FUNCTION STUDIES ON RING PROTEINS IN WHITE SPOT SYNDROME VIRUS FANG HE NATIONAL UNIVERSITY OF SINGAPORE 2009 FUNCTION STUDIES ON RING PROTEINS IN WHITE SPOT SYNDROME VIRUS FANG HE (B.Sc. Shanghai Jiao Tong University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY AND DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 Abstract White Spot Syndrome Virus, Nimaviridiae Whispovirus, is one of the major viral pathogens in the aquaculture industry responsible for high mortality in cultured shrimp. The infection mechanisms of WSSV have not been fully characterized at the molecular level due to the large size and uniqueness of its genome. This study was undertaken to advance our understanding of the specific function of RING-containing proteins in viral pathogenesis. A preliminary search for regulatory protein candidates in WSSV using functional domain determination identified four predicted viral proteins containing a RING-H2 domain. Among them, the three proteins WSSV222, WSSV249 and WSSV403 can be expressed in both E.coli and insect cells, suggesting their potential expression in shrimp. In this study, emphasis has been placed on the characterization of WSSV222 and WSSV403. WSSV222 exhibits RING-H2-dependent E3 ligase activity in vitro in the presence of the conjugating enzyme UbcH6. Mutations in the RING-H2 domain abolished WSSV222-dependent ubiquitination, displaying the importance of this domain. Yeast two-hybrid and pull-down analyses revealed that WSSV222 interacts with a shrimp tumor suppressor-like protein (TSL) sharing 60% identity with human OVCA1. A stable TSL-expressing cell line derived from the human ovarian cancer cell line A2780 was established, where a TSL-dependent prolonged G1 phase was observed. Based on this, WSSV222-mediated ubiquitination and MG132-sensitive degradation of TSL were detected both in the TSL-expressing cell line and in shrimp primary cell culture. Transient expression of TSL in BHK cells leads to apoptosis, which was rescued by the coexpression of WSSV222. Taken together, I these data suggest that WSSV222 acts as an anti-apoptosis protein by ubiquitinmediated proteolysis of TSL to ensure successful WSSV replication in shrimp. Overexpression of WSSV222 in SF9 and BHK cells could be silenced by specific anti-WSSV222 siRNA. Further, WSSV-challenged shrimp were treated with the anti-222 siRNA to knockdown WSSV222. The survival rate and the efficiency of WSSV replication were assessed to evaluate the efficacy of anti-222 siRNA to inhibit WSSV infection in shrimp. The anti-222 siRNA reduced the cumulative mortality in shrimp challenged with 103 copies of WSSV and delayed the mean time to death in shrimp challenged with the higher dosage of 106 copies. The results of real time quantitative PCR showed that virus replication was delayed and reduced in the WSSV-challenged shrimp treated with anti-222 siRNA in comparison to the challenged shrimp treated with random siRNA. Coimmunoprecipitation assays revealed that WSSV222 silencing inhibited the degradation of TSL in WSSV-challenged shrimp. These results indicate that WSSV222 is required for efficient replication of WSSV in shrimp. WSSV403 acts as a viral E3 ligase which can ubiquitinate itself in vitro in the presence of an E2 conjugating enzyme from shrimp. WSSV403 can be activated by a series of E2 variants. In RT-PCR and real time PCR, the transcription of WSSV403 was detected in specific-pathogen-free shrimp, suggesting its role as a latency-associated gene. Identified in yeast two-hybrid and verified by pull-down assays, WSSV403 is able to bind to a shrimp protein phosphatase, an interaction partner for another latent protein WSSV427. This study suggests that WSSV403 could be a regulator of latency state of WSSV by virtue of its E3 ligase function. II In summary, the studies presented here indicate that viral RING proteins are involved in ubiquitination events and interactions with a diverse range of shrimp proteins and play important roles as regulators of virus replication. In order to establish an effcient viral protein expression system, efforts have been made in the studies on WSSV immediate-early promoter one (IE1). The production of H5 HA of influenza virus by baculovirus was enhanced with WSSV IE1 promoter, especially compared with CMV promoter. This contributed to effective elicitation of HA-specific antibody in vaccinated chickens. This study provides an alternative choice for baculovirus based vaccine production. III Table of Contents List of Figures List of Table Acknowledgements VII VIII IX Chapter Introduction 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 WSSV AND ITS HOST RANGE…………………………………………………………………. PATHOLOGY AND TISSUE TROPISM OF WSSV…………………………………………… WSSV GENOME AND CLASSIFICATION…………………………………………………… MORPHOLOGY AND STRUCTURAL PROTEINS OF WSSV……………………… … . 