The Functional Study Of The Minor Structral Protein â…¨ In The Packaging Of Bovine Adenovirus Type3

Adenoviruses (Ad) are the most widely used viral vectors for gene therapy and vaccination.Currently, bovine adenovirus type3(BAdV-3) is being developed as potential vehicles forvaccination. It has reported a number of features of BAdV-3, including ease of manipulationof viral genome, growing to high titer, species specificity, overcoming the pre-existingimmunity against human adenovirus, better biodistribution and high biosafety, whichconfirmed it suitable as a vehicle for vaccine delivery (Zakhartchouk et al.,1999). The morecomprehensive understanding of the biology of the BAdV-3is essential to improve thefunction, safety, and versatility of the vector system. So far, the minor capsid proteins, proteinIX (pIX) has received considerable attention mainly as its multiple functions inHAdV2/5.However, there are few studies on the gene Ⅸof bovine adenovirus.In this study, we generated a series of BAdV-3mutants with deletions in the pIX gene orchemeric pⅨ gene. The modified pIX proteins were tested for their capacity to recombinationadenovirus rescue and to get more insight in the functional domain of pIX in BAdV-3lifecycle, which will have to be taken into account when designing BAdV3-based vectors fortherapeutic purposes.1.Polypeptide pⅨ is essential for Bovine adenovirus type3virions production.Intially, we generated full-length p Ⅸ deleted BAdV-3genome pFBAV304a.I-SceⅠ-△p Ⅸand pFBAV304a.I-SceⅠ-mATGpⅨ, in which theinitiation codon ATG of BAdV-3p Ⅸwasreplaced with CTG. After transfected the plasmids in VIDO DT1cells, we could observe veryweak fluorescence and no CPE appeared, even in subsequent passages, which meant pⅨdeletion (BAdV3△p Ⅸ) or initiation codon mutation (BAdV3-mATGpⅨ) abolished theproduction of progeny virions; then, we constructed plasmidpFBAV304a.I-SceⅠ-p Ⅸ/10GS/HA, in which the pⅨ/10GS/HA fragment was recombinedback in plasmid pFBAV304a.I-SceⅠ-△pⅨ. And the cells transfected with this plasmidshowed apparent cytopathic effects and strong expression of GFP, meaning the recombination adenovirus (BAdV3-p Ⅸ/10GS/HA) was successfully rescued. These results suggested thatpⅨ was essential for BAdV-3rescue and plays as a trans-acting factor.2. Identify the essential domain of pⅨ as trans-acting factor for BAdV-3rescueIn this study, we aligned the amino acid sequences of BAdV-3p Ⅸwith different humanadenoviruses using CLUSTAL W program and predicted the structure of BAdV-3pⅨ basedon the I-TASSER server and2-ZIP server, and identified three relatively conserved domains:the conserved N-terminus, coiled-coil domain (which contained a putative leucine-zipperelement) and C-terminus. Then we constructed several BAdV-3mutants with the differentdomain deletions in p Ⅸ. We confirmed that the conserved N-terminus and putative leucinezipper element (PLZP) were the essential domains, while the C-terminus following PLZP wasnon-essential.In addition, the swap of PLZP element and following region of BAdV-3pⅨ to correspondingdomains of human adenovirus type5(HAdV-5) or porcine adenovirus type3(PAdV-3) didn’taffect virions production while the swap of entire p Ⅸabolished the production of progenyvirions. Thus, we deduced that the failure of full-length p Ⅸswap might be due to the speciesspecificity of the N-terminus; while the function of coiled-coil domain of pⅨ encoded bydifferent members of Mastadenovirus depends on the secondary or tertiary structures than theprimary amino acid sequence.