THE HERPES SIMPLEX VIRUS TRANSCRIPTION-ACTIVATING PROTEIN ICP4. ROY H. PERSSON, B.Sc., M.Sc.

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1 USE OF EXPRESSER CELL LINES TO FUNCTIONALLY qharacterize THE HERPES SIMPLEX VIRUS TRANSCRIPTION-ACTIVATING PROTEIN ICP4 by ROY H. PERSSON, B.Sc., M.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfilment of the Requirements for the Degree Doctor of Philosophy McMaster University December, 1987

2 CELL LINES CONSTITUTIVELY EXPRESSING THE HSV-l lcp4 GENE

3 DOCTOR OF PHILOSOPHY (1987) (Medical Sciences) McMASTER UNIVERSITY Hpmilton, Ontari~ TITLE: Use of expresser cell lines to functionally ch~racterize the Herpes Simplex Virus transcription-activating prot~in ICP4 AUTHOR: Roy H. Fersson, B.Sc. (University of Calgary) M.Sc. (University of Calgary) SUPERVISOR: s. Bacchetti, Department of Pathology NUMBER OF PAGES: xii, 157 ii

4 ABSTRACT During Herpes Simplex Virus type 1 (HSV-l) infections, many viral proteins are synthesized and several have proven or suspected roles in regulating viral gene expression. To facilitate the study of the individual activity of one such protein, lcp4, the ICP4 gene was cloned in a plasmid vector, and expresser cell lines containing 5-30% of infected cell levels of ICP4 were established. The ICP4 is functional, correctly processed, and located in the cell nuclei. The endogenous ICP4 gene retained its capacity to respond to viral trans-acting factors, since its expression after superinfection with HSV-2 mimicked that of the viral gene. Although cells infected with lcp4 mutant viruses overproduce ICP4 and other immediate-early proteins, cell lines synthesizing a mutant form of ICP4 did not overproduce this protein, suggesting that autoregulation of the lcp4 gene requires more than 30% of the infected cell level of ICP4 or, alternatively, requires the pr~sence of other viral proteins. After superinfection in the presence of an inhi~itor of protein synthesis, the endogenous ICP4 is capable of transactivating viral early genes encoding thymidine kinase, lcp6, tep8, gb, gd, and ge. In contrast, the early gene for the viral alkaline exonuclease, the early-late ~ene for VPS and the late genes for p40 and gc, respond poorly or not at all. This demonstrates that most early genes can be induced by ICP4 in the absence of other viral immediate-early proteins, but that early-late and late genes require supplementary factors. iii

5 ACKNOWLEDGEMENTS For gifts of plasmids I thank D. R. Helinski (p~tk16)t R. McKinnon (ptk173), W.- C. Leung (plbjl1) and C. M. Preston (pgx58, pgx157 and pgx164). For gifts of cell lines t thank F. L. Graham (human 293), P. A. Schaffer (E5) and J. R. Smiley (N17BS3). For gifts of mutant viruses, t thank Y. Haj-Ahmad (AdTK4), P. A. Schaffer (HSV-l d120), J. R. Smiley (HSV-l d2) and C. M. Preston (HSV-l tsk). For gifts of monoclonal antibodies I thank K. L. Powell (Ql), P. G. Spear (II 481 B-2), L. Pereira (HI083), M. J. Svelegh (B2) and M. Zweig (195, 58S and 74S). For gifts of rabbit antisera I thank H. S~ ~arsden (IEI2/76), w. R. McClements (anti-icp4 antiserum) and W. C. Summers (anti-tk antiserum). I wish to thank my supervisor, Silvia Bacchetti, for her extraordinary generosity with her time, and for her support, tolerance and understanding. I am also especially grateful to Jim Smiley, who substituted for Silvia Bacchetti as my supervisor during her sabbatical leave, and who has continued to take an active and very helpful interest in my project. I also thank the remaining members of my supervisory committee, W. R. Rawls and L. V. Prevec, who have been generous with their time and patience, and who have provided many valuable insights. A number of the staff, students and technicians of the Molecular Virology and Immunology Program have pr~yided me ~'tth advice and assistance. In particular, I

