Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

AbstractOligonucleotides of a novel type containing 2′-O-β-ribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter-containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein-nucleic acid interactions

Prokaryotic expression, purification and immunogenicity in rabbits of the small antigen of hepatitis delta virus

Funding Information: Expression and purification of HDV antigen was supported by Russian Foundation for Basic Research (grant 16-04-01490a). Evaluation of serum by Western blot and confocal microscopy was supported by Russian Science Foundation (grant 14-14-01021). Experiments in rabbits were supported by the Swedish Institute grants 09272_2013 and 19806_2016. Cross-border collaboration of the partners, exchange of the materials and standard operation procedures used in the study, and dissemination of the data were supported by the EU Twinning project VACTRAIN, contract nr 692293. Publisher Copyright: © 2016 by the authors; licensee MDPI, Basel, Switzerland.Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.publishersversionPeer reviewe

Role of Polyamine-Induced Dimerization of Antizyme in Its Cellular Functions

Funding: This work was supported by grants from the Russian Science Foundation (grant # 17-74-20049—synthesis of C-methylated Spd analogues, ITC studies of dimerization of OAZ1, and frameshifting experiments), the Russian Science Foundation (grant # 19-74-10086—isolation of OAZ1, electrophoresis studies of dimerization of OAZ1), and the Academy of Finland (grants # 292574 and # 315487). Acknowledgments: The authors thank A. Karppinen, A. Korhonen, T. Reponen, M. Salminkoski, and S.D. Negrya for their skillful technical assistance.Peer reviewedPublisher PD

Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus

Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy

Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis

Cellular Immunogenicity of Novel Gene Immunogens in Mice Monitored by in Vivo Imaging

The efficient cell-mediated immune response clears cells expressing deoxyribonucleic acid (DNA) immunogens, but there are no methods to monitor this in vivo. We hypothesized that immune-mediated clearance can be monitored in vivo if DNA immunogens are coexpressed with reporter(s). To test this, we designed genes encoding human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) fused via its N- or C-terminus to 30–amino acid-long Gly-Ala-repeat of Epstein-Barr virus nuclear antigen 1 or via the N-terminus to the transport signal of invariant chain/Ii or inserted between the cytoplasmic and luminal domains of lysosome-associated membrane protein I (LAMP). DNA immunogens mixed with luciferase gene were injected into BALB/c mice with subsequent electroporation. Reporter expression seen as luminescence was monitored by in vivo imaging. When luminescence faded, mice were sacrificed, and their splenocytes were stimulated with RT-derived antigens. Fading of luminescence correlated with the RT-specific secretion of interferon-γ and interleukin-2. Both immune and in vivo imaging techniques concordantly demonstrated an enhanced immunogenicity of RT-LAMP and of the N-terminal Gly-Ala-RT fusion genes. In vivo imaging performed as an animal-sparing method to estimate the overall performance of DNA immunogens, predicting it early in the experiment. So far, in vivo imaging cannot be a substitute for conventional immune assays, but it is supplementary to them. Further experiments are needed to identify which arms of cellular immune response in vivo imaging monitors best

Some papers on Lyapunov exponents

The papers will appear in: L. Arnold and V. Wihstutz (eds.): Lyapunov Exponents. Proceedings of a workshop, Bremen, November 1984. Springer Lecture Notes in Mathematics, 1985SIGLETIB: RA 6154 (141) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman