Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry
2010; Elsevier BV; Volume: 9; Issue: 8 Linguagem: Inglês
10.1074/mcp.m900620-mcp200
ISSN1535-9484
AutoresGlen K. Shoemaker, Esther van Duijn, Sue E. Crawford, Charlotte Uetrecht, Marian Baclayon, Wouter H. Roos, Gijs J. L. Wuite, Mary K. Estes, B. V. Venkataram Prasad, Albert J. R. Heck,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoViral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP160 (3.36-MDa) and VP180 (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1180). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1180 particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP160 and VP180 species. Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP160 (3.36-MDa) and VP180 (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1180). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1180 particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP160 and VP180 species. Accounting for most cases of non-bacterial gastroenteritis, the norovirus represents an important human pathogen (1.Atmar R.L. Estes M.K. The epidemiologic and clinical importance of norovirus infection.Gastroenterol. Clin. 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Because of a lack of suitable animal models or in vitro cell culture systems, structural studies so far have been largely focused on recombinant norovirus-like particles (rNVLPs), 1The abbreviations used are:rNVLPrecombinant norovirus-like particleAFMatomic force microscopyccscollision cross-section(s)HBVhepatitis B virusIMMSion mobility MSVPviral proteinmbarmillibarsEMelectron microscopy. which are spontaneously assembled during the expression of recombinant VP1 and VP2 in insect cells (5.Xi J.N. Graham D.Y. Wang K.N. Estes M.K. Norwalk virus genome cloning and characterization.Science. 1990; 250: 1580-1583Crossref PubMed Scopus (452) Google Scholar). Importantly, these empty noninfectious particles have been demonstrated to be morphologically and antigenically similar to the genuine virion (9.Jiang X. Wang M. Graham D.Y. Estes M.K. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.J. Virol. 1992; 66: 6527-6532Crossref PubMed Google Scholar). recombinant norovirus-like particle atomic force microscopy collision cross-section(s) hepatitis B virus ion mobility MS viral protein millibars electron microscopy. The rNVLPs have been studied extensively using X-ray crystallography and electron microscopy (EM), which have provided a detailed image of the intact capsid, revealing the T = 3 icosahedral organization (6.Prasad B.V. Rothnagel R. Jiang X. Estes M.K. 3-Dimensional structure of baculovirus-expressed Norwalk virus capsids.J. Virol. 1994; 68: 5117-5125Crossref PubMed Google Scholar, 9.Jiang X. Wang M. Graham D.Y. Estes M.K. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.J. Virol. 1992; 66: 6527-6532Crossref PubMed Google Scholar, 10.Bertolotti-Ciarlet A. White L.J. Chen R. Prasad B.V. Estes M.K. Structural requirements for the assembly of Norwalk virus-like particles.J. Virol. 2002; 76: 4044-4055Crossref PubMed Scopus (150) Google Scholar, 11.Prasad B.V. Hardy M.E. Dokland T. Bella J. Rossmann M.G. Estes M.K. X-ray crystallographic structure of the Norwalk virus capsid.Science. 1999; 286: 287-290Crossref PubMed Scopus (734) Google Scholar). The VP1 monomer structure is principally composed of two domains, an S domain consisting of the 225 N-terminal residues and a C-terminal P domain. In the intact capsid, the S domain forms a contiguous protein shell with a diameter of ∼30 nm, whereas the P domain forms prominent protrusions, which give the rNVLPs a diameter of ∼40 nm. A remarkable feature of the rNVLPs is that a single protein is responsible for directing capsid assembly and host interactions. The rNVLPs thus represent a simple model to study the assembly of icosahedral viruses. Although the requirements for capsid assembly have been investigated previously (7.Ausar S.F. Foubert T.R. Hudson M.H. Vedvick T.S. Middaugh C.R. Conformational stability and disassembly of Norwalk virus-like particles: effect of pH and temperature.J. Biol. Chem. 2006; 281: 19478-19488Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 10.Bertolotti-Ciarlet A. White L.J. Chen R. Prasad B.V. Estes M.K. Structural requirements for the assembly of Norwalk virus-like particles.J. Virol. 