Oleg S. Morenkov

Distribution of B-cell epitopes on the pseudorabies virus glycoprotein B.

In order to map antigenically important regions of glycoprotein B (gB) of pseudorabies virus (PrV), a panel of recombinant fragments of gB expressed in E. coli and truncated fragments of gB generated by cleavage of purified native gB with trypsin and cyanogen bromide was analysed by using 26 monoclonal antibodies directed against gB. Three continuous epitopes were localized in the vicinity of the N terminus of gB, between amino acids (aa) 59 and 126. One continuous epitope mapped between residues 214 and 279. The residues involved in the assembly of eight discontinuous epitopes were located between aa 540 and 734. The constituents of two discontinuous epitopes were harboured in a segment encompassing aa 540-646. The clustering of continuous epitopes at the extreme N terminus of PrV gB and the locations of residues involved in the assembly of discontinuous epitopes of PrV gB are in good agreement with data on epitope locations in gB homologues from other herpesviruses.

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK-21) cells.

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.

Immunological characterisation of glycoprotein E of Aujeszky's disease virus

A panel of 14 monoclonal antibodies (MAbs) against glycoprotein E (gE) of Aujeszkys disease (pseudorabies) virus (ADV), which constitutes a representative sample of naturally occurring gE-specific antibodies in sera from infected animals, was produced and characterised. Eleven topologically distinct antigenic domains represented by one or more MAbs were identified on gE by using these MAbs and three additional gE-specific MAbs. Three of the MAbs available recognised conformation-independent epitopes on gE, while the other 14 MAbs bound to conformation-dependent epitopes. By using the recombinant protein encompassing the N-terminal part of gE, which was expressed in Escherichia coli, all the conformation-independent epitopes of gE were mapped within the first 125 amino-terminal amino acids of gE. The epitopes of gE were demonstrated to be conserved among gE-positive laboratory, field and vaccine ADV strains. Conformation-dependent epitopes were shown to contribute largely to the overall antibody response to gE in naturally infected swine and immunised mice. Most of the infected animals responded weakly to the identified conformation-independent epitopes of gE, while the group of immunodominant epitopes of gE was represented exclusively by conformation-dependent antigenic determinants from different antigenic domains. The results clearly demonstrated that conformation-dependent epitopes play a crucial role in inducing the humoral immune response to gE of ADV during the natural infection of swine and immunisation of mice. The application of MAbs of our panel as research and diagnostic tools is discussed.

Release of the glucose‐regulated protein 94 by baby hamster kidney cells

Glucose‐regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK‐21) cells into a serum‐free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi–dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl‐β‐cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK‐21 cells. The results suggest that BHK‐21 cells release grp94 into the serum‐free medium via the nonclassical secretory pathway in which lipid rafts play an important role. Copyright

Purification of the 90 kDa heat shock protein (hsp90) and simultaneous purification of hsp70/hsc70, hsp90 and hsp96 from mammalian tissues and cells using thiophilic interaction chromatography

Heat shock proteins (HSPs) hsp70/hsc70, hsp90 and hsp96 were separated from mammalian cells and tissues on a gel obtained by the reaction of beta-mercaptoethanol with divinyl sulfone-activated Sepharose CL-6B (thiophilic gel or T-gel). Hsp90 revealed a much higher affinity towards the T-gel than the other HSPs. One-step thiophilic interaction chromatography of proteins resulted in a more than 80% purity and 85% yield of hsp90. Based on this observation, a simple and efficient method for the purification of hsp90 and a procedure for the simultaneous purification of several HSPs (hsp70/hsc70, hsp90 and hsp96) using thiophilic interaction chromatography was developed. All the HSPs were recovered with a high yield and purity (90-99%). The results indicated that the thiophilic gel is a highly efficient affinity matrix for the purification of hsp90 and can be used in the protocols of purification of different HSPs from cells and tissues of various animal species.

A 2,5-Dihydroxybenzoic Acid-Gelatin Conjugate: The Synthesis, Antiviral Activity and Mechanism of Antiviral Action Against Two Alphaherpesviruses.

