ABSTRACT
Immune and tissue cells usually express pattern-recognition receptors (PRRs) to detect viruses and other microorganisms, thereby inducing signal cascade amplification and host innate immune responses. Since PRRs have strain-specific substrates and mechanisms of recognition, the identification of PRRs and mechanisms of PRRs-mediated responses is highly challenging. Besides, the research on RLRs-mediated immune responses has become more popular in cellular immunology recently. Accumulating evidence shows that post-translation modifications, such as ubiquitination, deubiquitination and ISGylation, play an important role in regulating host innate immune responses. In parallel, these approaches may be used by viruses to evade PRRs-mediated responses or to actively subvert these pathways for their own benefit. It was identified that STING (also called MITA/MPYS/ERIS) plays an important role in RIG-Ⅰ-like receptor(RLR) signaling as a type Ⅰ IFN stimulator, providing a special method for the research on complex host antiviral innate immune responses.
ABSTRACT
<p><b>OBJECTIVE</b>To analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.</p><p><b>METHODS</b>Two specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.</p><p><b>RESULTS</b>After reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.</p><p><b>CONCLUSIONS</b>Cleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.</p>