Structural and functional studies of the betanodavirus grouper nervous necrosis virus

論文翻譯標題: 石斑魚神經壞死病毒之晶體結構與?能研究
  • 陳 乃齊

學生論文: Doctoral Thesis


Structural and functional studies of the betanodavirus grouper nervous necrosis virus Nai-Chi Chen Chun-Jung Chen and Tzong-Yueh Chen Institute of Biotechnology College of Bioscience and Biotechnology SUMMARY Betanodaviruses belong to the family Nodaviridae and cause the mortality of numerous larval-stage fish species Here we report protein crystal structures of a piscine betanodavirus the grouper nervous necrosis virus in four different forms Highlights are two structural features that contribute to the viral molecular mechanisms of the T=3 and T=1 capsid assembly: a calcium-associated protrusion domain and a functional arginine-rich motif These results also shed insights into the structural basis for evolutionary lineage of the family Nodaviridae Key words: Nodaviridae nervous necrosis virus calcium-associated protrusion domain INTRODUCTION Aquaculture is one of the major global economic activities such as cultivation of marine fish and prawn in many countries In fact Taiwan an island surrounded by sea is one of the major distant water fisheries and aquaculture producers in the world In terms of production and value aquaculture has long been far ahead of offshore and coastal fisheries combined and been just the second to distant water fisheries (Chen and Qiu 2014) Today aquaculture in Taiwan including island and marine cultures is well correlation with Taiwanese economy and society such as income generation food source and food supply Furthermore several species such as oyster farmed fishes and grass carp have long been reared as an important food source in Taiwan Currently the major farmed fishes include milkfish tilapia grouper giant freshwater prawn and pacific white shrimp with the export economic importance in Taiwan (Chen and Qiu 2014) The stressful and crowded conditions of aquaculture might cause these most problematic impacts in infectious diseases Since the first half of the 20th century the more significant viral pathogens of finfish including aquabirnaviruses within the family Birnaviridae infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) within the family Rhabdoviridae infectious salmon anemia virus (ISAV) within the family Orthomyxoviridae epizootic hematopoietic necrosis virus (EHNV) within the family Iridoviridae and betanodavirus making up the family Nodaviridae are reported (Crane and Hyatt 2011) The family Nodaviridae is further classified as the two major genera including alphanodaviruses and betanodaviruses (Thiery et al 2004) and displays T=3 symmetry (180 subunits in the capsid) of approximately ~29?35 nm in diameter Basically one CP exhibits the typical canonical 8-stranded jelly roll β-barrel fold which consists of two pairs of antiparallel β-sandwich (Cheng and Brooks 2013) To elucidate the structural information and mechanisms of capsid conformation capsid assembly and viral infection by the genus betanodavirus of largely uncharacterized we have determined the crystal structures of the grouper nervous necrosis virus (GNNV) MATERIALS AND METHODS A DNA sequence corresponding to the full-length of orange-spotted grouper nervous necrosis virus (OSGNNV) RNA2 (GenBank accession no KT071606) was amplified with specific primers by PCR and cloned into the artificial vector between SfoI and XhoI restriction sites for the production of N-terminal hexa-histidine-SUMO-tagged fusion protein (Lee et al 2008) All constructs were transformed and over-expressed in Escherichia coli (E coli) BL21-CodonPlus(DE3)-RIL cells (Stratagene) Overexpression of the full-length GNNV CP were induced with 0 5 mM IPTG (isopropyl β-D-thio-galactopyranoside) for overnight at 18°C The SUMO-tag GNNV CP was then purified from the soluble supernatant by Ni2+-affinity chromatography (HiTrap HP Amersham Biosciences) Initial crystallization screening for the GNNV-LP was performed using the hanging-drop vapor diffusion method with a Mosquito liquid-handling robot (TTP Labtech) for high-throughput crystallization condition screening Crystals appeared within 1?2 weeks under the initial reservoir condition of 0 2 M sodium formate (pH 7 2) and 20% (w/v) PEG3350 The ab initio method by using icosahedral non-crystallographic symmetry (NCS) averaging with phase extension was performed to determine the initial phases of the T=3 GNNV-LP (Taka et al 2005) Finally the coordinates of the T=3 GNNV-LP were refined to a crystallographic Rcryst of 0 257 and Rfree of 0 295 at 3 6 ? resolution In the current model 97% of all residues were in the most favored region of the Ramachandran plot and 3% of that was in the allowed regions using MolProbity (Chen et al 2010) RESULTS AND DISCUSSION We determine the 3 6 ? resolution ab initio crystal structure of the T=3 GNNV-LP with non-crystallographic symmetry (NCS) averaging The manually modeling of residues 52?338 for subunits A and B and residues 34?338 for subunit C were fitting into the electron density of the icosahedral asymmetric unit (iASU) of the T=3 GNNV-LP One T=3 GNNV-LP consists of sixty trimeric S-domains that participate in inter-subunit contacts to form a continuous shell of the capsid with an empty inner cavity Three neighboring P-domains per iASU constitute the predominantly β-strand portion of the protein at the quasi three-fold (Q3) axes to form 60 protrusions on the particle surface The 180 neighboring monomeric S-domains from subunits A B and C make up the icosahedral shell in dimeric trimeric and pentameric interactions along the I2 I3 and icosahedral five-fold (I5) axes (Fig 1) The present structures of the genus betanodavirus GNNV provides the important mechanistic insights into the processes of capsid assembly and viral infection Actually despite conservations about a viral genome encoding three major proteins and a compatible geometry of the T=3 architecture in the family Nodaviridae the atomic striking features of the GNNV-LP described here facilitate to delineate the key structural components that trigger the CP oligomerization and stabilize the capsid assembly The molecular organizations and assembly mechanisms of the genus betanodavirus GNNV reveal that the family Nodaviridae may lead to the novel viral evolutional approaches among the Tombusviridae Caliciviridae and Birnaviridae families which are compatible with the independent evolution linage previously Structural mapping of the GNNV P-domain might facilitate the engineering of the vaccine development in the fish aquaculture industry Figure 1 Surface domain-colored diagram (left) and central cavity (right) representations of the T=3 GNNV-LP The tip-to-tip distance is ~350 ? the diameter of the central cavity is ~228 ? and the spike protrusion on the capsid surface is ~47 ? The S-domains of the subunits A B and C are shown in orange blue and red respectively and the P-domains are shown in cyan The structure of the GNNV-LP is viewed along the I2 I3 and I5 axes CONCLUSION In this study we report the ab initio structure of a T=3 grouper nervous necrosis virus-like particle (GNNV-LP) at 3 6 ? resolution determined by non-crystallographic symmetry averaging method In summary this work provides several important structural insights into the genus betanodavirus GNNV Despite conservation of a viral genome encoding three major proteins and a compatible geometry of the T=3 architecture in the family Nodaviridae the structure of the GNNV-LP obtained here allows us to delineate the key structural components that trigger the oligomerization and stabilize the capsid assembly Furthermore structural mapping of the GNNV P-domain might be useful for the development of vaccine strategies in the fish aquaculture industry
獎項日期2016 7月 25
監督員Tzong-Yueh Chen (Supervisor)