Various natural and synthetic polyanionic polymers with different chemical structures are known to exhibit potent antiviral activity in vitro toward a variety of enveloped viruses and may be considered as promising therapeutic agents. A water-soluble conjugate of 2,5-dihydroxybezoic acid (2,5-DHBA) with gelatin was synthesized by laccase-catalyzed oxidation of 2,5-DHBA in the presence of gelatin, and its antiviral activity against pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1), two members of the Alphaherpesvirinae subfamily, was studied. The conjugate produced no direct cytotoxic effect on cells, and did not inhibit cell growth at concentrations up to 1000 µg/mL. It exhibited potent antiviral activity against PRV (IC50, 1.5–15 µg/mL for different virus strains) and BoHV-1 (IC50, 0.5–0.7 µg/mL). When present during virus adsorption, the conjugate strongly inhibited the attachment of PRV and BoHV-1 to cells. The 2,5-DHBA–gelatin conjugate had no direct virucidal effect on the viruses and did not influence their penetration into cells, cell-to-cell spread, production of infectious virus particles in cells, and expression of PRV glycoproteins E and B. The results indicated that the 2,5-DHBA–gelatin conjugate strongly inhibits the adsorption of alphaherpesviruses to cells and can be a promising synthetic polymer for the development of antiviral formulations against alphaherpesvirus infections.

Cell-binding properties of glycoprotein B of Aujeszky's disease virus

The glycoprotein B (gB) of Aujeszkys disease virus (ADV) has a role in virus entry and cell-to-cell spread. In this report we examined the cell-binding properties of native ADV gB purified from the virus envelope by affinity chromatography. The binding of gB to the surface of susceptible cells BHK-21 and MDBK was specific, dose-dependent, and nearly saturable, which is characteristic of conventional receptor-ligand interactions. The purified gB was shown to specifically bind to immobilised heparin. The addition of soluble exogenous heparin and heparinase treatment of cells inhibited the binding of gB to the cells. Cell-associated gB could also be dissociated from the cells by soluble heparin. The results indicated that ADV gB binds specifically to cellular heparan sulphate. The binding of gB to cells inhibited the attachment of virus to cells and thus the formation of viral plaques. The results suggest that ADV gB may have a function in the initial attachment of ADV to the surface of susceptible cells.

Development of a competitive double antibody lateral flow assay for the detection of antibodies specific to glycoprotein B of Aujeszky's disease virus in swine sera

Three lateral flow assays (LFAs) for the detection of antibodies against glycoprotein B (gB) of Aujeszkys disease virus (ADV) in swine sera: a competitive double antibody sandwich LFA without a preincubation step (CDAS-gB-LFA), a CDAS-gB-LFA with a preincubation step (pCDAS-gB-LFA), and a competitive direct gB-LFA have been developed and were compared with each other and with a gB-ELISA. The assays are based on monoclonal antibodies to immunodominant epitopes of ADV gB. The pCDAS-gB-LFA proved to be the most specific and sensitive assay to detect antibodies directed to ADV gB. The specificity and sensitivity of the pCDAS-gB-LFA with the use of an LFA reader for test line intensity measurements were 97.6 and 94.9%, respectively. The lower diagnostic sensitivity of the pCDAS-gB-LFA compared to a gB-ELISA reflects its reduced analytical sensitivity, which was shown in titration experiments with positive sera. The pCDAS-gB-LFA, using the reader-based and visual detection modes, showed good agreement in respect to specificity; however, the LFA reader detection provided a higher diagnostic and analytical sensitivity compared to visual detection. The developed pCDAS-gB-LFA is a rapid, sensitive, and specific method for the detection of antibodies to ADV gB and can be used for screening ADV-infected swine in unvaccinated herds.

A sensitive and specific lateral flow assay for rapid detection of antibodies against glycoprotein B of Aujeszky's disease virus

A direct double antibody lateral flow assay (DDA-gB-LFA) for the detection of antibodies against the glycoprotein B (gB) of Aujeszkys disease virus (ADV) in swine sera was developed. A native ADV gB was used for the preparation of a conjugate with colloidal gold particles and the immobilization on the strip membrane. The gB purified from ADV virions by immunoaffinity chromatography retained its native epitope structure after adsorption on the nitrocellulose membrane and the surface of colloidal gold particles. The diagnostic specificity and sensitivity of the DDA-gB-LFA were evaluated using 236 field swine sera. The diagnostic specificity and sensitivity of the DDA-gB-LFA compared to a commercially available gB-based ELISA were 98.0% and 98.6%, respectively, when determined with the use of the reader-detection mode, and 98.0% and 93.5%, respectively, when determined using visual detection. The DDA-gB-LFA provides a rapid, sensitive, and specific determination of ADV gB-directed antibodies in sera and can be used for the detection of ADV-exposed swine.

Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane.

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90β, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90β are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90β to the plasma membrane.