MANIPULATION OF THE CUL4-DCAF1 UBIQUITIN LIGASE BY THE PARALOGOUS PRIMATE LENTIVIRAL ACCESSORY PROTEINS VPR AND VPX by Patrick Cassiday A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Department of Pathology The University of Utah December 2014 Copyright © Patrick Cassiday 2014 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Patrick Cassiday has been approved by the following supervisory committee members: Vicente Planelles , Chair 7/31/2014 Date Approved Diane M. Ward , Member 7/31/2014 Date Approved Roland D. Tantin , Member 7/31/2014 Date Approved Kael F. Fischer , Member 7/31/2014 Date Approved Adam M. Spivak , Member 7/31/2014 Date Approved and by Peter Jensen , Chair/Dean of the Department/College/School of Pathology and by David B. Kieda, Dean of The Graduate School. ABSTRACT Upon infecting a host, viruses immediately face restriction by the host immune system, including innate and adaptive responses. To mediate efficient replication, viruses have evolved a number of mechanisms to subvert and bypass the host immune responses. Among the earliest immune protections encountered by viruses are a group of cell- intrinsic immunity proteins called “restriction factors.” Restriction factors have been identified that, if not counteracted, are capable of inhibiting viral replication throughout the viral life cycle. One of the primary mechanisms utilized to counteract these restrictions is the manipulation of the cellular ubiquitin ligase system to induce the directed and specific degradation of these cellular factors. In the case of primate lentiviruses (HIVs and SIVs), four proteins (Vif, Vpu, Vpr, and Vpx) have been shown to alter the specificity of this cellular degradation machinery to target restriction factors. In this study, we explore the molecular interaction between the paralogous proteins Vpr (encoded by all primate lentiviruses) and Vpx (encoded by HIV-2 and some SIVs), the cellular ubiquitin ligase composed of Cul4-Roc1-DDB1- DCAF1 and the restriction factors they target for degradation (Mus81 in the case of Vpr and SAMHD1 in the case of Vpx). Through mutation of DCAF1, the substrate specificity factor for the ubiquitin ligase complex, to which Vpr and Vpx are known to directly interact, we show that although they share a high degree of homology, Vpr and Vpx interact with DCAF1 differently. In addition, through the generation of chimeric Vpr- Vpx proteins, we explore the molecular determinants of Vpr and Vpx substrate specificity. To this end, we demonstrate that manipulation of Cul4-DCAF1 substrate specificity by Vpr and Vpx is mediated by nonlinear determinants within the respective proteins, in contrast to previously proposed models. Finally, we demonstrate that Vpr induces the degradation of Mus81 in a manner independent of the induction of G2 arrest, in contrast to recent reports. iv Dedicated to those who came before & those who will come after. TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii LIST OF FIGURES ....................................................................................................................... vii ACKNOWLEDGEMENTS ............................................................................................................ ix CHAPTERS 1. INTRODUCTION ........................................................................................................................ 1 Cell Intrinsic Restriction of Viral Replication .............................................................................. 1 Vpr and Vpx: HIV/SIV’s Fraternal Twins ................................................................................... 3 Evolutionary Perspective on Vpr and Vpx Function .................................................................. 14 Molecular Insights into the Manipulation of the Cul4-DCAF1 Ligase by Vpr and Vpx ........... 18 References ................................................................................................................................... 30 2. UNDERSTANDING THE MOLECULAR MANIPULATION OF THE CUL4-DDB1-DCAF1 UBIQUITIN LIGASE BY THE PARALOGOUS LENTIVRIAL ACCESSORY PROTEINS VPR AND VPX .............................................................................................................................. 42 Abstract ....................................................................................................................................... 42 Introduction ................................................................................................................................. 43 Materials and Methods ................................................................................................................ 45 Results ......................................................................................................................................... 48 Discussion ................................................................................................................................... 56 References ................................................................................................................................... 70 3. ANALYSIS OF THE STRUCTURAL DETERMINANTS OF G2 ARREST AND SAMHD1 DEGRADATION BY THE SIV(AGM) VPR ................................................................................ 76 Abstract ....................................................................................................................................... 76 Introduction ................................................................................................................................. 77 Materials and Methods ................................................................................................................ 79 Results ......................................................................................................................................... 82 Discussion ................................................................................................................................... 89 References ................................................................................................................................. 102 4. DISCUSSION ........................................................................................................................... 106 Future Perspectives ................................................................................................................... 110 References ................................................................................................................................. 114 LIST OF FIGURES 1.1: Overview of Vpr/Vpx phylogeny ............................................................................................ 27 1.2: Mechanisms of viral manipulation of cellular ubiquitin ligases .............................................. 28 1.3: Alignment of Vpr from HIV-1 and SIVagm and Vpx of SIVmac .......................................... 29 2.1 DCAF1 domain architecture ..................................................................................................... 59 2.2: DCAF1 truncations DDB1 co-IP ............................................................................................. 60 2.3: DCAF1 mutation strategy ........................................................................................................ 62 2.4: Analysis of DCAF1 mutations for their ability to facilitate Vpx-mediated SAMHD1 degradation ..................................................................................................................................... 63 2.5: Analysis of DCAF1 truncations and point mutant ability to interact with Vpx ...................... 64 2.6: DCAF1 determinants of Vpr interaction are different than those of Vpx ............................... 65 2.7: DCAF1 WD40 mutants inhibit Vpr induced G2 arrest ........................................................... 66 2.8: Vpx fails to compete Vpr mediated G2 arrest ......................................................................... 67 2.9: Vpr functions dominantly to Vpx ............................................................................................ 68 2.10: Effect of Vpr and Vpx expression of the DCAF1-DDB1 interaction ................................... 69 3.1 HIV-1 Vpr C-terminal unstructured terminus is necessary ...................................................... 92 3.2: Alignment of Vpr from HIV-1 and SIVagm and Vpx of SIVmac .......................................... 93 3.3 Schematic of HIV-1 Vpr/SIVmac Vpx chimeras ..................................................................... 94 3.4 Cell cycle analysis of HIV-1 Vpr/SIVmac Vpx chimeras ........................................................ 95 3.5 Schematic of HIV-1 Vpr/SIVagm Vpr chimeras ...................................................................... 96 3.6 Cell cycle analysis of HIV-1 Vpr/SIVagm Vpr chimeras ........................................................ 97 3.7 Analysis of SAMHD1 degradative ability of HIV-1 Vpr/SIVagm Vpr chimeras .................... 98 3.8 Vpr induces the degradation of Mus81 ..................................................................................... 99 3.9 The G2 arrest defective