10 NON-STRUCTURAL PROTEINS IN WSSV…………………………………………………. 12 VACCINE STRATEGIES FOR CONTROL OF WSSV INFECTION .…………………… 15 UBIQUITINATION IN VIRUS INFECTION…………………………………………………… 20 VIRUS-RELATED APOPTOSIS IN HOST CELLS…………………………………………. 21 RING-CONTAINING PROTEINS IN WSSV…………………………………………………… 22 RESEARCH OUTLINE AND OBJECTIVES………………………………………………… .23 Chapter WSSV222 encodes a viral E3 ligase and mediates degradation of a host tumor suppressor via ubiquitination 2.1 2.2 2.2.1 2.2.2 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 2.4 26 INTRODUCTION………………………………………………………………………………… 27 MATERIALS AND METHODS…………………………………………………………… … 29 RACE PCR, wild type and mutants cloning…………………………………………………….29 Construction of shrimp cDNA library…………………………………………………………….30 Yeast two-hybrid assays………………………………………………………………………….31 Expression, purification of proteins and antibody preparation………………………………. 32 Pull-down assays…………………………………………………………………………………. 33 Cell culture, immunofluorescence and confocal microscopy………………………………… 33 Ubiquitination assays in vitro and in vivo………………………………………………………. 35 DNA Fragmentation Assays…………………………………………………………………… 36 FACS Analysis……………………………………………………………………………………. 36 RESULTS…………………………………………………………………………………………. 37 WSSV222 is a RING-H2 E3 ligase…………………………………………………………… 37 TSL, a shrimp orthologue for OVCA1, is a WSSV222 target……………………………… . 40 WSSV222 interacts with and ubiquitinates TSL in vitro……………………………………… 43 TSL is ubiquitinated for degradation by WSSV222 in vivo………………………………… . 46 TSL is subjected to ubiquitination and degradation in WSSV-infected shrimp cells……… 49 WSSV222 rescues apoptosis induced by transient expression of TSL in BHK cells…… . 51 DISCUSSION………………………………………………………………………………………53 Chapter Viral ubiquitin ligase WSSV222 is required for efficient WSSV replication in shrimp 58 IV 3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.4 INTRODUCTION…………………………………………………………………………………. 59 MATERIALS AND METHODS………………………………………………………………… 61 Synthesis of siRNAs……………………………………………………………………………… 61 Shrimp culture, WSSV infection and siRNA injection………………………………………… 61 In vitro silencing of WSSV222………………………………………………………………… . 62 Reverse transcription PCR and Real time quantitative PCR………………………………… 62 Co-immunoprecipitation and western blot analysis…………………………………………… 63 Fluorimetric assay of caspase activity…………………………………………………………. 64 Statistical analysis……………………………………………………………………………… . 65 RESULTS……………………………………………………………………………………… 66 WSSV222 silencing in cultured cells and WSSV infected shrimps…………………………. 66 WSSV222 silencing delayed death time in WSSV infected shrimp………………………… 70 Delayed and reduced WSSV replication in shrimp with WSSV222 silencing……………… 72 WSSV222 is required for TSL degradation in WSSV infected shrimp……………………… 74 WSSV222 contributes to the regulation on WSSV associated apoptosis in shrimp……….76 DISCUSSION………………………………………………………………………………………78 Chapter Identification and characterization of WSSV403 as a viral E3 ligase involved in virus latency 4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3 4.3.1 4.3.2 4.3.3 4.4 83 INTRODUCTION…………………………………………………………………………………. 84 MATERIALS AND METHODS………………………………………………………………… 86 Reverse transcription PCR and real time PCR……………………………………………… .86 Expression, purification of proteins and antibody preparation………………………………. 86 Pull-down assays…………………………………………………………………………………. 87 Ubiquitination assays in vitro……………………………………………………………………. 87 Yeast two-hybrid assays………………………………………………………………………… 88 RESULTS…………………………………………………………………………………………. 89 WSSV403 is a RING-H2 E3 ligase…………………………………………………………… . 