Hepatocellular carcinomas (HCC) are the most common form of malignant tumor in the world. The principal risk factors are chronic hepatitis B and C virus infections, mainly at the cirrhotic stage of these infections (1, 2). The high prevalence of hepatitis C virus (HCV) (about 3% of the world population) makes it a major cause of HCC. In the developed world, this prevalence is of the order of 1% (780,000 people affected in France), so chronic HCV infection is the primary cause of more than half the cases of HCC (3).
At present there are no vaccines against HCV. Drugs for this chronic infection are very costly and unsatisfactory due to their poor tolerance and limited effectiveness, especially in patients infected with genotypes 1 or 4: about 50% of sustained viral response rates are obtained with the best treatment regimen available (a combination of pegylated interferon and ribavirin) (4). Genotype 1 is responsible for 70 % of known cases of hepatitis C in the United States, Japan and Western Europe. There is therefore a pressing need for the development of new, more effective and less toxic drugs, especially for genotype 1.
Approved anti-HCV therapy is non-specific, whereas new strategies are targeting the hepatitis C virus directly. This is the concept of specifically targeted antiviral therapy against hepatitis C (5). At the cellular level, HCV hijacks host functions by introducing its genome, a DNA molecule, into liver cells. These then produce viral proteins, notably enzymes that catalyze the multiplication of the viral genome. There are many potential targets in this process, but the main molecules developed so far target RNA and viral enzymes or host-virus interactions (Figure 1). Considerable effort has therefore been aimed at developing NS3 protease and NS5b polymerase inhibitors.
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Figure 1: Current state of research into inhibitors of the HCV virus cycle in pre-clinical and clinical development (from ref. 5). * indicates molecules abandoned due to their toxicity.
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Numerous molecules, as outlined in Figure 1, function in vitro at the preclinical stage, but their development to the clinical stage on patients currently stops at phase I or II, either because they are ineffective or due to their toxicity. Knowledge of the virus itself, its mechanisms of multiplication and in particular the determinants of replication are needed more than ever when researching more effective drugs with fewer side effects.
Progress on acquiring greater knowledge of this virus has been slowed by the lack of available small animal models and especially by the absence of productive cell culture systems, the basic tools of virology (6). A major result in fundamental HCV research in the last few years has therefore been the development of cell systems permitting, first the study of viral replication (replicons), then, very recently, the complete virus cycle. A new era in HCV study started with the establishment of the first subgenomic replicon (Con1: genotype 1 consensus) (7).These cell systems, plus the genomic replicons, have led to major advances in the understanding of cellular and molecular mechanisms involved in HCV replication (8). With these, it has now become possible to study the active replication complex of HCV and show that only a small minority of non-structural proteins are actually associated with this complex (9).
Figure 2: Crystal structure of a hepatitis C virus polymerase (sub-type 1a) in a complex with the nucleotide initiator of replication (S. Bressanelli & F. A. Rey, unpublished). The three catalytic core subdomains (“Fingers”, “Palm” and “Thumb”) are colored red, yellow and blue, respectively. The "linker” extending the third subdomain and covering the catalytic crevice is in light brown. a), primary structure describing the linking of subdomains in the sequence, b) and c), two orthogonal views of the complex, with the enzyme represented as a ribbon and the linked nucleotides as sticks. d), Close-up view of c). The elements contacting one of the two nucleotides in the catalytic region (marked 2’) are represented as sticks.
However, even genomic replicons do not lead to the production of viral particles, even after the correct expression of the viral genes. It was not until 2005 that infectious viral particles could be produced in cell culture, due to the unique properties of the JFH1 isolate (Japanese fulminant hepatitis 1) of genotype 2a (10). This strain was isolated from a patient who died from fulminant hepatitis (unlike other forms of HCV which usually produce a persistent infection that only slowly damages the liver). A study of this strain showed that the particular properties of JFH1 depend on non-structural proteins of the replication complex, mainly NS5b (11).
We have been working for several years on the structural characterization of NS5b, focusing on the main hypothesis that the inhibition and activation of polymerase function are closely linked to the C-terminal part of the molecule (40 residue linker + 21 residue transmembrane anchor, see Figure 2). The positioning of the linker in the catalytic cleft would be critical for initiating RNA synthesis; its removal from the cleft would lead to NS5b opening up, thereby permitting the elongation of the neo-synthesized RNA strand.
We study the conformational changes in NS5b and the regulatory role played by its C-terminal part, mostly by two biophysical methods. The first is X-ray crystallography, which gives a static image of the molecule on the atomic scale and allows its structure and its interactions with natural substrates or inhibitors to be determined precisely (Figure 2). The second method is small-angle X-ray scattering (SAXS), which permits low resolution dynamic studies in solution, notably the visualization of significant conformational changes (Figure 3). These two methods require high performance X–rays and, in this respect, the SOLEIL synchrotron is one of the best available sources in the world. We have thus been able to determine the crystal structure of JFH1-NS5b with data obtained on the high brilliance beamline PROXIMA1 (Figure 4). The structure is of a sufficiently high quality that we are able to draw several important conclusions: first, the conformation of this NS5b is significantly more closed than other NS5bs whose structures are known. As the closed form is associated with the initiation of replication of the HCV genome, the replicative advantage of this JFH1 strain would seem to be linked to an easier start of this replication; and secondly, this particular conformation seems to be associated with a series of mutations disseminated throughout the NS5b structure. We would therefore like to carry out mutagenesis experiments directed at verifying the effects of these mutations on the NS5b conformation. This study will be performed, amongst others, using strain Con1, for which much data are available in the replicon system, notably the appearance of resistant mutants under antiviral treatment, and also strain J6 of subtype 2a, which is very close to JFH1 and yet incapable of replicating effectively in cell culture. We will also study conformational variations of these strains by SAXS, a method for which SOLEIL also has a cutting–edge beamline, the SWING beamline.
Figure 3: Analysis in solution by small angle X-ray scattering of a purified solution of hepatitis C virus polymerase (sub-type 1b). On the left, scattering intensity as a function of angle. In blue, experimental points and margins of error; in green and red, spectrum calculated based on the crystal structure of the same polymerase, without and with explicit modeling of the disordered parts, respectively. On the right, the ab initio reconstruction of the envelope in solution (S. Bressanelli and J. Perez, unpublished).
Figure 4: Polymerase mutations of the JFH1 strain of hepatitis C virus that contribute to its closed conformation (P. Simister and S. Bressanelli, article in preparation). The enzyme is in the same orientation as in Figure 2b, but here it is colored according to its rigid elements (moving parts of the molecular machine), with one color per element.
If these experiments confirm our conclusions, we will be able to explain the molecular mechanisms involved in the regulation of HCV replication. Here is an example, common in biology, of the fundamental study of an abnormal case (the excessive replicative ability of the JFH1 strain), opening the door to greater understanding of the normal mechanism (persistent controlled replication of other HCV strains).
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