FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Flaviviridae :T he Viruses and Their Replication

Infection with the hepatitis C virus (HCV) is a major cause of chronic liver disease. HCV is an enveloped plus-strand RNA virus closely related to flavi- and pestiviruses. The first cloning of the HCV genome, about 10 years ago, initiated research efforts leading to the elucidation of the genomic organization and the definition of the functions of most viral proteins. Despite this progress the lack of convenient animal models and appropriate in vitro propagation systems have hampered a full understanding of the way the virus multiplies. This review summarizes our current knowledge about HCV replication and describes attempts pursued in the last few years to establish efficient and reliable cell culture systems.

Replication of the hepatitis C virus

Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide.

https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1002/hep.20032

Structural biology of hepatitis C virus

/pdf/replication-of-hepatitis-c-virus-153kot0zbp.pdf

Replication of hepatitis C virus.

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 28-A resolution This enzyme is a key target for developing specific antiviral therapy The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H, Chopra, R, Verdine, G L & Harrison, S C (1998) Science 282, 1669-1675] This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds

https://www.pnas.org/content/pnas/96/23/13034.full.pdf

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus.

The processing at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions in the non-structural region of the hepatitis C virus (HCV) polyprotein is performed by a viral serine protease activity contained within the N-terminal 180 amino acids of the NS3 protein. Full protease activity is only achieved upon the interaction of a region at the N terminus of NS3 with the NS4A protein, this region is also involved in the modulation of the protease activity. Using the rabbit reticulocyte expression system, we have defined the minimal domain of NS4A that is necessary to increase the cleavage efficiency of NS3. A synthetic peptide containing the same region, NS4A amino acids 21 to 32, stimulates the proteolytic activity of NS3 at all the trans-cleavage sites.

A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease.

The RNA-dependent RNA polymerase activity of hepatitis C virus is carried out by the NS5B protein. The full-length protein was previously purified as a non-fusion protein from insect cells infected with a recombinant baculovirus. The characterization is now described of a C-terminal hydrophobic domain deletion mutant of NS5B purified from E. coli. In addition to increased solubility, deletion of this sequence also positively affected the polymerase enzymatic activity. The efficiency of nucleotide polymerization of both the full-length and the C-terminal truncated enzymes were compared on homopolymeric template-primer couples as well as on RNA templates with heteropolymeric sequences. The largest difference in the polymerase activity was observed on the latter. On all the templates, the increased activity could be ascribed, at least in part, to enhanced template turnover of the deletion mutant with respect to the full-length enzyme. The elongation rates of the two enzyme forms were compared under single processive cycle conditions. Under these conditions, both the full-length and the deletion mutant were able to incorporate about 700 nt/min.

Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence

Corrigendum: A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease (Journal of General Virology (1996) 77 (1065-1070))

The present invention relates to serine protease NS3 of hepatitis C virus, and in particular to the observation that the NS3 serine protease domain, in its native conformation, binds a Zn2+ ion and that bivalent metallic ions are necessary to the structural integrity of the protein and to the activity of the enzyme. The present invention further relates to recombinant polypeptides which comprise sequences of the NS3 protease and are characterised by a tail of at least three lysines at their C-terminal ends, to increase its solubility. A further subject of the present invention is a new process which allows the expression of said polypeptides, as metalloproteins, with the proteolytic activity of the HCV NS3 protease, in a soluble form and in a quantity sufficient to allow research to identify inhibitors and to determine the three-dimensional structure of the NS3 protease. Figure 4 shows the effects of the zinc ion on the production of the HCV NS3 protease as a soluble protein in E. Coli in a minimum culture medium.

Rosa Letizia Vitale

Papers

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus.

A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease.

Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence

Corrigendum: A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease (Journal of General Virology (1996) 77 (1065-1070))

Soluble polypeptides with activity of the ns3 serine protease of hepatitis c virus, and process for their preparation and isolation