Molecular analyses of untranslated regions of hibiscus latent singapore virus

MOLECULAR ANALYSES OF UNTRANSLATED REGIONS OF HIBISCUS LATENT SINGAPORE VIRUS CAO SHISHU (MSc, Huazhong Agri. Uni., China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements I am greatly indebted to my supervisor, Professor Wong Sek Man, for his professional guidance, continuous encouragements and support. I would like to thank my thesis committee members A/P Zhang Lian Hui from Institute of Molecular and Cell Biology and Dr. Song Jianxing from Biochemistry Department for their guidance and support. I would also like to thank Dr. Ichiro Maruyama, former Principal Investigator of Genome Institute of Singapore for his guidance during my study in his lab. My thanks also go to A/P Li Tianhu, Chemistry Department of NUS, who guided me to carry out some of my experiments in his lab. I would like to thank Professor Peter Palukaitis, Scottish Crop Research Institute, U.K., for his suggestions and encouragements during his visit to Singapore. I appreciate the following friends and members in my lab who have helped me a lot during my study: Li Weimin, Meng Chunying, Luo Qiong, Cheng Ao, Lim Chinchin, Yang Jing, Zhang Xin, Srinivasan KG, Pang Junxiong, Vincent, Bak Xiao Ying, Teh Yufen, Sunil KT and all those who have helped me and those I forgot to mention here. Sincere appreciation goes to Mr. Chong Ping Lee for providing necessary items for my research work and those persons in-charge of the DNA sequencing facilities in DBS. Lastly and most importantly, I would like to thank the National University of Singapore for awarding me a research scholarship, which makes my dream to study in Singapore possible. TABLE OF CONTENTS Acknowledgements ----------------------------------------------------------------------------2 Table of contents -----------------------------------------------------------------------------3 List of publications-----------------------------------------------------------------------------9 List of abbreviations ---------------------------------------------------------------------------10 List of Figures-----------------------------------------------------------------------------------13 List of Tables------------------------------------------------------------------------------------15 Summary-----------------------------------------------------------------------------------------16 CHAPTER LITERATURE REVIEW 1.1 Introduction-------------------------------------------------------------------------------------17 1.1.1 Hibiscus latent Singapore virus-----------------------------------------------------------17 1.1.2 The genome organization of HLSV-------------------------------------------------------19 1.2 Roles of viral untranslated regions----------------------------------------------------------20 1.2.1 Function as untrtanslated regions of mRNAs------------------------------------------ -20 1.2.1.1 Regulation of RNA stability-------------------------------------------------------------20 1.2.1.2 Modulation of translational expression-------------------------------------------------22 1.2.1.2.1 Translational control mechanisms--------------------------------------------------- 22 1.2.1.2.2 Regulation of translation by 5’UTR and its binding factors----------------------23 1.2.1.2.3 Regulation of translation by 3’-UTR and its binding factors---------------------23 1.2.1.2.4 Translational activation via poly(A) tail interaction with PABP----------------26 1.2.1.3 Targeting of RNA to specific subcellular sites---------------------------------------28 1.2.2 The translational regulation roles of viral 5’ and 3’UTRs---------------------------29 1.2.3 Other potential roles that viral UTRs---------------------------------------------------30 1.2.4 Communication between the 5’ and 3’ end of mRNAs or viral RNAs enhancing translation------------------------------------------------------------------------------------------31 1.2.5 Mechanism of viral IRES-driven translation and its interaction with 3’UTR------36 1.3 Mechanisms of translation of positive strand RNA viruses-----------------------------38 1.4 Methods used in the function analysis 5’, 3’UTR of viruses--------------------------- 40 1.4.1 Nucleotides deletion or mutation of UTRs to analyze its function in the infection or translational process--------------------------------------------------------------------------------40 1.4.2 Fusion with a reporter gene to analyze the UTRs as translational regulators--------41 1.5 Objectives and