The role of ORF3 protein in the molecular pathogenesis of porcine circovirus 2 infection

THE ROLE OF ORF3 PROTEIN IN THE MOLECULAR PATHOGENESIS OF PORCINE CIRCOVIRUS INFECTION ANBU KUMAR KARUPPANNAN NATIONAL UNIVERSITY OF SINGAPORE 2011 THE ROLE OF ORF3 PROTEIN IN THE MOLECULAR PATHOGENESIS OF PORCINE CIRCOVIRUS INFECTION ANBU KUMAR KARUPPANNAN (B.V.Sc., Madras Veterinary College, India, M.Sc., University of Kentucky, USA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 2011 TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE i ACKNOWLEDGEMENTS I am most thankful to my supervisor Professor Jimmy Kwang for providing me the precious opportunity to work in his lab, his valuable guidance and support throughout my stay in his laboratory. His wide knowledge, experience and interesting ideas have always amazed, educated and motivated me. His constant encouragement has always made me confident and has been a guiding beacon towards the goals of my work. I would like to thank my thesis committee, Dr. Vincent Chow, Dr. Cai Yu and Dr Toshiro Ito for their valuable comments and suggestions. Their diverse backgrounds and guidance has led my work in the proper direction. I also express my sincere thanks to all the past and current members of the Animal health biotechnology lab, especially, Dr. Liu Jue, Jennifer Lau, Zhu Yu, Jia Qiang, Dr. He Fang, Sumathy, Dr. Beau Fenner, Meng Tao, Dr. Syed Musthaq, TLL animal facility, TLL microscopy unit for their help, technical inputs and support in various aspects. All of them were always there when I needed help and support. I would like to express my appreciation to Song Yu, Kian Hong, Peck Junwei, and Reetu for stimulating scientific discussions and friendship. I also thank my family, especially my wife, for enduring me during the course of my study. Above all, I thank the Temasek Life Sciences Laboratory and National University of Singapore for providing me the opportunity and privilege of this education and training. ii TABLE OF CONTENTS TITLE…………………………………………………………………………… i ACKNOWLEDGEMENTS…………………………………………………… . ii TABLE OF CONTENTS……………………………………………………… . iii SUMMARY………………………………………………………………………. vii LIST OF TABLES……………………………………………………………… ix LIST OF FIGURES……………………………………………………………… x LIST OF SYMBOLS AND ABBREVIATIONS………………………………. xii LIST OF PUBLICATIONS…………………………………………………… . xiv Chapter1: INTRODUCTION…………………………… .…………… . 1.1 Introduction…………………………………………………………. 1.2 PCV2 associated disease conditions (PCVAD) …………………… 1.3 Morphology and Replication cycle………………………………… 1.4 Transcriptome and proteome of PCV……………………………… 1.5 Morphogenesis ……………………………………………………. 1.6 Epidemiological history of porcine circovirus infections………… 1.7 Evolutionary aspects of circoviruses……………………………… 1.8 Transmission of the virus…………………………………………… 1.9 Model of the PCVAD development………………………………… 2 10 14 15 16 18 18 1.10 Host Virus interaction………………………………………………. 20 1.11 Thesis outline……………………………………………………… 24 Chapter 2: Ablation of ORF3 expression from porcine circovirus leads to the attenuation of its pathogenicity in SPF piglets …………… 2.1 Introduction………………………………………………………… 2.2 Materials and methods………………………………………………. 26 27 29 iii 2.2.1 Viruses and cell culture……………………………………… 29 2.2.2 Generation of Mutant viruses and their characterization.…… 29 2.2.3 Yeast two hybrid assays…………………………………… 30 2.2.4 Antibodies and recombinant proteins………………………. 30 2.2.5 Experimental design………………………………………… 30 2.2.6 Serological analysis…………………………………………. 32 2.2.7 Quantitative real time PCR…………………………………. 32 2.2.8 Histology…………………………………………………… 33 2.2.9 Flow cytometry……………………………………………… 2.3 34 Results……………………………………………………………… 35 2.3.1 Mutations and genetic stability of the virus.………………… 35 2.3.2 Molecular interaction between ORF3 and Pirh2…………… 39 2.3.3 Characterization of the double mutant virus in vivo………… 43 2.3.4 Serum viremia and virus specific antibody response……… 43 2.3.5 Lymphocyte counts………………………………………… 48 2.3.6 Histological findings………………………………………… 2.4 50 Discussion…………………………………………………………… 54 Chapter 3: Porcine circovirus type ORF3 protein competes with P53 in binding to Pirh2 and mediates the deregulation of P53 homeostasis… 3.1 Introduction………………………………………………………… 3.2 Materials and methods……………………………………………… 60 61 65 3.2.1 Cell culture and transient transfections………………………. 65 3.2.2 Plasmids and recombinant proteins…………………………… 65 3.2.3 Binding assays……………………………………………… 66 3.2.4 Co-immunoprecipitation assays……………………………… 67 iv 3.2.5 Immunofluorescence assays…………………………………. 68 3.2.6 Estimation of protein turnover rates………………………… 68 3.2.7 MTT assay for cell viability………………………………… 69 3.2.8 Flowcytometry……………………………………………… 69 3.2.9 In vitro ubiquitination assay………………………………… 3.3 70 Results………………………………………………………………… 71 3.3.1 The ORF3 protein prevents p53 from binding pPirh2 in vitro and in vivo…………………………………………………… 3.3.2 ORF3 protein alters the subcellular localization of pPirh2…. 