Functional characterization of 100K protein of bovine adenovirus type 3
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Bovine adenovirus (BAdV)-3, a Mastadenovirus was isolated from the healthy and sick cattle (Darbyshire et al., 1965; Zhu et al., 2011). Like other adenoviruses, BAdV-3 replication is characterized by the temporally regulated expression of genes characterized by early, intermediate and late gene expression. Genus-common, non-structural protein 100K is encoded by late region L6 of BAdV-3. The objective of the present study was to characterize the BAdV-3 100K protein and identify cellular and viral proteins interacting with 100K. Although BAdV-3 100K encoded as 850 amino acid polypeptide (Reddy et al., 1998), rabbit antisera raised against peptides representing N-terminus or C-terminus recognized a protein of 130 kDa at 12-24 hrs post infection, and proteins of 130 kDa, 100 kDa, 95 kDa and 15 kDa at 36-48 hrs post infection. The 100K appeared to be localized to the nucleus and cytoplasm of BAdV-3 infected cells. In contrast, 100K localized predominantly to cytoplasm of transfected cells. However, BAdV-3 infection of cells transfected with 100K-EYFP expressing plasmid detected fluorescent protein in nucleus of the cells suggesting that another viral protein may be required for the nuclear localization of 100K. Using yeast two-hybrid and GST pull-down assays, 100K protein was shown to interact with BAdV-3 33K protein. These results were validated using bimolecular fluorescence complementation (BiFC) assay. Although, 100K protein interacts with 33K protein, co-expression of both proteins in transfected cells did not alter the cytoplasmic localization of 100K. Using GST-pull down assay and BiFC assay, 33K interacting region of 100K was localized to a stretch of 13 amino acids (624-637). Repeated attempts were not successful in rescuing a recombinant BAdV-3 expressing mutant 100K (containing deletion of amino acids 624-637). The interaction of cellular protein(s) with 100K was determined by mass spectrometric analysis of immunoprecipitated 100K. Mass spectrometry of immunoprecipitate obtained by immunoprecipitating 100K protein from BAdV-3 infected cells harvested at 48 hrs post infection identified six proteins including dynein light chain (DYNLT)1. The initial identified interaction of 100K with DYNLT1 was confirmed by the yeast two-hybrid assay, co-immunoprecipitation assay and BiFC assays. Furthermore, DYNLT1 interacting domain of 100K protein of BAdV-3 was found to be located between 499-587 amino acids. Co-expression of BAdV-3 100K-EY fusion protein with myc epitope tagged DYNLT1 protein did not alter the localization of 100K-EY fusion protein. The investigation into the differences in the subcellular localization of the 100K protein in the transfected and infected cells lead to identification of the cleavage by adenoviral protease. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (amino acid 740-745 and 781-786) in transfected or BAdV-3 infected cells. Although protease encoded by human adenovirus (HAdV)-5 or porcine adenovirus (PAdV)-3 also cleaved BAdV-3 100K at potential identified protease cleavage sites, no such cleavage of 100K encoded by HAdV-5 or PAdV-3 could be detected in cells expressing virus specific protease. Successful isolation of recombinant BAdV-3 expressing mutant protease (substitution of alanine for glycine in potential protease cleavage site) suggested that cleavage of BAdV-3 100K by viral protease is not essential for viral replication. However, further analysis observed less virus in the supernatant of cells infected with mutant BAdV-3 compared to WT BAdV-3 suggesting a possible role for cleaved C-terminal fagment in lysis of infected cells. Co-expression of BAdV-100K with other late viral proteins suggested that the 100K-EYFP fusion protein localized to the nucleus in cells co-expressing BAdV-3 protease-DsRed fusion protein. Interestingly, only C-terminal cleaved fragment of 100K localizes to the nucleus in BAdV-3 protease expressing cells. Further analysis suggested that C-terminal fragment localizing to the nucleus contains a bipartite nuclear localization signal, which is recognized by importin α3. Our results suggest that the N-terminal part of 100K may be retained in the cytoplasm by interaction with Tctex1 (DYNLT1). Our study provides for the first time a plasmid co-transfection system for the study of the protease cleavage of viral proteins. Moreover, this is the first report of cleavage of any non-structural viral proteins by adenoviral protease in infected cells.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorTikoo, Suresh K.
CommitteeChelico, Linda; Dar, Arshud; Zhou, Yan
Copyright DateDecember 2013
100K protein, bovine adenovirus type 3, BAdV-3