The Screening And Verification Of Antiviral Dugs Targeting Host Target

Influenza virus(IV), the causative agent of the homtavirus, is a worldwide communicable disease, and causes a broad range of illness, from symptomless infection through various respiratory syndromes, disorders affecting the lung, heart, brain, liver, kidneys, and muscles, to fulminant primary viral and secondary bacterial pneumonia, it is a so serious health problem. Two unprecedented epizootic avian influenza A (H5N1) virus and porcine influenza A virus (H1N1) are highly pathogenic had caused many human fatalities and poses an increasing pandemic threat. Moreover, existing vaccines and therapy for influenza virus infection have a limited value. The brand new antiviral thrathy is needing in urgent. The emergenting new biotechniques make inhibit virus propagation by inhibit a gene expression be a possible, but there is a big disadvantage, the genome of virus is simple and have so little tagets to use for drug design.Influenza viruses (IV) can trigger a series of signaling events inside the host cells and complex cellular responses. Understand the intricacies of host-pathogen interactions is a help to find the host molecular involved in the virus infection. Accumulating evidences suggested that host proteins have an essential role and represent novel targets for attenuation of viruses. So we can regulate the gene's expression of the molecular to inhibit the virus breeding in host cells. The new antimicrobial strategy has proved to be effective in laboratory and clinic, and pose a great potential for the future.Proteomics global analysis the moity, express level and modification changes of intracellular proteins, to research protein-protein interaction and to reveal the protein function and the regular pattern of life. It can study the changes of functional protein involved in virus-host interaction roundly. And subcellular proteomics provides a method to understand the protein expression level from regional.ASODNs pairs with special gene to inhibit the gene expression at gene level, have high specific and effective, been a majoy way of drug research. ASODN is a polymer of anions with a high molecular weight, hadro-polarity and strong electronegativity, which make cellular uptake being a hard problem. Even it is reported that only 1%~2% of the ASODNs can entry into cell. So it is a chanllge to strengthen the cellular uptaken of ASODNs, the present method is conjugate polycation, lipofectamine or transmembrane-peptide and ASODNs to get conjugates. But the clinical application of those conjugates were restrained by the potential cytotoxicity, so, to make ASODNs a suitable medicine a more suitable molecular is need for the modification to enhance the cellular uptake. In the research not only the length of ASODN sequence was optimized but also the rimantadine and lipoid were choosing for the modifier.In the research the subcellular proteomic technology was taken to find the host protein molecules involved in IV infection.Two- dimensional gel electrophoresis (2-DE) combined with mass spectrum (MS) was performed on protein extracts from nuclear, cytoplasm, mitochondria of infected and control human lung cells (A549). And the hosts proteins involved in influenza virus infection reported by other researchers and the prioteins may involve in the host-virus interaction calculated by computer were selected to validate to be relevant to the disease cause by the influenza virus infection.The following content were included in the study:a) The screening of host proteins involved in influenza virus infenctionTotally 112 differentially expressed protein molecules were found (40, 52 and 20 spots from nuclear, cytoplasm and mitochondria), 30, 37 and 13 protein spots of them were successfully identified by MS. Three proteins and 31 proteins by bioinformatics and other researchers report were selected.b) The identification of differential expressive proteinsThe differential expression of ISG15, MIF, PDCD5 and UCHL1 were authenticated by western blot. The specific expressions of them were proved to be have related with influenza virus infection.c) The design, synthesis and screening of ASODNsThe antisense oligodeoxyribonucleotide (ASODN) targeting ISG15, MIF, PDCD5, and UCHL1 were design by omputer software, and significantly alleviated IV propagation in A549 cells and the cytopathic effect (CPE) induced by IV infection, as detected by qRT-PCR and cell viability assay.The highest CPE inhibition was get from the inhibition of PDCD5, and the follow experiment proved that the CPE cause by H1N1, H3N2 (including rimantadine resistant isoforms) influenza virus were inhibited by inhibit PDCD5's mRNA expression. It give a message that: PDCD5 paly an important role in the apoptosis caused by influenza virus infection, it is a potential host target to anti influenza virus infection.d) The length optimization of ASODNThe length of ASODN is changed into different nucleotide number and the antiviral activity were taken on A549 cells challged with influenza virus, the sequence with less nucleotide number and higher antiviral ability was selected to be the best sequence (easy to sythensis and have high activity), can work with a low does.e) The effect on influenza virus infections of the enhancement of PDCD5 expressionThe plasmid were constructed carring PDCD5 gene CDS, the protein PDCD5 was exprssied in E.coli, the purified protein was added to the cell culture medium, and the PDCD5 gene CDS was constructed in eukaryon carrier plasmid, the plasmid was transfected A549 cells with liposomes, the results of the experiment show that improve the expression of PDCD5 the apoptosis induced by influenza challenge was enhanced.f) The antivial effect of ASODN in vivoA pilot study of antiviral activity of ASODN targeting PDCD5 was carried out in vivo. A highly pathogenic H1N1 influenza virus infected model was established in mice. Drugs were administrated by nasal cavity to mice infected with H1N1 virus. For 12 days this systemic treatment resulted in the improvement of survival rate, mean survival days (MSDs) and body weight loss in a dose-dependent manner. Meanwhile the lung index were slightly decreased. The work proved that the enrichment in the nuclear of PDCD5 paly an important role in the IV infenction and pathopoiesis.g) The effect of rimantadine and lipoid modificationRimantadine a drug of antiviral but influenza virus have drug resistance to it by antigenic drift. Lipoid is a chemical composition has high affinity to cell membrane. So, our work is modified ASODN sequence with rimantadine or lipoid to get an ASODN-rimantadine or ASODN-lipod conjugates, the result show that the CPE inhibit activity of ASODN was improved and ASODN- rimantadine and ASODN-lipoid conjugates can significantly improve the cellular uptake of ASODN.The results showed that high expression and enrich in the nucleus of PDCD5 is important for the infection and pathopoiesis of influenza virus, and the ASODN targeting PDCD5 hopefully appears to be a supplementary antiviral therapy approach to treatment of influenza A virus infection.In conclusion, the differential proteins identified in this study may provide clues for better understanding of the influenza virus-host interaction. PDCD5 paly an important role in the apoptosis caused by influenza virus infection, it maybe a potential target to anti influenza virus infection, and two methods to improve the cellular uptake of ASODN were found.