89 WSSV403 is a latency-associated gene……………………………………………………… 91 WSSV403 interacts with shrimp phosphatase………………………………………………… 93 DISCUSSION………………………………………………………………………………………95 Chapter WSSV ie1 promoter is more efficient than CMV promoter to express H5 from influenza virus in baculovirus as a chicken vaccine 98 5.1 5.2 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.3.6 5.3.7 5.3.7 5.3.8 5.4 5.4.1 5.4.2 5.4.3 ABSTRACT………………………………………………………………………………………. 99 INTRODUCTION………………………………………………………………………………. 100 MATERIALS AND METHODS……………………………………………………………… 102 Viruses and cells. … ………………………………………………………………………… 102 Luciferase activity assay………………………………………………………………………. 102 Construction of recombinant baculoviruses…………………………………………………. 103 Recombinant baculovirus purification………………………………………….……………. 104 Animal experiments…………………………………………………………………………… 104 Serological assays…………………………………………….……………………………… 105 Immunofluorescence assays……………………………….………………………………… 106 Immunohistochemistry……………………………….…………… .………………………… 106 Statistical analysis……………………………….……………………………… …………… 107 RESULTS………………………………………………………………………………………. 108 WSSV ie1 promoter mediates efficient protein expression in SF9 cells……………… . 108 WSSV ie1 promoter stimulates strong H5 hemagglutinin expression in baculovirus… . 110 Immunogenicity of H5 hemagglutinin expressed by WSSV ie1 promoter in chickens… 115 V 5.4.4 5.5 Significant antigen expression in chicken tissue by HA-VSVG coexpression constructs 119 DISCUSSION…………………………………………………………………………………… 120 Chapter General Discussion 6.1 6.2 6.2 123 ON THE ROLE OF RING PROTEINS IN WSSV………………………………………… 124 IN THE LIGHT OF NEW FINDINGS…………………………………………………………. 126 THAT WHICH REMAINS……………………………………………………………………… 128 Chapter Bibliography 131 VI List of Figures Figure 1. WSSV222, 249 and 403 contain RING-H2 domains…………………………………… 25 Figure 2. WSSV222 is a RING-containing E3 ligase………………………………………………… .39 Figure 3. Shrimp tumor suppressor-like (TSL) protein is functionally similar to human OVCA1… 42 Figure 4. WSSV222 interacts with & ubiquitinates shrimp tumor-suppressor–like protein in vitro .45 Figure 5. WSSV222 ubiquitinates and mediates degradation on shrimp TSL in vivo…………….…48 Figure 6. TSL is degraded and ubiquitinated in WSSV-infected shrimp cells…………………….… 50 Figure 7. WSSV222 antagonizes TSL-induced apoptosis in BHK cells………………………….… .52 Figure 8. Specific WSSV222 siRNA induces WSSV222 silencing in cultured cells……………… 68 Figure 9. Specific WSSV222 siRNA induces WSSV222 silencing in WSSV challenged shrimp… 69 Figure 10. Efficacy of 222 siRNA in WSSV-challenged shrimp……………………………………… 71 Figure 11. WSSV222 silencing results in the delay and reduction of WSSV gene expression in shrimp challenged with WSSV 73 Figure 12. Co-immunoprecipitation and western blot showed TSL degradation in normal and WSSV-challenged shrimp treated with 20 uM MG132……………………………………………… .75 Figure 13. WSSV222 silencing has effects on cell apoptosis in shrimp during WSSV infection… .77 Figure 14. WSSV403 is a viral E3 ubiquitin ligase 90 Figure 15. Detection of WSSV403 transcript in shrimp……………………………………………… 92 Figure 16. WSSV403 can interact with a shrimp protein phosphatase……………………………… 94 Figure 17. Comparison of promoter activity of WSSV ie1 and CMV promoter in luciferase assays in different cell lines……………………………………………………………………………………… .109 Figure 18. Schematic representation of the construction of variant baculoviruses in the study 113 Figure 19. Efficient production of activated HA protein of influenza virus by WSSV ie1 promoter in baculovirus …………………………………………………………………………………………… .114 Figure 20. Immunogenicity of HA-expressing baculoviruses………………………………… ………118 VII List of Table Table 1. 