significance of this study---------------------------------------------------41 CHAPTER MATERIALS AND METHODS 2.1 MATERIALS-----------------------------------------------------------------------------------43 2.1.1 Bacterial and agrobacteria strains----------------------------------------------------------43 2.1.2 Cloning vectors-------------------------------------------------------------------------------43 2.1.3 Media------------------------------------------------------------------------------------------43 2.2 METHODS-------------------------------------------------------------------------------------44 2.2.1 HLSV purification-------------------------------------------------------------------------- 44 2.2.2 Isolation of viral RNA----------------------------------------------------------------------44 2.2.3 cDNA synthesis------------------------------------------------------------------------------45 2.2.4 Purification of PCR fragments-------------------------------------------------------------45 2.2.5 Dephosphorylation of vector---------------------------------------------------------------45 2.2.6 End-filling of DNA fragments-------------------------------------------------------------46 2.2.7 Bacterial competent cell preparation and transformation-----------------------------46 2.2.8 Construction of full length HLSV cDNA clones---------------------------------------46 2.2.9 Construction of different poly(A) lengths and putative polyadenylation signal cDNA mutants-------------------------------------------------------------------------------------47 2.2.10 Construction of different clones fused with 5’UTR and 3’UTR--------------------47 2.2.11 Construction of bicistronic vectors for testing the 3’UTR on IRES-driven translation-------------------------------------------------------------------------------------------53 2.2.11.1 Constructs for in vitro assays----------------------------------------------------------53 2.2.11.2 Constructs for Agrobacterium infiltration assays-----------------------------------53 2.2.12 Nucleotide sequencing--------------------------------------------------------------------55 2.2.13 RNA gel electrophoresis------------------------------------------------------------------57 2.2.14 Northern blot analyses--------------------------------------------------------------------57 2.2.15 Generation of DIG-labeled cRNA probes---------------------------------------------58 2.2.16 Polyacrylamide gel electrophoresis and western blot--------------------------------58 2.2.17 In vitro transcription----------------------------------------------------------------------60 2.2.18 In vitro translation-------------------------------------------------------------------------60 2.2.19 Coupled in vitro transcription and translation-----------------------------------------61 2.2.20 Isolation of protoplasts--------------------------------------------------------------------61 2.2.21 PEG inoculation of protoplasts-----------------------------------------------------------62 2.2.22 Luciferase assay----------------------------------------------------------------------------63 2.2.23 Preparation of electro-competent Agrobacterium cells-------------------------------63 2.2.24 Electroporation of Agrobacterium-------------------------------------------------------64 2.2.25 β-Glucuronidase (GUS) fluorimetric assay--------------------------------------------64 2.2.26 RNA secondary structure prediction----------------------------------------------------66 2.2.27 RT-PCR analysis---------------------------------------------------------------------------66 2.2.28 Observation of leave symptoms----------------------------------------------------------66 CHAPTER THE LENGTH OF INTERNAL POLY(A) TRACT AND PUTATIVE POLYADENYLATION SIGNAL SEQUENCE INFLUENCE HLSV COAT PROTEIN EXPRESSION AND SYSTEMIC MOVEMENT IN N. BENTHAMIANA 3.1 Introduction------------------------------------------------------------------------------------67 3.2 Transcripts derived from three full-length cDNA clones with different lengths of poly(A) tract are able to infect and move systemically in N. benthamiana ---------------68 3.3 Lack of systemic movement of transcripts less than 77 nt poly(A) tract in N. benthamiana and coat protein expression in upper leaves-----------------------------------71 3.4 Symptoms of upper leaves from the N. benthamiana with transcripts of different internal poly(A) lengths---------------------------------------------------------------------------75 3.5 Putative polyadenylation signal