J. 15:1203-1210.[Medline]
    19. 19
  19. Martinez, M. A., J. P. Vartanian, and S. Wain-Hobson. 1994. Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations. Proc. Natl. Acad. Sci. USA 91:11787-11791.[Abstract/Free Full Text]
    20. 20
  20. Pessi, A. 2001. A personal account of the role of peptide research in drug discovery: the case of hepatitis C. J. Peptide Sci. 7:2-14.
    21. 21
  21. Ptashne, M. 1986. A genetic switch. Cell Press, Cambridge, Mass.
    22. 22
  22. Reed, K. E., A. Grakoui, and C. M. Rice. 1995. Hepatitis C virus-encoded NS2-3 protease: cleavage site mutagenesis and requirements for bimolecular cleavage. J. Virol. 69:4127-4136.[Abstract]
    23. 23
  23. Rosenberg, S. 2001. Recent advances in the molecular biology of hepatitis C virus. J. Mol. Biol. 313:451-464.[CrossRef][Medline]
    24. 24
  24. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    25. 25
  25. Shimizu, Y., K. Yamaji, Y. Masuho, T. Yokota, H. Inoue, K. Sudo, S. Satoh, and K. Shimotohno. 1996. Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease. J. Virol. 70:127-132.[Abstract]
    26. 26
  26. Sices, H. J., and T. M. Kristie. 1998. A genetic screen for the isolation and characterization of site-specific proteases. Proc. Natl. Acad. Sci. USA 95:2828-2833.[Abstract/Free Full Text]
    27. 27
  27. Sices, H. J., M. D. Leusink, A. Pacheco, and T. M. Kristie. 2001. Rapid genetic selection of inhibitor-resistant protease mutants: clinically relevant and novel mutants of the HIV protease. AIDS Res. Hum. Retrovir. 17:1249-1255.[CrossRef][Medline]
    28. 28
  28. Taremi, S. S., B. Beyer, M. Maher, N. Yao, W. Prosise, P. C. Weber, and B. A. Malcolm. 1998. Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease. Protein Sci. 7:2143-2149.[Medline]
    29. 29
  29. Tomei, L., C. Failla, E. Santolini, R. De Francesco, and N. La Monica. 1993. NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J. Virol. 67:4017-4026.[Abstract/Free Full Text]
    30. 30
  30. Tomei, L., C. Failla, R. L. Vitale, E. Bianchi, and R. De Francesco. 1996. A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease. J. Gen. Virol. 77:1065-1070.[Abstract/Free Full Text]
    31. 31
  31. Urbani, A., G. Biasiol, M. Brunetti, C. Volpari, S. Di Marco, M. Sollazzo, S. Orru, F. D. Piaz, A. Casbarra, P. Pucci, C. Nardi, P. Gallinari, R. De Francesco, and C. Steinkuhler. 1999. Multiple determinants influence complex formation of the hepatitis C virus NS3 protease domain with its NS4A cofactor peptide. Biochemistry 38:5206-5215.[CrossRef][Medline]

Antimicrobial Agents and Chemotherapy, May 2003, p. 1760-1765, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1760-1765.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sabariegos, R., Picazo, F., Domingo, B., Franco, S., Martinez, M.-A., Llopis, J. (2009). Fluorescence Resonance Energy Transfer-Based Assay for Characterization of Hepatitis C Virus NS3-4A Protease Activity in Live Cells. Antimicrob. Agents Chemother. 53: 728-734 [Abstract] [Full Text]  
  • Fernandez, G., Clotet, B., Martinez, M. A. (2007). Fitness Landscape of Human Immunodeficiency Virus Type 1 Protease Quasispecies. J. Virol. 81: 2485-2496 [Abstract] [Full Text]  
  • Parera, M., Fernandez, G., Clotet, B., Martinez, M. A. (2007). HIV-1 Protease Catalytic Efficiency Effects Caused by Random Single Amino Acid Substitutions. Mol Biol Evol 24: 382-387 [Abstract] [Full Text]  
  • Sabariegos, R., Gimenez-Barcons, M., Tapia, N., Clotet, B., Martinez, M. A. (2006). Sequence Homology Required by Human Immunodeficiency Virus Type 1 To Escape from Short Interfering RNAs. J. Virol. 80: 571-577 [Abstract] [Full Text]  
  • O'Boyle, D. R. II, Nower, P. T., Lemm, J. A., Valera, L., Sun, J.-H., Rigat, K., Colonno, R., Gao, M. (2005). Development of a Cell-Based High-Throughput Specificity Screen Using a Hepatitis C Virus-Bovine Viral Diarrhea Virus Dual Replicon Assay. Antimicrob. Agents Chemother. 49: 1346-1353 [Abstract] [Full Text]  
  • Parera, M., Clotet, B., Martinez, M. A. (2004). Genetic Screen for Monitoring Severe Acute Respiratory Syndrome Coronavirus 3C-Like Protease. J. Virol. 78: 14057-14061 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martinez, M. A.
Right arrow Articles by Clotet, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martinez, M. A.
Right arrow Articles by Clotet, B.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»