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The morphogenesis of HCV belongs to steps of the viral cycle that have not yet been targeted by antiviral strategies. With the lack of an efficient and reliable culture system able to produce and secrete HCV virions, we use two complementary approaches to study the effect of c~-gluensidase inhibitors on HCV morphogenesis, secretion and entry. These two approaches have allowed us to demonstrate tile mechanism of tile antiviral effect of O,-glucosidase inhibitors on HCV glyenprotein folding and assembly, morphogenesis, viral secretion, and viral infectivity.

HCV morphogenesis is a key step of the viral cycle and represents an original target for the development of new antivirals that could complement more conventional antiviral strategies based on inhibition of viral enzymes. As proof of concept, the inhibition of morphogenesis was demonstrated using the BVDV, a pestivirus related to HCV, as a surrogate model. Using this model, it was shown that alpha-glucosidase inhibitors, i.e. deoxynojirimycin (DNJ) derivatives, could inhibit viral morphogenesis in cellulo via the perturbation of the N-glycosylation pathway. Due to the importance of N-glycosylation for HCV glycoprotein folding and assembly, it was hypothesized that such inhibitors could also affect HCV morphogenesis.

Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P=0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 [corrected] and PD-1 levels in liver correlated with clearance or chronicity. CONCLUSION: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.

Novel anti-HCV molecules are still needed to better combat HCV infection. Molecules targeting specifically viral activities are the most attractive in terms of drug development and are therefore the most studied. The antiviral properties of glucosidase inhibitors were established using the most relevant models currently available.

Background. Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post-translationally inducible Cre-Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells. Methods. To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses β-galactosidase only upon Cre-mediated recombination. This was used together with the ROSA26-R ES cell Cre-reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti-Cre polyclonal antibody, and by monitoring the expression of β-galactosidase. Results. Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone-inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre-PR (fusion of Cre and progesterone receptor) or β-galactosidase. Cre-mediated recombination in more than 60% of ROSA26-R ES cells was achieved when infected by a VSV-G-pseudotyped MESV retrovirus at MOI of 50. Conclusions. The MESV-based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells. Copyright © 2004 John Wiley & Sons, Ltd.