6 am grateful to Mike Evelegh, Claudio Sartori, Bev Leslie, Helen Rudzroga, David Johnson and Frank Graham. The research was funded partially by a grant to S. Bacchetti from the National Cancer Institute of Canada, and partially by a research allowance to R. H. P. from the Alberta Heritage Foundation for Medical Research. During the period of the research R. H. P. was supported by a research studentship from the Alberta Heritage Foundation for Medical Research. v

7 TABLE OF CONTENTS Page B. Herpes Simplex Virus. 6 I. Introduction. A. Mechanisms ~nd Control of Transcription 1. Prokaryotes 2. Eukaryotea. C. Regulation of H5V Gene Expression 8 1. Regulation of immediate-early genes Regulation of early genes Regulation of late genes. 20 D. Post-translational Processing of ICP4 22 E. Subcellular Location of ICP4. 24 F. Project Rationale 26 II. Materials and Methods Cloning Transfection and selection of cell lines Cells Viruses. '3S Labeling of proteins with S-methionine SD5-polyacrylamide gel e~ectrophoresis Immunoprecipitation of proteins Western blotting Cell fractionation 33 a. NP-40 lysis 33 b. Fractionation into subnuclear fractions Indirect immunofluorescence. 36 U. 51 nuclease mapping. 37 III. Results. 39 A. Development of Cell Lines Cloning Transfection and screening 44 B. Characterization of Cell Lines Comp1ementation of an ICP4 deletion mutant virus Amount of ICP4 per cell Response of the endogenous ICP4 gene to viral regulatory signals Post-translational processing of ICP4 in expresser cells. 65 vi

8 S. Subcellular location of ICP4 iu expresser cells Detection of ICP47 in Z4 cells 83 C. Transactivation of HSV-1 Genes by the ICP4 of Expresser Cells ICP4 is sufficient to induce early gene transcription Transactivation by ICP4 is reproducible in other cell lines Early gene context dramatically affects its inducibility 107 IV. Discussion A. Cell Biology of the ICP4 Protein B. Regulation of the Endogenous ICP4 Gene in Expresser Cells. 115 C. ICP4 Activity ICP4 sufficiency in early gene induction Induction of early ~. late genes The importance of target gene context ICP4 mechanism References vii

9 LIST OF FIGURES Figure Number Figure Title Page 1 Restriction endonuclease cleavage luap of plasmid plbjll 41 2 Restriction endonuclease cleavage map of plasmid prhp Restriction endonuclease cleavage map of plasmid prhp Screening of four cell lines. established by cotransfection with pn17bs3 and prhp6. for ICP4 synthesis 49 5 Comparison of the amount of ICP4 in Z4 cells with the amount in infected LTA cells ICP4 mrna levels in cells grown at 39 0 and o at ICP4 st~bllity i~ cells maintained at 39 0 and Time course of rate of synthesis of the endogenous ICP4 in Z4 c~lls after superin.~ection with USV Post-translational processing of the endogenous ICP4 in Z4 cells The effect of HSV-2 superinfection on the location of wt ICP4 and ts ICP4 in expresser cells The effect of HSV-2 superinfection on the solubility of ICP4 in expresser cells The effect of HSV-2 superinfection on the subnuclear compartmentalization of ICP4 in Z4 cells Assay for ICP47 in Z4 cells TK mana levels induced by ICP VP5 mrna leve3s induced by ICP4. 93 viii

10 16 Assay for early and late proteins translated from mrnas induced by ICP Induction of gb and TK gene expression at 39 0 and 32 0 cells containing ts ICP Induction of the TK gene at 39 0 and 32 0 in cells containing ts ICP4: TK mrna levels in LTA and K4 cells infected with AdTK4 110 ix

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