The Mitochondrial Short Circuit: Regulation of Bax- and Bak-Mediated Cell Death By Murine Cytomegalovirus Open Access
Crosby, Lynsey Nicole (2013)
Abstract
Cell death is a host defense strategy, limiting viral pathogenesis. Activation of cell death can be initiated extrinsically at the plasma membrane or from intrinsic stress signals, leading to apoptosis or necrosis. Cytomegaloviruses (CMV), members of Herpesviridae, efficiently subvert both death pathways, facilitating lifelong persistence within the host. In particular, CMVs encode conserved suppressors that localize to the mitochondria and subvert cell death signals transmitted through this organelle. Stress signals are relayed to the mitochondrial membrane via members of the B cell lymphoma 2 (Bcl-2) family of proteins. Effector Bcl-2 proteins, Bax and Bak, simultaneously permeabilize the mitochondrial membrane upon activation, allowing for the release of proapoptotic proteins that drive apoptosis. Human CMV (HCMV), an opportunistic pathogen and the leading infectious cause of congenital disease, and murine CMV (MCMV), an important model for HCMV, subvert mitochondrial cell death by regulating Bax and Bak. HCMV encodes a single inhibitor, the viral mitochondrial inhibitor of apoptosis (vMIA), from UL37 exon 1 that blocks activation of both Bax and Bak. MCMV, on the other hand, encodes separate inhibitors of Bax and Bak from m38.5, encoding vMIA, and m41.1, encoding the viral inhibitor of Bak oligomerization (vIBO), respectively. Individually, vIBO promotes efficient replication and dissemination in the host, similar to m38.5 (vMIA); however, the combined impact of these two proteins is greater than their individual contributions. Mutant viruses lacking the ability to suppress both Bax and Bak were largely attenuated in terms of replication in macrophages as well as during replication and dissemination in the host, revealing an essential contribution of mitochondrial cell death in limiting pathogenesis. Individual disruption of upstream Bim, Bid, or PUMA did not reveal a single direct activator of Bax and Bak during infection of macrophages; however, elimination of Bid-dependent pathways throughout the host did provide a modest benefit to viral replication. Thus, Bid may activate Bax and Bak in certain cell types infected throughout the host. All together, these results demonstrate that mitochondrial cell death is a potent antiviral host defense mechanism and viral proteins subverting Bax and Bak, such as HCMV vMIA, are crucial to replication and pathogenesis.
Table of Contents
Chapter 1: Introduction ........................................................................................................... 1
1.I. Cytomegalovirus Subversion of Mitochondrial Cell Death..................................................... 2
1.I.i. General Mechanisms of Host Defense and the Intersection with Cytomegaloviruses...... 2
1.I.ii. Human Cytomegalovirus, A Member of Herpesviridae.............................................. 3
1.I.iii. Clinical Impact of Human Cytomegalovirus............................................................ 4
1.I.iv. Murine Cytomegalovirus as a Model for Human Cytomegalovirus.............................. 6
1.I.v. Murine Cytomegalovirus Replication and Pathogenesis............................................. 7
1.I.vi. Cytomegalovirus Suppression of Apoptosis and Necrosis......................................... 9
1.I.vii. Communicating Death Signals to the Mitochondria............................................... 13
1.I.viii. Viral Suppression of Mitochondrial Cell Death..................................................... 15
1.I.ix. Cytomegalovirus Suppression of Mitochondrial Cell Death...................................... 17
1.II. Figures (1.1-1.8), Tables (1.1-1.2), and Legends............................................................ 23
Chapter 2: Gene Products of the Embedded m41/m41.1 Locus of Murine Cytomegalovirus
Differentially Influence Replication and Pathogenesis ........................................................... 34
2.I. Abstract..................................................................................................................... 35
2.II. Introduction.............................................................................................................. 36
2.III. Results.................................................................................................................... 40
2.IV. Discussion................................................................................................................ 49
2.V. Materials and Methods................................................................................................. 53
2.VI. Figures and Figure Legends 2.1-2.6............................................................................. 58
2.VII. References.............................................................................................................. 67
Chapter 3: Inhibition of Bax- and Bak-Mediated Cell Death by Murine Cytomegalovirus is
Crucial to Viral Replication and Pathogenesis......................................................................... 71
3.I. Abstract.................................................................................................................... 72
3.II. Introduction............................................................................................................. 73
3.III. Results................................................................................................................... 78
3.IV. Discussion............................................................................................................... 87
3.V. Materials and Methods................................................................................................ 91
3.VI. Figures and Figure Legends 3.1-3.5............................................................................ 96
3.VII. References............................................................................................................. 103
Chapter 4: Conclusions and Future Directions ...................................................................... 108
4.I. Cytomegalovirus Subversion of Mitochondrial Cell Death................................................. 109
4.I.i. General Overview............................................................................................ 109
4.I.ii. Cell Death Suppression in Monocytic Cell Populations........................................... 110
4.I.iii. Bax and Bak Activation During Murine Cytomegalovirus Infection.......................... 113
4.I.iv. Relating Murine Cytomegalovirus vMIA/vIBO Back to Human
Cytomegalovirus vMIA.............................................................................................. 115
4.I.v. Golgi-Localized m41: A Possible Role in Conjunction with vIBO.............................. 122
4.II. Figures and Figure Legends 4.1-4.8............................................................................. 126
References............................................................................................................................ 134
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