Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Via Pontina Km 30.600, 00040 Pomezia (Roma), Italy1
Institut für Medizinische Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Str. 6, D-06097 Halle (Saale), Germany2
Author for correspondence: Cinzia Traboni. Fax +39 06 91 09 32 25. e-mail traboni{at}irbm.it
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Abstract |
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Main text |
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A key molecule in the life-cycle of HCV is the NS3 protein, which shows different enzymatic activities organized in two domains. The N-terminal domain is responsible for the proteolytic cleavages that lead to the mature non-structural proteins, whereas the C-terminal domain shows helicase and NTPase activity (De Francesco, 1999 ). A similar molecule is encoded by the GBV-B genome that shows limited amino acid identity (about 40%) to the HCV protein (Muerhoff et al., 1995
). However, a high level of conservation of specific sequence motifs is present in both the protease (Scarselli et al., 1997
) and helicase (Muerhoff et al., 1995
; Zhong et al., 1999
) domains. In a previous study, we described the purification of an active recombinant GBV-B NS3 protease domain and demonstrated that this enzyme is able to cleave HCV substrates in the form of synthetic peptides or in vitro-translated proteins. We also showed that the addition of an HCV NS4A activator peptide, Pep4A2134, did not modify the activity of the GBV-B enzyme (Scarselli et al., 1997
).
In order to characterize further the substrate specificity and cofactor requirements of GBV-B NS3 protease under more physiological conditions, we have analysed the full-length NS3 activity by transient expression in a hepatoma cell line. Experiments were performed both by transfecting plasmids that spanned NS3 and its cognate substrates (in cis) and by co-transfecting plasmids that encoded NS3 and its substrates separately (in trans). Experiments were also performed in vitro to confirm the identification and specificity of the cofactor.
The constructs used in this study, outlined in Fig. 1(a), were produced by RTPCR amplification of specific portions of the GBV-B genome from total RNA of infected tamarin sera, as described previously (Sbardellati et al., 1999
). PCR products were cloned in the pCite-2b vector downstream of the T7 polymerase promoter. The construct NS33'UTR, which spanned the region encoding NS3, NS4A, NS4B, NS5A and NS5B and part of the 3' UTR, was used to transfect Hep3B hepatoma cells infected with recombinant vaccinia virus encoding T7 polymerase as described previously (Failla et al., 1994
). Transfected cells were metabolically labelled with [35S]methionine and proteins were extracted and immunoprecipitated as described previously (Failla et al., 1994
). Specific GBV-B proteins were immunoprecipitated by using rabbit antisera raised against recombinant purified GBV-B antigens NS3 (Scarselli et al., 1997
), NS5BdeltaC23 (L. Tomei and C. Traboni, unpublished), NS4BHis and NS5AHis (S. Falcinelli, unpublished) and analysed by SDSPAGE and autoradiography.
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The experiments described above indicate that mature protein products were obtained from a construct encoding both NS3 and its cognate substrates upon expression in mammalian cells. They do not, however, provide any indication of the involvement of a protease cofactor, which is known to be required by the NS3 protease of HCV (Failla et al., 1994 ). Moreover, the detection of a mature GBV-B protein product corresponding to NS4A was hampered by the lack of a suitable specific antibody. To test the hypothesis that GBV-B NS3 is activated by its cognate NS4A protein, we compared the activity of the full-length NS3 protein in the presence or absence of NS4A by co-transfecting constructs that encoded substrates with constructs that encoded either NS3 or NS34A. A tentative identification of the NS4A protein boundaries was achieved by comparing the complete polyprotein sequences of GBV-B and HCV (using programs of the Genetics Computer Group package, Madison, WI, USA). In spite of a very low similarity at the level of NS4A protein, the homology was high enough in the sequences flanking NS4A, that is the NS3 helicase region and the boundary between NS4A and NS4B. We constructed two plasmids, NS3/pCite and NS34A/pCite (see Fig. 1a
), on the basis of this analysis. Each of the two plasmids was introduced into Hep3B cells infected with recombinant vaccinia virus encoding T7 polymerase in combination with one of the plasmids described below that encoded GBV-B substrates. Specific labelled protein products were immunoprecipitated and analysed as described previously (Failla et al., 1994
). NS4B3'UTR/pCite was chosen to detect the cleavage between NS4B and NS5A and between NS5A and NS5B; NS4A4B/pCite was used to detect the cleavage between NS4A and NS4B.
The experiment demonstrated that mature NS3 protein was produced when either the NS3/pCite or NS34A/pCite construct was transfected (Fig. 1d, lanes 1 and 2). Unfortunately, the lack of specific reagents for the NS4A moiety did not allow us to discriminate between the NS3 and NS34A proteins, which have a very small size difference, and hence to ascertain whether cis cleavage occurred between NS3 and NS4A. However, specific NS5A and NS5B products were clearly detectable when NS4B3'UTR/pCite was used as a substrate and NS34A/pCite as an enzyme source (Fig. 1d
, lanes 9 and 12). They were instead not distinguishable from the background when the NS3/pCite construct was used (Fig. 1d
, lanes 8 and 11), as well as when no protease construct was co-transfected (data not shown).