71 76 3.3.3 Mapping of the minimal domain of ORF3 protein that binds with pPirh2………………………………………………… . 3.3.4 Interaction of ORF3 protein with pPirh2 up-regulates cellular p53 levels…………………………………………………… 3.3.5 ORF3 interferes with the in vitro ubiquitination of p53…… 3.4 80 86 88 Discussion……………………………………………………………. 90 Chapter 4: ORF3 of porcine circovirus enhances the in vitro and in vivo spread of the virus .……………………………………… 4.1 Introduction………………………………………………………… 4.2 94 95 Materials and methods……………………………………………… 98 4.2.1 Cell culture and viruses………………………………………. 98 4.2.2 Quantitative real-time PCR………………………………… 99 4.2.3 Plasmids and transfection……………………………………. 99 4.2.4 Western blot analysis………………………………………… 99 4.2.5 Assay for caspase activity……………………………………. 100 4.2.6 Mice infections studies………………………………………. 4.3 100 Results……………………………………………………………… 103 4.3.1 Growth kinetics of wild-type PCV2 and ORF3-deficient PCV2 …………………………………………………… … 4.3.2 Role of ORF3-induced apoptosis in the spread of the virus in cell culture……………………………………………………. 103 106 v 4.3.3 Mixed culture of ORF3-deficient PCV2 with a chimeric PCV1-2 virus………………………………………………… 4.3.4 Role of ORF3-induced apoptosis in the in vivo spread of the virus………………………………………………………… . 4.3.5 Role of macrophages in the spread of PCV2 viremia……… 4.4 109 112 114 Discussion…………………………………………………………… 118 Conclusion…………………………………………………………………… 123 5.1 The role of ORF3 in the pathogenicity of PCV2 infection and the molecular mechanism behind the cellular pathogenesis…………… . 124 Future directions……………………………………………………… 129 REFERENCES…………………………………………………………………. 131 5.2 vi SUMMARY Porcine circovirus (PCV2) of the Circoviridae family is a nonenveloped, single stranded DNA virus with a circular genome of 1.7 kilobases. It is a major pathogen of porcine species causing growth retardation, lymphadenopathy, multiorgan inflammation and immune suppression, especially affecting weanling piglets. The PCV2 open reading frame (ORF3) codes a 104 amino acid protein that causes apoptosis of PCV2 infected cells, and is not essential for virus replication. This thesis describes the characterisation of the role of ORF3 in the molecular and the systemic pathogenesis during the PCV2 infection in cell culture, mice model and in natural infection in piglets. Mutant PCV2 lacking the expression of ORF3 are infectious and replicate in cells in vitro, but not cause apoptosis of the infected cells. The ORF3 of PCV2 has been shown to be involved in the pathogenesis of the virus in mice model. In PCV2 infected piglets, B and CD4 T lymphocyte depletion and lymphoid organ destruction are generally observed; however, the ORF3 deficient PCV2 is attenuated in its pathogenicity in infected piglets. The mutant virus does not cause any observable disease or perturbation of the lymphocyte count in the inoculated piglets and elicits an efficient immune response. When compared with the wildtype virus infection, the ORF3 mutant PCV2 infection is characterized by mild viremia and absence of pathological lesions. In infected cells, the ORF3 protein interacts with the porcine homologue of Pirh2 (pPirh2), a p53-induced ubiquitin-protein E3 ligase and causes the accumulation of p53 by disrupting the physiological association of p53 and pPirh2. The ORF3 protein competes with p53 in binding to pPirh2. The amino vii acid residues 20 to 65 of the ORF3 protein are essential in this interaction of ORF3 protein with pPirh2, which leads to an alteration in the cellular localization and a significant reduction in the stability of pPirh2. These events contribute to the deregulation of p53 by pPirh2, leading to increased p53 levels and apoptosis of the infected cells. In addition to its role in causing the apoptosis of the immune cells, characteristic of the PCV2 infection associated disease conditions, the ORF3 also plays a role in the systemic dissemination of the PCV2 infection. The ORF3 expedites the spread of the virus by inducing the early release of the virus from the infected cells. Further, in PCV2 infected mice, the ORF3 induced apoptosis also aids in recruiting macrophages to phagocytise the infected apoptotic cells leading to the systemic dissemination of the infection. The apoptotic activity of the ORF3 of PCV2 hence lends advantage to the spread of the virus. viii List of Tables Table 1. Functional domains of ORF1……………… ………………………….11 Table 2. List of cellular proteins reported to interact with PCV2 proteins… … 23 Table 3. List of ORF3 mutations in the mutant PCV2………………… …….…37 Table 4. 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Virology 87, 500– 512. 