Dimerization-driven interaction of hepatitis C virus core protein with NS3 helicase.

Hepatitis C virus (HCV) infects over 130 million people causing a worldwide epidemic of liver cirrhosis and hepatocellular-carcinoma. Because current HCV treatments are only partially effective, molecular mechanisms involved in HCV propagation are actively being pursued as possible drug targets. Here, we report on a new macromolecular interaction between the HCV capsid core protein and the helicase portion of HCV non-structural protein 3 (NS3h), confirmed by four different biochemical methods. The protease portion of NS3 is not required. Interaction between the two proteins could be disrupted by two types of specific inhibitors of core dimerization, the small molecule SL201 and core106, a C-terminally truncated core protein. Cross-linking experiments suggest that the physical interaction with NS3h is probably driven by core oligomerization. Moreover, SL201 blocks the production of infectious virus, but not the production of a subgenomic HCV replicon by hepatoma cells. Time-of-addition experiments confirm that SL201 has no effect on entry of the virus. These data underline the essential role of core as a key organizer of HCV particle assembly, confirm the importance of oligomerization, reveal the interaction with viral helicase and support a new molecular understanding of the formation of the viral particle at the level of the lipid droplets, before its migration to the site of release and budding.

Peptide inhibitors of hepatitis C virus core oligomerization and virus production.

Hepatitis C virus (HCV) nucleocapsid assembly requires dimerization of the core protein, an essential step in the formation of the virus particle. We developed a novel quantitative assay for monitoring this protein-protein interaction, with the goal of identifying inhibitors of core dimerization that might block HCV production in infected Huh-7.5 hepatoma cells. Two core-derived, 18-residue peptides were found that inhibited the dimerization of a fragment of core comprising residues 1-106 (core106) by 68 and 63%, respectively. A third, related 15-residue peptide displayed 50% inhibition, with an IC50 of 21.9 microM. This peptide was shown, by fluorescence polarization, to bind directly to core106 with a Kd of 1.9 microM and was displaced by the unlabelled peptide with an IC50 of 18.7 microM. When measured by surface plasmon resonance, the same peptide bound core169 with a Kd of 7.2 microM. When added to HCV-infected cells, each of the three peptides blocked release, but not replication, of infectious virus. When measured by real-time RT-PCR, the RNA levels were reduced by 7-fold. The 15-residue peptide had no effect on HIV propagation. Such inhibitors may constitute useful tools to investigate the role of core dimerization in the virus cycle.

Localization and quantification of carbon-centered radicals on any amino acid of a protein.

A general strategy to localize and quantify carbon-centered radicals within proteins is described. The methodology was first exemplified on amino acids and then on a peptide. This method is applicable to any protein system regardless of size, and the site of hydrogen abstraction by *OH on all residues within proteins is easily and accurately detected.

Statistical models for renal micropuncture studies.

Statistical issues relating to data analysis of re-collection micropuncture experiments are presented. In the presence of significant animal-treatment interaction, namely, differential response of each animal at different levels of the treatment, the conventional paired or unpaired t testing would not be entirely appropriate. Accordingly, two analysis of variance (ANOVA) models have been derived for the appropriate paired and unpaired designs of micropuncture experiments. Interactive computer programs have been written for both these analyses, and the results are illustrated with experimental data. An example is presented in which the results are statistically significant with paired t testing and by analysis of variance for unequal number of tubules but not when the animal-treatment interaction is included in the analysis of variance model. To investigate linkage in renal transport mechanisms, we propose the use of partial correlation analysis. Experimental results from our laboratory are used to illustrate these techniques.