2002; 76: 4044-4055Crossref PubMed Scopus (150) Google Scholar), there is little information regarding intermediates along the (dis)assembly pathway. Obtaining such information can be quite difficult because of the inherent heterogeneity of capsid assembly. An emerging technique for interrogating such heterogeneous protein assemblies is native electrospray ionization mass spectrometry (ESI-MS). Long regarded as a tool for small molecule analysis and more recently proteomics investigations, the utility of mass spectrometry in structural biology is increasingly applied and accepted (12.Heck A.J. 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High-resolution mass spectrometry of viral assemblies: Molecular composition and stability of dimorphic hepatitis B virus capsids.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9216-9220Crossref PubMed Scopus (181) Google Scholar, 17.Uetrecht C. Versluis C. Watts N.R. Wingfield P.T. Steven A.C. Heck A.J. Stability and shape of hepatitis B virus capsids in vacuo.Angew. Chem. Int. Ed. Engl. 2008; 47: 6247-6251Crossref PubMed Scopus (101) Google Scholar) reported ESI-MS data on the hepatitis B virus (HBV) capsid. In these studies, sufficient mass resolution was obtained to determine the accurate mass and stoichiometry of the T = 3 and T = 4 HBV capsids despite their large mass of 3 and 4 million Da, respectively (15.Uetrecht C. Versluis C. Watts N.R. Roos W.H. Wuite G.J. Wingfield P.T. Steven A.C. Heck A.J. High-resolution mass spectrometry of viral assemblies: Molecular composition and stability of dimorphic hepatitis B virus capsids.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9216-9220Crossref PubMed Scopus (181) Google Scholar, 17.Uetrecht C. Versluis C. Watts N.R. Wingfield P.T. Steven A.C. Heck A.J. Stability and shape of hepatitis B virus capsids in vacuo.Angew. Chem. Int. Ed. Engl. 2008; 47: 6247-6251Crossref PubMed Scopus (101) Google Scholar). In addition to being able to measure the mass and stoichiometry of protein assemblies, the capacity of native MS to analyze simultaneously a heterogeneous population of assembly intermediates makes it a powerful technique to study virus assembly (27.Morton V.L. Stockley P.G. Stonehouse N.J. Ashcroft A.E. Insights into virus capsid assembly from non-covalent mass spectrometry.Mass Spectrom. Rev. 2008; 27: 575-595Crossref PubMed Scopus (43) Google Scholar). In the work described here, the disassembly of rNVLPs was monitored over a range of solution conditions using native ESI-MS, providing insights into their stability and factors that govern icosahedral assembly for this model calicivirus. Unraveling the details of these complex structures and the associated self-assembly pathways that lead to their efficient and precise construction may play an important role in the development of antiviral therapeutics and in the field of nanotechnology where there is much interest in the fundamentals of particle self-assembly. The native ESI-MS measurements were carried out using recombinant VP1, which was expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus expression system and purified as described previously (9.Jiang X. Wang M. Graham D.Y. Estes M.K. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.J. Virol. 1992; 66: 6527-6532Crossref PubMed Google Scholar). Briefly, Sf9 cells were harvested 5–7 days after baculovirus infection and then purified using centrifugation with a CsCl gradient of 1.362 g/cm3. The purified rNVLPs (400 μm VP1) were stored in water at 4 °C. For ESI-MS analysis, the rNVLPs were exchanged into an aqueous ammonium acetate buffer (50–500 mm) at various pH values using an Amicon Ultra 0.5-ml centrifugal filter (Millipore, Billerica, MA) with a molecular mass cutoff of 10 kDa. The pH of the aqueous ammonium acetate solution was adjusted by using an aqueous solution of either ammonia or acetic acid. All measurements at the different experimental conditions were repeated at least three times on different days. Additionally, results from two different preparations of reconstituted rNVLPs yielded consistent results. High resolution and tandem mass spectra were recorded on a modified Q-ToF 1 instrument (Waters) in positive ion mode (28.van den Heuvel R.H. van Duijn E. Mazon H. Synowsky S.A. Lorenzen K. Versluis C. Brouns S.J. Langridge D. van der Oost J. Hoyes J. Heck A.J. Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry.Anal. Chem. 2006; 78: 7473-7483Crossref PubMed Scopus (205) Google Scholar). To enhance the transmission of the large ions corresponding to rNVLPs, xenon, at a pressure of 2 × 10−2 mbar, was used in the collision cell (29.Lorenzen K. Versluis C. van Duijn E. van den Heuvel R.H. Heck A.J. Optimizing macromolecular tandem mass spectrometry of large non-covalent complexes using heavy collision gases.Int. J. Mass Spectrom. 2007; 268: 198-206Crossref Scopus (59) Google Scholar). The voltages and pressures were also optimized for large non-covalent protein complexes (30.Sobott F. Hernández H. McCammon M.G. Tito M.A. Robinson C.V. A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies.Anal. Chem. 2002; 74: 1402-1407Crossref PubMed Scopus (440) Google Scholar). Briefly, the capillary and cone voltages were kept constant at 1450 and 165 V, respectively. The voltage before the collision cell was varied from 10 to 400 V but generally left at 50 V for the accumulation of native ESI mass spectra. Ions were introduced into the source under an elevated pressure of 10 mbar. Ion mobility measurements were performed on a Synapt HDMS (Waters) (31.Pringle S.D. Giles K. Wildgoose J.L. Williams J.P. Slade S.E. Thalassinos K. Bateman R.H. Bowers M.T. Scrivens J.H. An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument.Int. J. Mass Spectrom. 2007; 261: 1-12Crossref Scopus (666) Google Scholar). To generate intact gas phase ions from large protein complexes in solution, the source was maintained at 6.1 mbar, and voltages of 1400 and 164 V were applied to the capillary and sample cone, respectively. Xenon was used as the background gas in the trap and transfer ion guides at a flow rate of 4 ml/min. The voltages in the trap and transfer were 20 and 25 V, respectively. The gas in the ion mobility cell was nitrogen at a flow rate of 25 ml/min and a ramped wave height of 10–30 V with a velocity of 250 m/s. Ramped wave heights were shown previously to provide better results for protein complexes in the megadalton range (17.Uetrecht C. Versluis C. Watts N.R. Wingfield P.T. Steven A.C. Heck A.J. Stability and shape of hepatitis B virus capsids in vacuo.Angew. Chem. Int. Ed. Engl. 2008; 47: 6247-6251Crossref PubMed Scopus (101) Google Scholar). Collision cross-sections (ccs) were determined from the measured drift times through calibration using proteins of known cross-sections as described previously (17.Uetrecht C. Versluis C. Watts N.R. Wingfield P.T. Steven A.C. Heck A.J. Stability and shape of hepatitis B virus capsids in vacuo.Angew. Chem. Int. Ed. Engl. 2008; 47: 6247-6251Crossref PubMed Scopus (101) Google Scholar, 32.Ruotolo B.T. Benesch J.L. Sandercock A.M. Hyung S.J. Robinson C.V. Ion mobility-mass spectrometry analysis of large protein complexes.Nat. Protoc. 2008; 3: 1139-1152Crossref PubMed Scopus (854) Google Scholar, 33.van Duijn E. Barendregt A. Synowsky S. Versluis C. Heck A.J. Chaperonin complexes monitored by ion mobility mass spectrometry.J. Am. Chem. Soc. 2009; 131: 1452-1459Crossref PubMed Scopus (131) Google Scholar). Denatured ESI-MS analysis of the VP1 monomers was performed on an LCT instrument (Waters). The performance of the instruments was tested with an aqueous CsI solution, and a calibration was applied where necessary. ESI tips were prepared in house from borosilicate glass tubes of 1.2-mm outer diameter and 0.68-mm inner diameter (World Precision Instruments, Sarasota, FL) by using a P-97 micropipette puller (Sutter Instruments, Novato, CA). The ESI tips were gold-coated using a Scancoat six Pirani 501 sputter coater (Edwards Laboratories, Milpitas, CA). Atomic force microscopy (AFM) experiments were conducted in aqueous ammonium acetate solutions with a Nanotec Electronica (Madrid, Spain) instrument operating in jumping mode (34.de Pablo P.J. Colchero J. Gomez-Herrero J. Baro A.M. Jumping mode scanning force microscopy.Appl. Phys. Lett. 1998; 73: 3300-3302Crossref Scopus (152) Google Scholar) using rectangular gold-coated cantilevers (Olympus, Zoeterwoude, The Netherlands) with a tip apex nominal value of <20 nm. The cantilevers were calibrated as described previously (35.Sader J.E. Chon J.W. Mulvaney P. Calibration of rectangular atomic force microscope cantilevers.Rev. Sci. Instrum. 