The use of the construct NS4A4B/pCite as a substrate to visualize the NS4B product gave a more complex result. A specifically immunoprecipitated NS4B product, clearly missing in control experiments without the enzyme source (Fig. 1d, lane 4), was obtained even in the absence of the NS4A gene in the NS3 construct (Fig. 1d
, lane 5), although it was more abundant with NS34A/pCite as an enzyme source (Fig. 1d
, lane 6). This was expected, since an enhancing activity may be exerted by an NS4A protein either encoded in cis (as in the NS34A/pCite construct) or supplied in trans, i.e. the NS4A moiety of the NS4A4B precursor and the free NS4A obtained eventually by cleavage of the precursor. Nonetheless, this part of the experiment is an important internal control that shows that the NS3/pCite (without NS4A) construct produces an enzyme that, by itself or upon activation by NS4A, is able to display proteolytic activity. This implies that the lack of cleavage products when other substrates were used cannot be attributed to a lack of activity of the enzyme produced by the NS3/pCite plasmid.
All of the specific products immunoprecipitated in this co-transfection experiment were also detectable when an HCV NS34A/pCite construct was used in place of the GBV-B homologous plasmid (Fig. 1d, lanes 3, 7, 10, 13). This indicates that the HCV enzyme is capable of processing GBV-B substrates, thus confirming that the two proteases share substrate specificity, as we have shown already for the purified protease domains tested on in vitro-translated HCV substrates (Scarselli et al., 1997
).
In these transfection experiments, in which a full-length form of the enzyme was produced, NS3 was apparently active only in the presence of the cofactor, at least for the NS4B5A and NS5A5B cleavage sites, in contrast to what occurs with the protease domain when tested on HCV substrates (Scarselli et al., 1997 ) and on the predicted GBV-B NS4A4B cleavage site peptide (A. Sbardellati, unpublished). This is not surprising, since it is known that the HCV NS3 protease domain shows baseline NS4A-independent activity whereas the cofactor is essential for the full-length enzyme (Gallinari et al., 1998
, 1999
; Hamatake et al., 1996
; Steinkühler et al., 1996a
).
In order to identify the core region of NS4A that makes contact with NS3 and is essential for the enzyme enhancement, we constructed a structural homology model of GBV-B protease by using the program Insight II (Molecular Simulation Inc.) (Dayringer et al., 1986 ) and manual refinement (Amati & Tramontano, 1997
). The model is based on the similarity of the primary sequences of the HCV and GBV-B NS3 protease domains (about 30% identity) and on information about the three-dimensional structure of the HCV NS3 protease domain complexed with an HCV NS4A cofactor peptide (Kim et al., 1996
; Yan et al., 1998
). HCV NS4A participates in the formation of the eight-
-strand sheet in the N-terminal part of NS3. Some mutation-sensitive NS4A residues, Val-23, Ile-25, Ile-29 and Leu-31 (Bartenschlager et al., 1995
; Lin et al., 1995
; Shimizu et al., 1996
), protrude into hydrophobic holes of the NS3 structure, contributing to the formation of the stabilized core of the complex (Kim et al., 1996
; Yan et al., 1998
). By comparing the sequences of HCV and GBV-B NS4A proteins, it appears that the homology in the region of the activator peptide is not greater than in the rest of the molecule (see Fig. 2
). Even the four critical hydrophobic residues of the HCV cofactor are not conserved in the GBV-B NS4A protein. However, the alignment before and after that region suggests the correspondence shown in Fig. 2
. In this hypothesis, the hydrophobic character of the interaction with the protease would be conserved, although different local alignment obviously cannot be excluded. Correspondingly, in the predicted structure of the GBV-B NS3 protease, the hydrophobicity of the residues forming the pockets in which NS4A residues are positioned is maintained (Amati, 1999
). We have then modelled the GBV-B NS4A putative core peptide into the model of NS3 protease to check its structural compatibility (Fig. 2
). The peptide is well inserted into the NS3 structure; non-favourable contacts between amino acids and discontinuities in the structure are absent.
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During the revision process of this manuscript, a paper was published (Butkiewicz et al., 2000 ) that reported the characterization of the activity of purified full-length GBV-B NS3 protease and its cofactor requirement by using in vitro-translated GBV-B substrates. That study led to similar conclusions to our own, although some differences were apparent concerning GBV-B NS3 protease activity at the level of specific cleavage sites. Butkiewicz et al. (2000)
did not observe cleavage of the NS4B5A substrate (with full-length NS3 in vitro), whereas we did (in vivo). They obtained complete cleavage for NS4A4B only in the presence of the cofactor (in vitro with full-length NS3), whereas we noticed a baseline activity even in the absence of the cofactor in experiments in vitro (with NS3 protease domain). These differences might be explained by the different experimental systems used by Butkiewicz et al. (2000)
and by us. With the exception of these differences, the data shown by Butkiewicz et al. (2000)
are in overall agreement with our results and confirm that NS4A is required as a cofactor for GBV-B NS3 protease and that this cofactor is virus-specific.
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Acknowledgments |
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Footnotes |
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References |
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Received 9 February 2000;
accepted 31 May 2000.