Functions of the viral chitinase (CHIA) in the processing, subcellular trafficking and cellular retention of proV-CATH from Autographa californica multiple nucleopolyhedrovirus by Jeffrey James Hodgson A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Molecular and Cellular Biology Guelph, Ontario, Canada © Jeffrey James Hodgson, December, 2011 ABSTRACT FUNCTIONS OF THE VIRAL CHITINASE (CHIA) IN THE PROCESSING, SUBCELLULAR TRAFFICKING AND CELLULAR RETENTION OF PROV- CATH FROM AUTOGRAPHA CALIFORNICA MULTIPLE NUCLEOPOLYHEDROVIRUS Advisor: Jeffrey James Hodgson Dr. Peter J. Krell University of Guelph co-advisor: Dr. Basil M. Arif The baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, thereby enhancing dissemination of progeny virions in nature. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the intracellular processing and trafficking of the baculovirus v-cath expressed cathepsin (V-CATH) is poorly understood and a mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not been determined. The cathepsins of Autographa californica multiple nucleoplyhedrovirus (AcMNPV) and most other group I alphabaculoviruses have well-conserved chymotrypsin cleavage (Y11) and myristoylation sites (G12) suggestive of proteolytic cleavage to generate proV-CATH, and subsequent acylation which could promote membrane anchoring in order to foster cellular retention of the protein. Proteolytic ii N-terminal processing of baculoviral procathepsin was determined by fusing HA epitope-coding tags to the 5’ and/or 3’ ends of v-cath, indicating that the gene is expressed as a pre-proenzyme. However no evidence for myristoylation of proV- CATH was found, suggesting that another mechanism is responsible for retaining proV-CATH in cells. Prior evidence suggested that CHIA is a proV-CATH folding chaperone and that lack of chiA expression causes proV-CATH to become insoluble and unable to mature into V-CATH enzyme. A putative CHIA chaperone activity for assisting in proV-CATH folding implies that proV-CATH and CHIA interact in the ER of infected cells. Fluorescence microscopy demonstrated co-localization of CHIA-GFP and proV-CATH-RFP fusion proteins in the ER. An mRFP-based bimolecular fluorescence complementation (BiFC) assay helped to determine not only that AcMNPV proV-CATH interacts directly with the full-length viral CHIA, but also that it independently binds to the N-terminal chitin-binding domain (CBD) and C-terminal active site domain (ASD) of CHIA, in the ER during virus replication. Moreover, reciprocal Ni/HIS pull-downs of HIS-tagged proteins confirmed the proV-CATH interactions with CHIA, or with the CBD and ASD biochemically. The reciprocal co-purification of proV-CATH with all three polypeptides (CHIA, CBD, ASD) suggests proV-CATH specifically interacts with each of them, and corroborates the BiFC data. Furthermore, CHIA KDEL deletion allowed for premature secretion of not only CHIA but also of proV-CATH, suggesting that the CHIA/proV-CATH interaction in the ER aids cellular retention of proV-CATH. In contrast to prior reports, it was also determined that CHIA is iii dispensable for correct folding of proV-CATH since proV-CATH produced by a chiA-deficient virus was soluble, prematurely secreted from cells and could mature into V-CATH enzyme. Taken together, these data indicate that the viral chitinase plays a major role in ensuring that proV-CATH is neither prematurely secreted nor activated to V-CATH enzyme. iv Acknowledgements I thank Monique van Oers (Wageningen University) for kindly supplying the chiA/v-cath deletion bacmid (AcΔCCBAC), to Jeffrey Slack for providing the anti-V-CATH serum and to Joachim F. Uhrig (Max Planck Institute for Plant Breeding Research) for his contribution of the split mRFP constructs pBatTL- smRFPN and pBatTL-smRFPC. Thanks are also given to Éva Nagy for editorial suggestions (Chapter 2) and to David Leishman for technical support. The support of grants to PJK from the Natural Sciences and Engineering Research Council of Canada (STPGP 365213 and RGPIN 8395) is gratefully acknowledged. v Table of contents ABSTRACT ........................................................................................................... ii Table of contents ................................................................................................. vi List of Tables ....................................................................................................... ix List of figures ........................................................................................................ x List of abbreviations ............................................................................................ xii Virus Abbreviations ............................................................................................. xiii Chapter 1: Introduction and review of the literature .............................................. 1 1.1 Baculoviruses .............................................................................................. 1 1.2 Baculovirus infection and pathogenesis ...................................................... 2 1.3 Baculovirus temporal regulation of gene expression ................................... 7 Immediate early and early transcription ......................................................... 9 Late and very late transcription .................................................................... 10 1.4 Chitinases and cathepsin proteases .......................................................... 11 Chitin and chitinases .................................................................................... 11 Cathepsin proteases .................................................................................... 16 1.5 Baculovirus chiA and v-cath genomic organization and expression .......... 18 AcMNPV chiA expression ............................................................................ 22 AcMNPV v-cath expression ......................................................................... 25 1.6 CHIA and V-CATH co-dependence in host liquefaction ............................ 29 1.7 Rationale, hypotheses and experimental design ....................................... 32 Chapter 2: Autographa californica multiple nucleopolyhedrovirus and Choristoneura fumiferana multiple nucleopolyhedrovirus v-cath genes are expressed as pre-proenzymes ............................................................................ 34 Abstract ........................................................................................................... 34 2.1 Introduction ................................................................................................ 35 2.2 Materials and Methods .............................................................................. 40 Cells and virus ............................................................................................. 40 V-cath constructs ......................................................................................... 40 Molecular cloning ......................................................................................... 41 V-CATH–DsRED/mGFP5 co-localization .................................................... 45 Western blot analysis ................................................................................... 46 2.3 Results and Discussion ............................................................................. 46 vi Analysis of the N-linked glycosylation state of AcMNPV and CfMNPV proV- CATH ........................................................................................................... 46 Proteolytic co-translational removal of the N-terminal signal peptides ......... 48 Subcellular localization of proV-CATH-DsRED ............................................ 50 Chapter 3: Interaction of Autographa californica multiple nucleopolyhedrovirus proV-CATH with CHIA as a mechanism for proV-CATH cellular retention .......... 53 3.1 Introduction ................................................................................................ 55 3.2 Materials and Methods .............................................................................. 59 Cell and virus ............................................................................................... 59 Cloning methodology for generating viral constructs ................................... 60 Manipulation of viral genomes and generation of engineered viruses ......... 69 Northern blot analysis .................................................................................. 71 Western blot analyses ................................................................................. 73 Co-localization of CHIA and proV-CATH in virus-infected cells ................... 73 GFP ER-translocation assay ........................................................................ 75 mRFP-based bimolecular fluorescence complementation assay................. 76 Ni-agarose affinity co-purification of CHIA and proV-CATH ......................... 76 Temporal analysis of CHIA and proV-CATH co-retention/co-secretion ....... 78 3.3 Results .....................................................................................................