1999; 70: 3967-3969Crossref Scopus (1698) Google Scholar) and were found to have a spring constant of 0.052 ± 0.004 newton/m. Glass coverslips were cleaned by immersion in an 86% (v/v) ethanol solution saturated with KOH for 14 h. After being rinsed thoroughly with deionized water and left to dry, the coverslips were rendered hydrophobic by incubating them under a saturated hexamethyldisilazane (Fluka, Zwijndrecht, The Netherlands) vapor for 14 h (36.Ivanovska I.L. de Pablo P.J. Ibarra B. Sgalari G. MacKintosh F.C. Carrascosa J.L. Schmidt C.F. Wuite G.J. Bacteriophage capsids: tough nanoshells with complex elastic properties.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7600-7605Crossref PubMed Scopus (285) Google Scholar). For imaging, a droplet containing VP1 (0.2–10 μm) in an aqueous ammonium acetate buffer (500 mm) at various pH values was placed on the hydrophobic glass coverslip and analyzed after a short (5-min) incubation. To first investigate whether the intact capsid can be detected using ESI-MS, the rNVLPs were analyzed at neutral pH, a condition that is known from previous EM studies to be favorable for the formation of intact capsid (7.Ausar S.F. Foubert T.R. Hudson M.H. Vedvick T.S. Middaugh C.R. Conformational stability and disassembly of Norwalk virus-like particles: effect of pH and temperature.J. Biol. Chem. 2006; 281: 19478-19488Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 9.Jiang X. Wang M. Graham D.Y. Estes M.K. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.J. Virol. 1992; 66: 6527-6532Crossref PubMed Google Scholar). A mass spectrum at pH 7 in a 250 mm aqueous ammonium acetate buffer is presented in Fig. 1a. A broad distribution of ions is present in the high m/z region of the mass spectrum, corresponding to intact rNVLPs as well as a small amount of VP1 dimers (around m/z 5000). Because the individual charge states of the capsid ions are unresolved, the mass cannot be precisely assigned. Importantly, however, the ions corresponding to rNVLPs are detected in the expected region of the mass spectrum. To demonstrate this, an inset of two other virus capsids analyzed by ESI-MS is included in Fig. 1a. This spectrum corresponds to the two HBV capsids, which exhibit T = 3 and T = 4 symmetry, that possess masses of 3 and 4 MDa, respectively (15.Uetrecht C. Versluis C. Watts N.R. Roos W.H. Wuite G.J. Wingfield P.T. Steven A.C. Heck A.J. High-resolution mass spectrometry of viral assemblies: Molecular composition and stability of dimorphic hepatitis B virus capsids.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9216-9220Crossref PubMed Scopus (181) Google Scholar, 17.Uetrecht C. Versluis C. Watts N.R. Wingfield P.T. Steven A.C. Heck A.J. Stability and shape of hepatitis B virus capsids in vacuo.Angew. Chem. Int. Ed. Engl. 2008; 47: 6247-6251Crossref PubMed Scopus (101) Google Scholar). These data are also represented as a plot of charge state versus the square root of the mass for several protein and protein assemblies analyzed by ESI-MS that is expected to be linear according to the charge residue model for electrospray ionization (37.Felitsyn N. Peschke M. Kebarle P. Origin and number of charges observed on multiply-protonated native proteins produced by ESI.Int. J. Mass Spectrom. 2002; 219: 39-62Crossref Scopus (125) Google Scholar, 38.Heck A.J. Van Den Heuvel R.H. Investigation of intact protein complexes by mass spectrometry.Mass Spectrom. Rev. 2004; 23: 368-389Crossref PubMed Scopus (506) Google Scholar). As can be seen in Fig. 1b, the plot demonstrates excellent linearity, and the rNVLPs are detected around the expected charge state. With a mass of ∼10.1 MDa, the rNVLPs are among the largest protein assemblies analyzed by native ESI-MS. We attempted to subject these capsid ions to high energy CID to enhance desolvation, but unfortunately, the individual charge states remained unresolved. At even higher voltages in the collision cell, dissociation of the capsid was observed that proceeded with the loss of VP1 monomers and concomitant high mass fragments, which could not be well resolved (data not shown). To confirm the identity and homogeneity of the major capsid protein (VP1), the rNVLPs were also analyzed with ESI-MS under denaturing conditions. The rNVLPs were denatured in an acetonitrile/water solution that was acidified using formic acid (Fig. 1c). The denatured mass spectrum revealed the existence of two nearly equally abundant VP1 monomer species with masses of 56,077 ± 2 and 55,813 ± 2 Da, respectively. The relative abundance of each monomer was
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