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Molecular Characterization of Bovine Adenovirus-3 IVa2 Protein



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ABSTRACT Adenovirus is a naked icosahedral viral particle enclosing a double stranded DNA genome. Adenoviral genes are classified as early, intermediate and late based on their duration of expression. Adenoviruses encode two intermediate gene products, pIX and IVa2. Adenovirus IVa2 is one of the conserved proteins encoded by members of the family of Adenoviridae. Prior studies indicate that IVa2 localizes to the nucleus\nucleolus and, is involved in adenovirus genome packaging and activation of major late promoter (MLP) thus enhancing the expression of late adenoviral proteins. Moreover, IVa2 has been suggested to perform multiple functions by interacting with different viral proteins including 33K and DBP (DNA binding protein), 52K and pVIII. Positional homologs encoded by members of Mastadenovirus genus are different in structure and function. Since Bovine adenovirus (BAdV) -3 IVa2 displays limited homology compared with homologs encoded by other members of Mastadenovirus genus, the present study was undertaken to characterize IVa2 of BAdV-3, to identify viral protein interacting with IVa2 and to determine the role of IVa2 in the virus life cycle. As part of characterization, BAdV-IVa2, detected as a 50kDa protein in BAdV-3 infected cells (12-18 hrs post infection) and plasmid DNA transfected cells (48 hrs post transfection). Although IVa2 could be detected both in the nucleus and nucleolus of virus infected or DNA transfected cells, IVa2 appeared predominantly located in the nucleolus. Analysis of IVa2 by cNLS mapper program predicted the nuclear localization signals (NLSs) of IVa2 interacting with importin. Analysis of mutant IVa2 suggested that N- terminus amino acids 4-18 appear essential for the nuclear localization of IVa2. Several lines of evidence suggest that IVa2 N-terminus amino acids 1-25 appear to contain functional NLS. First, IVa2 N-terminus amino acids 4-18 are not sufficient to translocate a cytoplasmic fusion protein GFP/β gal to the nucleus of transfected cells. Secondly, GST pull-down assay suggested that amino acid 4-18 does not bind to importin α-1. Thirdly, N-terminus 25 amino acids bind to importin α-1 and translocate GFP/β gal to the nucleus of the transfected cells. Analysis of mutant IVa2 proteins also suggested that C-terminal amino acids 373-448 appear essential for the nucleolar localization of IVa2 in transfected cells. While amino acids 373-448 appear to contain multiple nucleolar localization signals (NoLSs), the NLS and NoLS of IVa2 does not appear to be overlapping. The identified nuclear localization signal of IVa2 contains clusters of basic amino acids, (6RRK8), similar to that of classical nuclear localization signal. The substitution of arginine or lysine residues to glycine did not alter the nuclear localization of IVa2 in DNA transfected cells or mutant BAdV-3 infected cells indicating that 6RRK8) residues are not essential for nuclear localization of BAdV-3 IVa2. To identify the viral proteins interacting with IVa2, initially we attempted to use affinity tag purification method. However, repeated attempts to isolate recombinant BAdV-3 expressing IVa2 containing Step Tag were not successful. Instead, using Bi molecular Fluorescence Complementation (BiFC) assay, we identified several viral proteins including pV that interact with IVa2. Mass spectrometry also indicated that pV interacts with IVa2. Finally, the interaction of IVa2 with pV was confirmed by co-immunoprecipitation (Co-IP) assay in DNA transfected and BAdV-3 infected cells. Further analysis identified IVa2 amino acids 121-140 as the domain involved in the interaction with pV. Several lines of evidences suggest that IVa2 protein is sensitive to any addition \deletion. To assess the role of IVa2 in the virus life cycle, we attempted to isolate IVa2 deleted BAdV-3 in non-IVa2 complementing cells. First, transfection of cells with full-length BAdV-3 genomic clone containing deletion of IVa2 region overlapping polymerase region did not result in the isolation of viable virus. Second, transfection of cells with full-length BAdV-3 genomic clone containing addition of Cre recombinase recognition loxP sequence in-frame to IVa2 coding sequence did not result in the rescue of viable virus. Third, transfection of cells with individual full length BAdV-3 genomic clone containing insertion of Strep tag at amino acids 146,242 or 448 did not result in the isolation of viable virus. However, transient expression of BAdV-3 IVa2 complemented the defect of IVa2 in BAV304a.dIVa2 in cotton rat lung (CRL) cells suggesting that IVa2 is essential for replication of BAdV-3.



Bovine adenovirus IVa2



Doctor of Philosophy (Ph.D.)


Veterinary Microbiology


Veterinary Microbiology


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