Who is killing whom? Hantaviruses vs programmed cell death
Hantaviruses belong to the Bunyaviridae family of negative stranded RNA viruses. They carry a tri segmented genome and consist of four structural proteins. The four structural proteins are two glycoproteins Gn and Gc, a nucleocapsid (N) protein and an RNA dependent RNA polymerase. An additional nonstructural protein can be expressed by some hantaviruses. Hantavirus infection cause two severe diseases in humans with potential deadly outcome, namely hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) One main target for hantavirus infection is the endothelial cells and vascular leakage is a hallmark for both HFRS and HCPS. In patients strong cytotoxic lymphocyte responses are seen. Cytotoxic lymphocytes, such as natural killer (NK) cells and cytotoxic T lymphocytes, cause apoptosis in virus infected cells via the cytotoxic granule pathway or the death receptor pathway. The cytotoxic granule pathway uses granzyme B to facilitate programmed cell death (PCD) in the target. The death receptor pathway uses death ligands, among them tumor necrosis factor related apoptosis inducing ligand (TRAIL) binds to death receptor (DR) 4 or 5, to induce PCD. One potential mechanism regarding hantavirus pathogenesis might be killing of infected endothelial cells by cytotoxic lymphocytes, thus causing leakage of the endothelium. This is contradicted by findings showing that in patient autopsy, hantavirus infected cells are intact. The aim of this PhD thesis is to give a possible explanation of this dichotomy and to better understand hantavirus pathogenesis. The first part of this thesis (paper I and II) shows that hantavirus infection protects cells from cytotoxic lymphocytes via inhibiting granzyme B activity and by down regulating DR5 from the cell surface. Granzyme B and caspase 3, enzymes needed for apoptosis (a type of PCD) both interacts with hantavirus N protein, and they are both inhibited by the N protein. Further, hantavirus infection of primary endothelial cells causes miss localization of DR5. In infected cells DR5 is found in the nucleus instead of on the cell surface. Taken together, hantavirus infection blocks the two major pathways used by cytotoxic lymphocytes to induce cell death, suggesting that hantavirus pathogenesis is not due to killing of infected cells by cytotoxic lymphocytes. The last part of this thesis (paper III) focuses on hantavirus activated NK cell mediated killing of uninfected endothelial cells. NK cells co incubated with hantavirus infected endothelial cells are activated. This activation is contact dependent and was attributed to IL 15 and IL 15Ra expression on hantavirus infected cells' surface. Interestingly, these activated NK cells induce cell death in uninfected cells with normal HLA class I levels, indicating that hantavirus might cause NK cell mediated killing of uninfected bystander cells. Taken together, the papers I, II and III included in this thesis shows that hantavirusinfection protects cells from cytotoxic lymphocyte mediated killing, while infected cells can cause NK cell activation and possibly subsequent NK cell killing of uninfected cells. LIST OF SCIENTIFIC PAPERS I. Gupta S, Braun M, Tischler ND, Stoltz M, Sundstrom KB, Bjorkstrom NK, Ljunggren HG, Klingstrom J. Hantavirus infection Confers Resistance to Lymphocyte Mediated Apoptosis. PLoS Pathogens, 2013 9(3):e1003272. II. Gupta S, Sola Riera C, Braun M, Bjorkstrom NK, Ljunggren HG, Klingstrom J. Hantavirus Causes Nuclear Translocation of Death Receptor 5 and Protects Infected Cells from TRAIL Induced Apoptosis. Manuscript. III. Braun M, Bjorkstrom NK, Gupta S, Sundstrom K, Ahlm C, Klingstrom J, Ljunggren HG. NK Cell Activation in Human Hantavirus Infection Explained by Virus Induced IL 15/IL 15Ra Expression. PLoS Pathogens, 2014 20;10(11):e1004521. CONTENTS INTRODUCTION 1 ABOUT HANTAVIRUSES 1 The history 1 Hosts and distribution 2 Diseases 3 Structure 3 Hantavirus proteins. 4 Life cycle 6 IMMUNOLOGY AND HANTAVIRUSES 7 Pattern recognition receptors 7 Nuclear factor kappa light chain enhancer of activated B cells 8 Interferons 8 Dendritic cells 9 Natural killer cells 10 Cytotoxic T Lymphocytes 11 PROGRAMMED CELL DEATH 12 Apoptosis 12 Intrinsic apoptosis 13 Extrinsic apoptosis 13 Caspases 13 Viruses and apoptosis 14 Granzyme B mediated cell death 15 TRAIL induced cell death 16 HANTAVIRUS PATHOGENESIS 19 AIM. 21 RESULTS AND DISCUSSION 23 Inhibition of chemically induced apoptosis 23 Inhibition of cytotoxic granule mediated apoptosis 24 Inhibition of a death ligand/receptor pathway 26 Aberrant localization of death receptor 5 27 NK cells activated by hantaviruses 27 Activation through interleukin 15. 29 An unexpected function of hantavirus activated NK cells 29 CONCLUSIONS 32 ACKNOWLEDGEMENTS 35 REFERENCES 37 LIST OF ABBREVIATIONS ACD Accidental cell death ANDV Apaf 1 Bcl 2 Bid Andes virus Apoptotic protease activating factor 1 B cell leukemia/lymphoma 2 BH3 interacting domain death agonist CD Cluster of differentiation CMV Cytomegalovirus CrmA CTL DAXX DC DcR1 DcR2 DISC DOBV DR4 DR5 ER FADD HCPS HFRS HLA Cytokine response modifier A protein Cytotoxic T lymphocyte Death domain associated protein 6 Dendritic cell Decoy receptor 1 (also known as TRAIL receptor 3) Decoy receptor 2 (also known as TRAIL receptor 4) Death inducing signaling complex Dobrava virus Death receptor 4 (also known as TRAIL receptor 1) Death receptor 5 (also known as TRAIL receptor 2) Endoplasmic reticulum Fas associated death domain Hantavirus cardio pulmonary syndrome Hemorrhagic fever with renal syndrome Human Leukocyte Antigen HTNV IFN IL MDA 5 MOMP mRNA N Hantaan virus Interferon Interleukin Melanoma differentiation associated gene 5 Mitochondrial outer membrane permeability Messenger ribonucleic acid Nucleocapsid NFkB NK NLR PAMP PCD PHV PRR PUUV RCD RIG I RLR RNA STAT SNV STS tBid TLR TNF TRAF TRAIL TULV VEGF Nuclear factor kappa light chain enhancer of activated B cell Natural Killer Nucleotide binding oligomerization domain receptors Pathogen associated molecular pattern Programmed cell death Prospect Hill virus Pattern recognition receptor Puumala virus Regulated cell death Retinoic acid inducible gene I RIG I like receptor Ribonucleic acid Signal transducer and activator of transcription Sin Nombre virus Staurosporine Truncated Bid Toll like receptor Tumor necrosis factor TNF receptor associated factor TNF related apoptosis inducing ligand Tula virus Vascular endothelial growth factor