We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-Å 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.
Neoantigens (nAgs) are promising tumor antigens for cancer vaccination with the potential of inducing robust and selective T cell responses. Genetic vaccines based on Adenoviruses derived from non-human Great Apes (GAd) elicit strong and effective T cell-mediated immunity in humans. Here, we investigate for the first time the potency and efficacy of a novel GAd encoding multiple neoantigens. Prophylactic or early therapeutic vaccination with GAd efficiently control tumor growth in mice. In contrast, combination of the vaccine with checkpoint inhibitors is required to eradicate large tumors. Gene expression profile of tumors in regression shows abundance of activated tumor infiltrating T cells with a more diversified TCR repertoire in animals treated with GAd and anti-PD1 compared to anti-PD1. Data suggest that effectiveness of vaccination in the presence of high tumor burden correlates with the breadth of nAgs-specific T cells and requires concomitant reversal of tumor suppression by checkpoint blockade.
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.
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.
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