[Affect of deoxynojirimycin derivatives on hepatitis C virus morphogenesis].

Viral hepatitis C is one of the wide-spread and dangerous human diseases. The choice of drugs for treatment of chronic hepatitis C virus (HCV) infection is limited and prophylactic vaccines do not exist. Thus, the development of new antiviral strategies and substances are of great importance. The targeting of viral morphogenesis might be used as an alternative approach to existing strategies of HCV blocking. The glycosylation of viral envelope proteins is an important step of viral particle morphogenesis that determines the correct assembly of HCV virions. The derivatives of glucose analog deoxynojirimycin (DNJ)--inhibitors of alpha-glucosidase can impair the assembly of structural proteins and HCV particle formation. In the present work the affect of alkylated derivatives of DNJ N-pentyl-DNJ and N-benzyl-DNJ to HCVmorphogenesis in a model system insect cells producing three viral structural proteins with formation of virus-like particles was studied. Intracellular N-glycosylation of HCV envelope glycoproteins was shown to be impaired by DNJ derivatives. At 1 mM concentrations of these substances the level of gpE1 and gpE2 glycoproteins increase and their electrophoretic mobility decrease which seems to be due to inhibition of a-glucosidase in endoplasmic reticulum and accumulation of hyperglycosylated N-glycans in HCV glycoproteins. The interaction of the latters with calnexin leads to formation of unproductive dimers and bloks productive assembly of virus-like particles.

[Baculovirus vectors for efficient gene delivery and expression in mammalian cells].

Baculovirus expression vectors are extensively used for the delivering foreign genes and expression of recombinant proteins in insect and mammalian cells. Modified baculoviruses containing mammalian promoter elements (BacMam viruses) for an efficient transient and stable transduction of diverse mammalian cells prove a high level of heterologous proteins' expression both in vitro and in vivo. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus promoter, green or red fluorescent protein gene, polyadenylate signal SV40pA, and polylinker MCS were constructed for the delivery of genes encoding hepatitis C virus structural proteins into mammalian cells. In Hek293T and Huh7 cells formation of glycoprotein complexes and HCV-like particles was observed. A high efficiency of the baculovirus-mediated gene transfer and expression of the virus envelope proteins in mammalian cells was demonstrated with using fluorescence, flow cytometry and immunoblot techniques.