sequence is important for infectivity of transcripts-75 3.6 Symptoms of the upper leaves inoculated with mutants containing different putative polyadenylation signals---------------------------------------------------------------------------80 3.7 Viral RNA accumulates but coat protein are not detected in the inoculated leaves with defect transcripts------------------------------------------------------------------------------------82 3.8 Discussion---------------------------------------------------------------------------------------85 CHAPTER THE 5’, 3’ UTR-UTR INTERACTION FACILITATES BOTH POLY(A) TAIL-DEPENDENT AND POLY(A) TAIL-INDEPENDENT TRANSLATION OF HIBISCUS LATENT SINGAPORE VIRUS 4.1 Introduction------------------------------------------------------------------------------------90 4.2 HLSV 5’UTR enhances translation---------------------------------------------------------94 4.3 HLSV 3’UTR enhances translation greater than the 5’UTR----------------------------97 4.4 Cap, 5’UTR, 3’UTR of HLSV enhance translation to the highest level---------------97 4.5 Disruption of base-pairing between 5’UTR and 3’UTR decrease translation efficiency-------------------------------------------------------------------------------------------102 4.6 Restored base-pairing between 5’UTR-D and 3’UTR-S enhance translation to the wild-type levels------------------------------------------------------------------------------------103 4.7 Different combinations of 5’UTR and 3’UTR or mutants enhance translation differentially---------------------------------------------------------------------------------------106 4.8 Both 5’UTR and 3’UTR of HLSV translational enhancements are cap-dependent-112 4.9 Discussion and Conclusion------------------------------------------------------------------114 4.9.1 5’UTRs as translational enhancers-------------------------------------------------------114 4.9.2 3’UTRs as translational enhancers-------------------------------------------------------115 4.9.3 Poly(A)-dependent translation------------------------------------------------------------117 4.9.4 Poly(A)-independent translation----------------------------------------------------------118 CHAPTER THE 3’TLS AND POLY(A) TRACT PROMOTE HLSV IRES TRANSLATION IN VITRO AND IN VIVO SEPARATELY 5.1 Introduction-----------------------------------------------------------------------------------123 5.2 HLSV IRES translation is less efficient than canonical cap-dependent translation in vitro-------------------------------------------------------------------------------------------------124 5.3 HLSV IRESCP134 translation is less efficient than TMV-Crucifer strain IRESCP148 translation in vitro--------------------------------------------------------------------------------125 5.4 The full length HLSV 3’UTR promotes HLSV CP and MP IRES translation in vitro-------------------------------------------------------------------------------------------------------128 5.5 Domain D1 in the 3’TLS functions more important than D2, D3 in promoting IRES translation in vitro--------------------------------------------------------------------------------128 5.6 HLSV 3’UTR promotes IRES translation in vivo---------------------------------------130 5.7 Discussion------------------------------------------------------------------------------------133 CHAPTER FUTURE DIRECTIONS 6.1 Finding possible interactions between the poly(A) tract and PABP and its effect on translation-----------------------------------------------------------------------------------------140 6.2 Analysis the mechanism of poly(A) extension in the 3’UTR of HLSV--------------141 6.3 Substitution of the poly(A) tract to analyze its functions-------------------------------142 6.4 Exploring binding factors between the 5’UTR and 3’TLS which may help promoting translation------------------------------------------------------------------------------------------143 REFERENCES-----------------------------------------------------------------------------------146 LIST OF PUBLICATIONS — Cao, SS and S M Wong, "5', 3' RNA-RNA interaction facilitates both poly(A) taildependent and poly(A) tail-independent translation of Hibiscus latent Singapore virus mRNA". International Conference on the Frontiers of Plant Molecular Biology (p50) (22 - 24 May 2006). Changsha, Hunan, China. — Cao SS, Wang HH, Luhur A and Wong SM. (2005). Yeast expression and characterization of SARS-CoV N protein. J. Virol. Methods 130: 83-88. LIST OF ABBREVIATIONS Viruses AMV Alfalfa mosaic virus BMV Brome mosaic virus BSMV Barley stripe mosaic virus BYDV Barley yellow dwarf virus DENV Dengue virus EMCV Encephalomyocarditis virus FMDV Foot and mouth disease virus HAV Hepatitis A virus HCRSV Hibiscus chlorotic ringspot virus HCV Hepatitis C virus HLSV Hibiscus latent Singapore virus ORSV-S1 Odontoglossum ringspot virus, Singapore isolate PVX Potato virus X RCNMV Red clover necrotic mosaic virus SHMV Sunn-hemp mosaic virus STNV Satellite Tobacco necrosis virus TBSV Tomato bushy stunt virus TEV Tobacco etch virus TMV-Cr Tobacco mosaic virus, strain crucifer TNV Tobacco necrosis virus ToMV Tomato mosaic virus 10 Switched Reluctance Motors,” IEEE PEDS 2003, Nov, 2003, Singapore. 