Mechanism Of Hexon-chimaeric RAd5Vectors Replication And Assembly

Because of many features, recombinant adenovirus type5(rAd5) vectors areconsidered the main vectors for cancer gene therapy and vaccine clinical research.However, the wide occurrence of a pre-existing humoral response in the humanpopulation will significantly reduce the transduction efficiency and transgeneexpression of rAd5vectors. Hexon is the most abundant adenoviral structural proteinwhich have seven hypervariable regions(HVRs) that form the exposed surface of thehexon protein. Manipulating the virus capsid components, especially HVRs, bygenetic engineering may provide some solutions to this problem. A major impedimentto the advancement of hexon-modified vectors is the hexon-chimaeric rAd5vectorsoften results in failure of rescuing viruses or poorly growing viruses. Therefore, studyon the mechanism of hexon-chimaeric rAd5vectors replication and assembly will bevery useful to know what regions of hexon can be modified without affecting viabilityof the Ad5viruses and has a strong scientific significance and application value.This paper detected the activity of the hexon-chimaeric rAd5vectors virus[Ad5-GFP、Ad5-37(5,7)-GFP、Ad5-43(5,7)-GFP] in vitro and the protein expressionlevel of these chimeric vectors plasmid [Ad5、Ad5-37(5,7)、Ad5-43(5,7)、Ad5-37(1-7)、Ad5-43(1-7)]. We found that the modification changed the amount and the form ofadenovirus hexon in cell and this change was the cause of the failure of rescuingviruses or poorly growing viruses in hexon-chimaeric rAd5vectors. Then, accordingto the five chimeric vectors plasmid [Ad5、Ad5-37(5,7)、Ad5-43(5,7)、Ad5-37(1-7)、Ad5-43(1-7)], we designed and constructed five chimeric-hexon eukaryoticexpression plasmid[5Hexon、5Hexon-37(5,7)、5Hexon-37(1-7)、5Hexon-43(5,7)、5Hexon-43(1-7)] and the rAd5hexon chaperone protein L4-100K eukaryoticexpression plasmid(5-100K). We tested the expression level, trimerization and nucleartrasport of these chimeric-hexon with or without L4-100K protein and discussed theaffection of the modifications in the hexon HVRs. We found that although the expression of chimeric-hexon was able to be increased with the help of5-100K,different retrofit scheme change the level of chimeric-hexon forming trimer and theefficiency of enter the nucleus. Finally, we changed the type of L4-100K from type5to group D, retested the trimerization of the chimeric-hexon which replaced the sevenHVR from group D[5Hexon-37(1-7)、5Hexon-43(1-7)] with D-100K. to analyzewhether the. Hexon-chimaeric rAd5vectors resulted in a severely diminished progenyvirus production not by changing serum type-specific of chimeric-hexon andprecluding the interaction between L4-100K and hexon, but subverting hexonbiogenesis.In summary, this study determine the chimeric transformation of the hexoninfluence the adenovirus packing efficiency and the level of replication by subvertinghexon biogenesis, clarified the mechanism of the relevant part of the adenoviruspackaging. From the study in this paper, we set up a effective evaluation of chimericmodified adenovirus strategy platform, through the detection of hexon interact withL4-100K to evaluate and predict the influence of chimeric modified adenovirusstrategy to viral packaging and replication. This method can help us effectivelyeliminate the strategy that may cause the failure of rescuing viruses and greatlyimprove the success rate of getting an ideal hexon-chimaeric rAd5vector.