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February 14, 2007 Crystal structure of the catalytic domain of the hepatitis C virus NS2-3 proteaseI.C. Lorenz, J. Marcotrigiano, T.G. Dentzer, and C.M. Rice Hepatitis C virus (HCV) is an important human pathogen leading to cirrhosis and liver cancer. The viral RNA genome encodes a polyprotein that is processed into 10 distinct proteins by action of host cellular and viral proteases. Recently, we have determined the crystal structure of the catalytic domain of the HCV NS2-3 protease, which mediates one cleavage reaction in the viral polyprotein essential for viral replication. NS2 contains a dimeric cysteine protease with a novel fold. Interestingly, the dimer contains a pair of composite active sites, with both monomers contributing residues to each active site. This unusual feature may enable the virus to regulate proteolytic processing and thus the onset of RNA replication. Moreover, the structure may serve as a basis for the development of new antiviral therapies.
An estimated 120 million people are infected worldwide with HCV. Chronic infection is common and causes severe liver damage and hepatocellular carcinoma. A vaccine has not yet been developed, and only 55-60% of the patients respond to the currently available therapies. Therefore, approaches to identify novel drug targets are sorely needed. The single-stranded, positive-sense RNA genome contains one open reading frame, which codes for ten viral proteins. Three proteins are structural components of the virus particle, whereas the remainder is involved in viral replication and host interactions. Synthesis of the HCV proteins occurs by translation of the viral genome into a polyprotein precursor, which is processed into individual proteins by two host cellular and two viral proteases (Fig. 1). One of them, the HCV NS2-3 protease, mediates cleavage of the viral polyprotein at the junction between nonstructural proteins (NS) 2 and 3. This step is required for replication of the viral RNA both in vivo and in vitro. We have recently determined the crystal structure of the catalytic domain of the NS2-3 protease, which spans the carboxy-terminal half of NS2 (NS2pro, residues 94-217). The functional unit of NS2pro appears to be a dimer, with each monomer consisting of an amino-terminal alpha-helical subdomain and a carboxy-terminal subdomain forming an antiparallel beta sheet (Fig. 2a and b). The two subdomains are connected by an extended linker, resulting in an exchange of the carboxy-terminal subdomains. Early mutagenesis experiments led to the identification of a putative catalytic triad consisting of amino acid residues histidine 143, glutamate 163, and cysteine 184. In the crystal structure, these residues form an active site with a geometry similar to those of other viral and cellular cysteine proteases. Surprisingly, each active site consists of residues from both monomers: histidine 143 and glutamate 163 are contributed by one monomer, whereas the nucleophilic cysteine 184 originates from the other chain (Fig. 3a and b). The dimeric form of the protease suggests a mechanism for regulated polyprotein processing and viral RNA replication.
The NS2pro structure corresponds to the post-cleavage form of the protease. The carboxy-terminal leucine 217 remains coordinated in the active site after cleavage, forming hydrogen bonds with the adjacent atoms of the catalytic cysteine and histidine residues (Fig. 3b). Binding of leucine 217 to the active site has two implications: (I) the protease becomes inaccessible for other substrates, and (II) NS2 is ‘locked’ as a dimer, which may have other functions in the viral life cycle after proteolytic processing. To demonstrate that NS2 can form dimers with composite active sites in vivo, we used two mutant NS2-3 polypeptides, either containing a histidine 143 to alanine or a cysteine 184 to alanine point mutation in the NS2 active site. Each of these NS2-3 variants expressed individually cannot undergo cleavage at the NS2/3 junction. However, if a composite active site can form, then mixing of the two NS2-3 mutants should lead to the reconstitution of a functional active site, resulting in partial proteolytic processing. This was indeed the case when these mutants were analyzed in a series of experiments in tissue culture. Thus, we were able to demonstrate the in vivo relevance of the mechanism we proposed based on the crystal structure.
Proteolysis through formation of a composite active site has never been observed in a cysteine or serine protease before. However, these features are reminiscent of human immunodeficiency virus (HIV), which encodes a dimeric aspartic protease with a single active site at the dimer interface. Thus, HCV and HIV may have evolved similar strategies to control the timing of events during their viral life cycles. BEAMLINES FUNDING PUBLICATION FOR MORE INFORMATION |
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