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RING containing proteins in WSSV 2 Determination of the transcription profile of the viral E3 ligases during WSSV infection 3 Identification and characterization of host/shrimp interaction partners of these viral E3 ligases 4 Functional studies on these viral E3 ligases and their interaction partners during WSSV infection in shrimp 5 Comparative studies on WSSV immediate early promoter 1 in recombinant... al., 2005) 1.9 RING- containing proteins in WSSV A previous study (Freemont, 2000) has revealed that the RING finger domains of E3 ubiquitin ligases are involved in specific ubiquination events It is now recognized that the RING finger domain is present in the largest known class of E3 ubiquitin ligases (Borden, 2000; Yang and Yu, 2003) It has functions involved in cell-cycle control, oncogenesis and... of RING proteins in viral regulation will certainly pave the way for an increased understanding of WSSV and other viral infection mechanisms Therefore, to fully determine the function of RING proteins in WSSV, emphasis in this study is placed on the WSSV222 and WSSV403 proteins and the following research objectives were established: 1 Identification of viral E3 ubiquitin ligase activity of these RING. .. other virus infection models For unique viruses with a large genome like WSSV, it is thus important to reveal its infection mechanism, which might throw a light on new regulatory pathways in virus- host interaction As suggested by previous findings on WSSV RING proteins (Wang et al., 2005), they might play key roles in WSSV pathogenicity and infection machinery Studies focusing on the potential function. .. domains (A) Schematic representation of WSSV222 and 403 proteins by SMART program WSSV222 RING domain is from 308aa to 358 aa , while WSSV403 from 329 aa to 370 aa Pink bars indicated segments of low compositional complexity on WSSV222 Green bars indicated coiled coil regions on WSSV 403 (B) Alignment of the RING portion from RING proteins identified in WSSV These WSSV RING domains are of the C3H2C3 type... pathogenicity by taking a principal role in limiting inflammatory reactions at the site of infection and by inducing specific immunity In contrast, apoptosis enhances HIV-1 pathogenicity by inducing massive cell death in indispensable organs, signaling the onset of disease (Cossarizza, 2008; Velilla et al., 2005) Although replication of most viruses is suppressed by apoptosis of infected cells, certain viruses... in natural populations, persistent viral life strategy has not received much attention Persistence has been defined as the state in which a virus maintains its capacity for either continued or episodic reproduction in an individual host, subsequent to an initial period of productive infection and occurrence of an antiviral host response This definition also includes the condition known as latency in. .. in China from 1993 to 1994 (Cai S., 1995) The virus from the People's Republic of China has also been called Chinese baculovirus (CBV) The virus has also been taxonomically affiliated as: China virus disease, red disease (Alapide-Tendencia E.V., 1997), white 7 spot disease, and white spot baculovirus However, presently the virus is referred to as white spot syndrome virus (WSSV) Virus classification... attenuation of viral pathogenicity in shrimp Despite these findings, there is still no clear conclusion as to 20 the relative contribution of apoptosis to viral pathogenicity and/or host defensive responses in shrimp 1.8 Ubiquitination in virus infection Ubiquitin-mediated proteolysis plays an important role in a variety of basic pathways and processes during cell life and death In the ubiquitin-dependent... protein mediates direct binding with the UEV (ubiquitin E2 variant ) domain in the N-terminus of Tsg101 (tumor susceptibility gene 101), and two E3 ligases have been identified as regulators of HIV budding (Klinger and Schubert, 2005; Li and Wild, 2005) In addition, ubiquitination of APOBEC3G by an HIV-1 Vif-Cullin5-Elongin B-Elongin C complex is essential for Vif (virion infectivity factor) function . understanding of the specific function of RING- containing proteins in viral pathogenesis. A preliminary search for regulatory protein candidates in WSSV using functional domain determination identified. OF WSSV INFECTION …………………… 15 1.7 UBIQUITINATION IN VIRUS INFECTION…………………………………………………… 20 1.8 VIRUS- RELATED APOPTOSIS IN HOST CELLS…………………………………………. 21 1.9 RING- CONTAINING PROTEINS IN WSSV……………………………………………………. ligase function. III In summary, the studies presented here indicate that viral RING proteins are involved in ubiquitination events and interactions with a diverse range of shrimp proteins