MOLECULAR CHARACTERIZATION OF 52K PROTEIN OF BOVINE ADENOVIRUS TYPE 3 A Thesis Submitted to the Faculty of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Veterinary Microbiology University of Saskatchewan Saskatoon By Carolyn Patricia Paterson © Copyright Carolyn P. Paterson, August 2010. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a postgraduate degree from the University of Saskatchewan, I agree that the libraries of this university may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, whole or in part, for scholarly purposes may be granted by the professors who supervised my thesis work or in their absence, the Head of the Department or the Dean of the college in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts therof for financial gain shall not be allowed without any written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Request for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Veterinary Microbiology University of Saskatchewan Saskatoon, Saskatchewan, S7N 5B4 i ABSTRACT Bovine adenovirus (BAdV)-3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans (Rasmussen et al., 1999; Zakhartchouk et al., 1999). Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV) -5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported (Reddy et al., 1998). Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin β and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin α/importin β pathway for nuclear import. In support of this, a specific interaction between 52K and importin α3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin α/importin β pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K ii and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells (Zhang and Arcos, 2005). Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFκB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed in vitro and in vivo. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K. iii ACKNOWLEDGMENTS First and foremost, I would like to thank my supervisor, Dr. Suresh Tikoo, for giving me the opportunity to pursue my graduate degree in his laboratory. Your support, encouragement, and advice during my graduate studies were greatly appreciated. I would also like to thank the members of my advisory committee, Dr. Lorne Babiuk, Dr. Lambert Loh, Dr. Alexander Zakhartchouk, and Dr. Vikram Misra for their valuable input and assistance during the course of my program. I would also like to thank the past and present members of the Vectored Vaccines group for their help, suggestions, and support. In particular, the technical assistance of Satyender Hansra, Jill Van Kessel, and Kyla Shea was invaluable. I also owe thanks to the multitude of support staff at the Vaccine & Infectious Disease Organization for assisting with everything from peptide synthesis to administrative paperwork. It has been a pleasure to work with all of you. I would like to thank my friends and fellow graduate students for their moral support and friendship. Our scientific discussions were always a great help in troubleshooting experiments, and our non-scientific ones were always a great help in maintaining my sanity. Amanda, Jean, Candice, Erin, Megan, Tara, Ellen, Patricia, and everyone else who has come and gone in our basement office, thank you for making my time as a graduate student much more enjoyable. Finally, I would like to thank my parents, Guy and Sharon, and my brother Greg. I am immensely grateful for all your love, support, and encouragement over the years. I couldn’t have done it without you. iv TABLE OF CONTENTS PERMISSION TO USE.................................................................................................................i ABSTRACT ...................................................................................................................................ii ACKNOWLEDGMENTS ...........................................................................................................iv TABLE OF CONTENTS.............................................................................................................. v LIST OF TABLES .......................................................................................................................ix LIST OF FIGURES ...................................................................................................................... x ABBREVIATIONS USED IN THIS WORK............................................................................xii 1.0 LITERATURE REVIEW....................................................................................................... 1 1.1 Adenoviruses .................................................................................................................. 1 1.1.1 Adenovirus taxonomy ......................................................................................... 1 1.1.2 Human adenovirus............................................................................................... 3 1.1.2.1 Virus structure and genome organization ............................................... 3 1.1.2.2 Virus entry and release............................................................................ 4 1.1.2.3 Early gene expression ............................................................................. 7 1.1.2.4 DNA replication...................................................................................... 9 1.1.2.5 Intermediate and late gene expression .................................................. 11 1.1.2.6 Virus assembly and release................................................................... 13 1.1.3 The 52/55-kilodalton protein............................................................................. 16 1.1.4 Bovine adenovirus............................................................................................. 18 1.1.4.1 Classification......................................................................................... 18 1.1.4.2 Bovine adenovirus type 3...................................................................... 18 1.1.4.2.1 Genome structure and organization.......................................... 19 1.1.4.2.2 Replication cycle ...................................................................... 26 1.2 Nuclear transport in Eukaryotes................................................................................ 28 1.2.1 Nuclear