[Role of N-linked glycans in HCV glycoprotein E1 in the folding of structural proteins and formation viral particles].

Envelope proteins of HCV play a major role in virus lifecycle. These proteins are main components of the virion. They are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation which is supposed to play a role in their stability, in the assembly of the functional HCV glycoprotein heterodimer, protein folding and viral entry. The role of N-linked glycosylation sites in HCV E1 protein in structural proteins assembly was analyzed by site-directed mutagenesis in a model system--insect cells producing three viral structural proteins with formation of virus-like particles. Removing of single N-linked glycosylation sites in HCV E1 protein does not affect the efficiency of its expression in insect Sf9 cells. E1 electrophoretic mobility is increasing in parallel with decreasing the number of glycosylation sites. The destroying of glycosylation sites N1 or N5 in E1 influences the assembly of noncovalent glycoprotein heterodimer E1E2--the prototype of natural complex incorporated in virion. The lack of glycans in N1 and N5 sites of E1 was shown to affect the efficiency of its expression in mammalian HEK293 T cells.

[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 expression systems for recombinant protein production in insect and mammalian cells].

The baculovirus vector systems has been extensively used for the expression of foreign gene products in insect and mammalian cells. New advances increase the possibilities and applications of the baculovirus expression system, which has the capability to express multiple genes simultaneously within a single infected insect cells and to use recombinant virus with mammalian cell-active expression cassettes to permit expression of recombinant proteins in mammalian cells in vitro and in vivo. Future investigations of the baculovirus expression system designed for specific target cells, can open wide variety of applications. This review summarizes the recent achievements in applications the baculovirus vector systems and optimization recombinant protein expression in both insect and mammalian cell lines.

[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.

[Characterization of hepatitis C virus structural proteins and HCV-like particles produced in recombinant baculovirus infected insect cells].

Three proteins, namely: "core" protein C and glycoproteins E1 and E2, are main structural proteins forming a hepatitis C vius (HCV) virion. The virus structure and assembly, a role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Using recombinant baculoviruses expressing HCV structural protein genes in insect cells the specific structural proteins at the level of 25-35% relative to a common cell protein content, heterodimers of the glcoproteins, and HCV-like particles have been obtained. It has been demonstrated that recombinant proteins C, E1, and E2 go through the posttranslation modification, the glycoproteins form the non-covalent heterodimer, and HCV-like particles are located in endoplasmatic reticulum membrains of infected cells. An ability of the expressed proteins for forming E1E2 dimers and HCV-like particles was used for studying the role of E1 protein glcosylation upon expression and processing of the glycoproteins.

[Expression of the human steroid 21-hydroxylase gene and its mutant variant C169R in insect cells and functional analysis of expression products].

Steroid 21-hydroxylase is a key enzyme of glucocorticoid and mineralocorticoid biosynthesis in the adrenal gland that belongs to the family of microsomal cytochrome P450. The steroid 21-hydroxylase deficiency is the most frequent cause of the congenital adrenal hyperplasia. The human steroid 21-hydroxylase (CYP21 A) and its mutant variant (C 169R) found previously in patient with the classical congenital adrenal hyperplasia were synthesized for the first time in the insect cell lines Sf9 and Hi5 infected by recombinant baculoviruses. Under optimal conditions the level of CYP21A2 production in insect cells achieves 28% of the total microsomal protein. C169R mutation does not effect the synthesis of CYP21 A2 in insect cells and does not prevent the incorporation of the enzyme into the membranes of endoplasmic reticulum. Functional analysis of the mutant enzyme in vitro suggested the virtually complete lack of catalytic activity towards two substrates - progesterone and 17-hydroxyprogesterone.

[Improved efficient gene transfer into insect and mammalian cells by baculovirus vectors].

The fragments of genomics DNA of the nuclear polyhedrosis virus (NPV) containing genes of late viral proteins p10, p35, p39, were cloned, the promoter regions of this genes were used to design baculovirus transfer vectors. A double-promoter and triple-promoter baculovirus transfer vectors were obtained. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus (CMV) promoter, the gene for green or red fluorescent protein, SV40pA and polylinker MCS were constructed for the delivery of foreign genes into mammalian cells.

[Expression of the mouse serotonin receptor (5HT1c) cDNA in cultured insect cells]. / Ekspressiia kDNK gena serotoninovogo retseptora myshi (5HT1c) v kul'ture kletok nasekomykh.

Baculovirus-mediated cloning and expression of the mouse serotonin receptor (5HT1c) cDNA in insect cells was proposed to obtain an alternative to an oocyte-based system, which is commonly employed in electrophysiological studies of ionic channels. A recombinant bacmid was constructed, and the 5HT1c cDNA transferred into the AcNPV genome to yield a recombinant baculovirus. Infected inset Sf9 cells produced recombinant 5HT1c.

[The role of the serotonin system in the stress response of various cells]. / Rol' serotoninovoi sistemy v reaktsii razlichnykh tipov kletok na stressornye signaly.

The recombinant mouse brain serotonin receptor (5HT1c) was used to study the response of plant cells and oocytes to a stress signal activated by the serotonin-serotonin receptor interaction and associated Ca2+ flow. Based on plant expression vectors, recombinant constructs were obtained to direct production of 5HT1c fused with the green fluorescent protein in plant cells. The mRNAs for hybrid proteins were synthesized in an in vitro transcription system. The expression and function of the hybrid protein and the function of the associated ion channels were electrophysiologically studied in Xenopus laevis oocytes injected with the hybrid mRNA. The hybrid protein was functional and changed the operation of the Ca2+ channel in oocytes. To study the expression of the hybrid constructs in plant cells, the in vitro transcription product was inoculated in tobacco leaves, which then fluoresced.

[A baculovirus expression system for insect cells]. / Bakulovirusnaia sistema ékspressii v kletkakh nasekomykh.

The review considers the biology of baculoviruses, construction of transfer vectors for the baculovirus expression system, selection of recombinant baculoviruses, approaches to expression of multimeric proteins, and the potentialities and prospects of the system.

[3'terminal nucleotide sequence of barley stripe mosaic virus RNA]. / Struktura 3'-kontsevogo uchastka RNK virusa shtrikhovatoi mozaiki iachmenia.

Barley stripe mosaic virus (BSMV) is a representative of the hordeiviruses which has a functionally fragmented RNA genom. 3'-terminal nucleotide sequence of the noncoding region of BSMV RNA 2 has been determined. This region contains the internal olygo(A) sequence (n = 21) and the tyrosine accepting tRNA-like structure at 3'-extreme end. tRNA-like sequence can fold into the two distinct secondary structure which strikingly similar to such a structures which has been proposed recently for cucumo- and bromoviruses.