171 Publications 172 3. S. K. Sahoo, S. K. Panda, and J. X. Xu, “Iterative Learning based Torque Ripple Minimization in Switched Reluctance Motors,” IEEE IECON 2003, Nov, 2003, USA. 4. S. K. Sahoo, S. K. Panda, and J. X. Xu, “Iterative Learning Control based Direct Instantaneous Torque Control of Switched Reluctance Motors,” IEEE PESC 2004, June, 2004, Germany. 5. S. K. Sahoo, S. K. Panda, and J. X. Xu, “Direct Torque Controller for Switched Reluctance Motor using Sliding Mode Control,” IEEE PEDS 2005, Nov, 2005, KualaLumpur, Malaysia. Accepted Conference 1. S. K. Sahoo, S. K. Panda, and J. X. Xu, “Application of Spatial Iterative Learning Control for Direct Torque Control of Switched Reluctance Motor Drive,” PES Annual meeting Tampa, Florida, June 2007 . Submitted for Review Transactions 1. S. K. Sahoo, S. K. Panda, and J. X. Xu“Torque Control of Switched Reluctance Motor Drive using Nonlinear Robust Tracking Control,”IEEE Trans. on Power Electronics Publications 173 2. S. K. Sahoo, S. K. Panda, and J. X. Xu“Application of Spatial Iterative Learning Control for Direct Torque Control of Switched Reluctance Motor Drive,”IEEE Trans. on Energy Conversion 3. S. K. Sahoo, S. K. Panda, and J. X. Xu“Novel Piecewise Cubic Polynomial model for SRM to implement real-time model-based controllers,”IEEE Trans. on Magneitcs Appendices 174 Appendices 175 Appendix A Measured phase flux-linkage (Wb-t) data with rotor locked at different positions and for different phase currents. Phase 1A current Rotor position 2A 3A 4A 5A 6A 7A 8A 9A 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 0.015183 0.015183 0.015026 0.015314 0.016286 0.017502 0.019972 0.023771 0.029004 0.034483 0.039889 0.045422 0.050993 0.056553 0.062452 0.068304 0.073903 0.079928 0.085442 0.091178 0.096317 0.10189 0.10745 0.11297 0.11813 0.12313 0.12827 0.13263 0.13731 0.14045 0.14045 0.023261 0.023261 0.023275 0.023752 0.024989 0.026825 0.030478 0.035922 0.043164 0.05097 0.059233 0.067645 0.075711 0.083543 0.091761 0.10006 0.10826 0.11656 0.1247 0.13303 0.14071 0.14858 0.15625 0.16371 0.17049 0.17683 0.1825 0.187 0.19021 0.19214 0.19214 0.032912 0.032912 0.033096 0.033901 0.035431 0.03787 0.042267 0.049046 0.057388 0.066519 0.075956 0.085921 0.095493 0.10475 0.11412 0.12368 0.13304 0.14278 0.15188 0.1612 0.16954 0.17798 0.18561 0.19241 0.19809 0.20287 0.20715 0.21013 0.21246 0.21378 0.21378 0.042862 0.042862 0.043076 0.044029 0.046069 0.049312 0.05483 0.062338 0.07134 0.080739 0.090489 0.10073 0.1108 0.12083 0.13102 0.14106 0.15097 0.16095 0.17069 0.1802 0.18856 0.19636 0.20322 0.20947 0.21485 0.21917 0.22281 0.22527 0.22748 0.22881 0.22881 0.052638 0.052638 0.053168 0.054497 0.056894 0.060497 0.066518 0.074542 0.083976 0.093576 0.10335 0.11356 0.12371 0.13367 0.14389 0.15397 0.16399 0.17403 0.18367 0.1931 0.20107 0.20875 0.21539 0.2219 0.22712 0.23167 0.2351 0.23751 0.23924 0.24031 0.24031 0.062502 0.062502 0.063075 0.064587 0.067393 0.071552 0.078007 0.086343 0.095816 0.10541 0.11516 0.12528 0.13535 0.14535 0.15556 0.16575 0.17559 0.18535 0.19436 0.20316 0.21086 0.2184 0.22515 0.2316 0.2369 0.24131 0.24465 0.24676 0.2484 0.24931 0.24931 0.072058 0.072058 0.072633 0.074287 0.077353 0.082124 0.089234 0.097994 0.10754 0.11709 0.12672 0.13684 0.14681 0.15667 0.16675 0.1766 0.18599 0.19515 0.20374 0.21223 0.21966 0.22678 0.23313 0.2393 0.24459 0.24904 0.25228 0.25433 0.25582 0.25667 0.25667 0.081972 0.081972 0.082753 0.084742 0.088172 0.093326 0.10071 0.10967 0.11926 0.12862 0.13805 0.14792 0.15788 0.16769 0.1774 0.18681 0.19562 0.20425 0.21229 0.2204 0.22767 0.23449 0.24055 0.24634 0.25146 0.25576 0.25895 0.26071 0.26195 0.26254 0.26254 0.0058061 0.0058061 0.0057113 0.0057822 0.0062539 0.0067684 0.0082337 0.010162 0.012915 0.015578 0.018316 0.02103 0.023239 0.025898 0.028455 0.031346 0.033803 0.036974 0.039472 0.042175 0.04473 0.047794 0.050802 0.053025 0.054964 0.05665 0.059123 0.061778 0.064176 0.065357 0.06535 [...]