161 [...]... status of herds, this study also reveals that certain aminoacids variations in ORF3 are also associated with PMWS in the herds Especially the positions 14, 29 , 41 and 1 02 of the ORF3 varied between the PCV2a and PCV2b groups This study lends clue to the role of the ORF3 protein in the pathogenesis of PCV2 Yet another interesting association with regard to the polymorphisms in ORF2 is the presence of the. .. proteins involved in the replication of the virus (Replicase proteins; Rep), of which the Rep and Rep’ are essential for the replication of the virus (Fig.5 & 6) (Steinfeldt et al., 20 01) The ratio of the Rep and the Rep’ transcripts and proteins are found to vary during the course of infection of the PCV1 and PCV2 virus, with a transient increase of the Rep’ compared to the Rep (Mankertz et al., 20 04... on the growth kinetics of PCV2 108 Figure 26 Role of ORF3 in the release of PCV2 from infected cells……… …111 Figure 27 Role of ORF3 induced apoptosis on the In Vivo spread of PCV… 113 Figure 28 Role of Macrophages in the In Vivo spread of PCV2… ………… 116 Figure 29 Induction of TNFα expression in Macrophages by ORF3 …… 117 xi List of Symbols and Abbreviations CD – Cluster of Differentiation CMV- Cytomegalovirus... for the Rep proteins The numbering of the nucleotide begins in the site of the “nicking” by Rep proteins and is followed as a convention in the field 9 1.4 Transcriptome and proteome of PCV The transcriptome of PCV1 and PCV2 have been thoroughly analysed in a series of studies using 5’ and 3’ RACE (Rapid amplification of cDNA ends) cloning (Cheung A K., 20 03 a, b) The transcription profiling of the. .. genome, and the ORF2 in the antisense strand, which codes for the capsid protein A third ORF, the ORF3, is ensconced in the antisense strand of the ORF1 The replication of the genome takes place in the nucleus The genome has a stem-loop forming sequence at the origin of replication, in between the two genes, ORF1 and ORF2, and a short intergenic region is present between the end of the ORFs (Fig 4) The stemloop... infection. ……….……………………… 52 Figure 15 InVitro Competetive binding assay: ORF3, p53, Pirh2………… … 73 Figure 16 InVivo Competetive binding: ORF3, p53, Pirh2……………… ……74 Figure 17 Effect of ORF3 protein on the subcellular-localization of pPirh2 ….77 Figure 18 Effect of ORF3 protein on the turnover of Pirh2…………… …… 78 Figure 19 Apoptosis induction by truncated and deletion mutants of ORF3 ….81 Figure 20 In Vitro and In Vivo... Particles xiii List of publications 1 Karuppannan AK, Kwang J ORF3 of porcine circovirus 2 enhances the in vitro and in vivo spread of the virus Virology 20 11 Feb 5;410(1) :24 8-56 2 Meng T, Jia Q, Liu S, Karuppannan AK, Chang CC, Kwang J Characterization and epitope mapping of monoclonal antibodies recognizing Nterminus of Rep of porcine circovirus type 2 J Virol Methods 20 10 May;165 (2) :22 2-9 3 Karuppannan... avian species, e.g Porcine circovirus, Bovine circovirus, Beak and feather disease virus, Pigeon circovirus, Goose circovirus, Canary circovirus, Starling circovirus, Finch circovirus, e.t.c (Firth et al., 20 09) To date there are two reported circoviruses that can infect porcine species, namely, the Porcine circovirus 1 (PCV1) and Porcine circovirus 2 (PCV2) The non-pathogenic PCV1 was initially identified... other hand, the PCV2 has been classified into many phylogroups based on the polymorphisms in the ORF2 gene coding for the capsid proteins (Cheung et al., 20 07, Firth et al., 20 10, Hughes et al., 20 08) Of these, the two major phylogroups are the PCV2a and PCV2b Cheung et al., (20 07) has defined short amino acid motifs which could be used to distinguish the PCV2a and PCV2b The motifs located from amino... of ORF3 deletion mutants……… ……83 Figure 21 Induction of cellular p53 by ORF3 mutants…………… ………… 87 Figure 22 Effect of ORF3 on the In Vitro ubiquitination of p53 by pPirh2 … 89 Figure 23 Accumulation kinetics of cell free and cell associated PCV2 virus 104 x Figure 24 Effect of caspase inhibitor zVAD on apoptosis induced by PCV2 107 Figure 25 Effect of caspase inhibitor zVAD on the growth kinetics of . This thesis describes the characterisation of the role of ORF3 in the molecular and the systemic pathogenesis during the PCV2 infection in cell culture, mice model and in natural infection in. PCV2 107 Figure 25 . Effect of caspase inhibitor zVAD on the growth kinetics of PCV2 108 Figure 26 . Role of ORF3 in the release of PCV2 from infected cells……… …111 Figure 27 . Role of ORF3 induced. causing the apoptosis of the immune cells, characteristic of the PCV2 infection associated disease conditions, the ORF3 also plays a role in the systemic dissemination of the PCV2 infection. The