Gintautas Zvirblis

Influenza Virus Ribonucleoprotein Complexes Gain Preferential Access to Cellular Export Machinery through Chromatin Targeting

In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration.

Production and characterization of monoclonal antibodies against vaginolysin: Mapping of a region critical for its cytolytic activity

Vaginolysin (VLY) is a protein toxin released by Gardnerella vaginalis. VLY belongs to the group of cholesterol-dependent cytolysins (CDCs). We have generated a panel of novel monoclonal antibodies (MAbs) against VLY. For the generation of MAbs, we have used recombinant VLY expressed in Escherichia coli. The functional activity of recombinant VLY was confirmed by an in vitro hemolytic assay using human erythrocytes. The MAbs raised against recombinant VLY were reactive with VLY from G. vaginalis both by Western blot and ELISA. The cross-reactivity of MAbs with other CDCs was investigated. For this purpose, recombinant cytolysins perfringolysin, listeriolysin, intermedilysin, pneumolysin and streptolysin were expressed in E. coli. The MAbs were specific exclusively to VLY and did not react with other CDCs. All MAbs were studied for the ability to neutralize hemolytic activity of VLY in vitro and several neutralizing MAbs were identified. The MAb produced by clone 9B4 showed the most potent neutralizing activity. The epitope for this MAb was localized near the N-terminus of VLY, between amino acid (aa) residues 112 and 268. The region recognized by the neutralizing MAb 9B4 includes the conserved motif (VAARMQYD, aa 189-196) supposed to be involved in VLY oligomerization. Selected MAbs were employed to develop a sandwich ELISA for VLY quantification. The MAb-based immunoassay was suitable for the detection of VLY in the cultures of G. vaginalis. In conclusion, the MAbs described in the current study may be useful for structural and functional studies of VLY as well as immunodetection of VLY in biological specimens.

Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis

BackgroundGardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance.ResultsSingle-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S)4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells.ConclusionsHybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis.

Construction, Purification, and Characterization of a Homodimeric Granulocyte Colony-Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) has found widespread clinical application, and modified forms with improved biopharmaceutical properties have been marketed as well. PEGylation, the covalent modification of G-CSF with polyethylene glycol (PEG), has a beneficial effect on drug properties, but there are concerns connected to the immunogenicity of PEGylated compounds and bioaccumulation of the synthetic polymer. To overcome challenges connected with chemical modifications, we developed fusion proteins composed of two G-CSF molecules connected via different peptide linkers. Three different homodimeric G-CSF proteins were purified, and their in vitro and in vivo activities were determined. A G-CSF dimer, GCSF-Lα, was constructed using an alpha-helix-forming peptide linker, and it demonstrated an extended half-life in serum with a stronger neutrophil response as compared to the monomeric G-CSF protein. The GCSF-Lα protein, therefore, might be selected for further studies as a potential drug candidate.