... 1.1.1 Hibiscus latent Singapore virus According to the serological relatedness, virus morphology, host range and genome organization, HLSV belongs to the genus tobamovirus which is one of the very well characterized groups of viruses (Srinivasan et al., 2003) HLSV caused no visible disease symptoms on hibiscus plant The virus was co-purified with another plant virus called Hibiscus chlorotic ringspot virus. .. length of the internal poly(A) tract and the polyadenylation signal sequence were important for infectivity of HLSV in Nicotiana benthamiana Lastly, HLSV 3’UTR was able to enhance IRES translation tested in vitro 16 CHAPTER 1 LITERATURE REVIEW 1.1 Introduction Hibiscus latent Singapore virus (HLSV) is a plant virus recently reported from Singapore (Srinivasan et al., 2002, 2005) The genome of the virus. .. acids (N and I) of the CP It may be a regulator for the latter internal poly(A) tract The whole genome organization of HLSV is shown as Fig.1.1 Instead of other elements in the genome, HLSV 5’and 3’UTRs were the main focus in this study In the following part of this review, the roles of UTRs will be focused on 1.2 Roles of viral untranslated regions 1.2.1 Function as untrtanslated regions of mRNAs 1.2.1.1... absence of the rest of the viral 3’UTR could inhibit translation of reporter RNAs (Li and Brinton, 2001) While it is clear that the flavivirus 3’UTR plays a role in the modulation of translation efficiency, the mechanism of action is still not clear and possibly regulated by many factors, including the genomic context of the 3’SL There are 33 conserved domains but not the 3’SL in the 3’UTR of flaviviruses... 1.2.1.1 Regulation of viral RNA stability Viral RNAs are a special kind of mRNA Their UTRs could have the same functions as the UTRs of mRNAs From the various studies on mRNAs, clues as to the function of UTRs could be found and similar functions of UTRs of viral RNA could be deduced Studies showed that the poly(A) tails of mRNAs could be involved in the turnover or degradation of mRNAs (Decker and... enhancer, including members of the flaviviridae family and rotavirus (Reoviridae) 32 However, several mechanisms regarding the interaction of the 5’ and 3’ ends enhancing of translational efficiency has been demonstrated for these viruses (Chiu et al., 2005; Holden and Harris, 2004; Piron et al., 1998) For example, The flaviviruses, such as the dengue virus (DENV) and West Nile virus (WNV), are not polyadenylated... 50 15 Summary Hibiscus latent Singapore virus (HLSV) is a new tobamovirus recently reported The genome contains a 5’- and a 3’ -untranslated region (UTR) In this study, the functions of 5’UTR and 3’UTR in regulating gene expression were analyzed by in vitro and in vivo assays In wheat germ extract and kenaf protoplasts, the presence of both 5’UTR and 3’UTR enhanced luciferase... of virus infection, multiplication and their survivals Modern molecular biological techniques have helped us to gain insights into the genome organization and expression strategies of different viruses, which in turn lead to the discovery of methods to overcome the crop losses resulting from virus epidemics as well as the exploitation of viruses as vectors for expressing therapeutic proteins (Hamamoto... -74 Fig 3.3 Obvious curly top disease symptom of upper leaves of N benthamiana inoculated with transcripts of 77 nt, 85 nt and 96 nt length of poly(A), same as the symptom of the virus infected leaves; while no symptom inoculated with the transcript less than 77 nt, which is the same as Mock -76 Fig 3.4 The significance of putative polyadenylation signal for HLSV transcripts... symptom of upper leaves of N benthamiana inoculated with transcripts of PS, MS, WT, SS1, SS2 infected plants, same as the disease symptom of HLSV infected leaves 81 Fig 3.6 Accumulation of viral RNA while no coat protein expression in the inoculated leaves with defect transcripts 84 Fig 4.1 Genome organization of HLSV and its characterization of . 1 MOLECULAR ANALYSES OF UNTRANSLATED REGIONS OF HIBISCUS LATENT SINGAPORE VIRUS CAO SHISHU (MSc, Huazhong Agri Introduction 17 1.1.1 Hibiscus latent Singapore virus 17 1.1.2 The genome organization of HLSV 19 1.2 Roles of viral untranslated regions 20 1.2.1 Function as untrtanslated regions of mRNAs -20 1.2.1.1. Hepatitis C virus HLSV Hibiscus latent Singapore virus ORSV-S1 Odontoglossum ringspot virus, Singapore isolate PVX Potato virus X RCNMV Red clover necrotic mosaic virus SHMV Sunn-hemp mosaic virus