Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry (2010)

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. 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. North Am. 2006; 35 (viii): 275-290Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 2.Estes M.K. Prasad B.V. Atmar R.L. Noroviruses everywhere: has something changed?.Curr. Opin. Infect. Dis. 2006; 19: 467-474Crossref PubMed Scopus (170) Google Scholar). It is the most predominant pathogen within the family Caliciviridae, which also includes Sapovirus, Vesivirus, and Lagovirus (3.Green K.Y. Ando T. Balayan M.S. Berke T. Clarke I.N. Estes M.K. Matson D.O. Nakata S. Neill J.D. Studdert M.J. Thiel H.J. Taxonomy of the caliciviruses.J. Infect. Dis. 2000; 181: S322-S330Crossref PubMed Scopus (409) Google Scholar). The prototypical strain of the genus Norovirus is the Norwalk virus. It is a small (7.7-kb genome) non-enveloped, single-stranded RNA virus. Its genome contains three open reading frames, encoding for the major capsid protein (VP1), the minor capsid protein (VP2), and a non-structural polyprotein (4.Jiang X. Wang M. Wang K. Estes M.K. Sequence and genomic organization of Norwalk virus.Virology. 1993; 195: 51-61Crossref PubMed Scopus (530) Google Scholar, 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). VP1 forms homodimers, and the mature Norwalk virus capsids (T = 3) are composed of 90 VP1 dimers (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, 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) and possibly a few copies of VP2 that are thought to stabilize the icosahedral structure as well as affect the expression of VP1 (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, 8.Bertolotti-Ciarlet A. Crawford S.E. Hutson A.M. Estes M.K. The 3′ end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein.J. Virol. 2003; 77: 11603-11615Crossref PubMed Scopus (166) Google Scholar). 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 which the rNVLPs a diameter of of the rNVLPs is that a protein is for capsid assembly and The rNVLPs a model to study the assembly of icosahedral Although the requirements for capsid assembly have been (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 is information regarding the (dis)assembly information be because of the inherent heterogeneity of capsid assembly. for protein is native mass spectrometry as a for small analysis and the of mass spectrometry in structural biology is and Native mass a and structural PubMed Scopus Google Scholar, K. A.M. for protein in the of PubMed Scopus Google Scholar, M. The of mass spectrometry in structure of protein 76: PubMed Scopus Google Scholar, mass spectrometry of viral and stability of hepatitis B virus PubMed Scopus Google Scholar). 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Scopus Google Scholar). intact protein complexes in the was at and of and were to the and was used as the in the and ion at a of The in the and were and The in the ion mobility cell was at a of and a of with a of were to for protein complexes in the range and of hepatitis B virus capsids in Chem. PubMed Scopus Google Scholar). were the using proteins of as and of hepatitis B virus capsids in Chem. PubMed Scopus Google Scholar, A.M. spectrometry analysis of protein PubMed Scopus Google Scholar, A. S. complexes by ion mobility mass Am. Chem. PubMed Scopus Google Scholar). analysis of the VP1 was on an The of the was with an and a was were in of diameter and diameter by using a The were using a Atomic force microscopy were in with a in J. J. A.M. force Scopus Google Scholar) using The with a of The were as of atomic force 1999; Scopus Google Scholar) and were found to have a of were by in an solution with for being with and to the were by them under a The for with PubMed Scopus Google Scholar). a VP1 in an at pH was on the and analyzed a investigate the intact capsid be using the rNVLPs were analyzed at pH, a that is studies to be 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). mass at pH in a is in of is in the of the mass to intact rNVLPs as well as a small of VP1 dimers Because the of the capsid are the mass be Importantly, the to rNVLPs are in the of the mass an of two virus capsids analyzed by is in to the two which T = 3 and T = that of 3 and mass spectrometry of viral and stability of hepatitis B virus PubMed Scopus Google Scholar, and of hepatitis B virus capsids in Chem. PubMed Scopus Google Scholar). data are also as a of the of the mass for protein and protein analyzed by that is to be to the model for M. and of observed on native proteins by J. 2002; Scopus Google Scholar, of intact protein complexes by mass PubMed Scopus Google Scholar). be in the excellent and the rNVLPs are the a mass of the rNVLPs are among the largest protein analyzed by native We to subject these capsid to to the At in the collision of the capsid was observed that with the of VP1 and mass which could be well the and of the major capsid protein (VP1), the rNVLPs were also analyzed with under The rNVLPs were in an solution that was using The mass revealed the of two VP1 monomer species with of and Da, The of monomer was and an monomer mass of was used in for the VP1 The two monomer species in mass by Da, which is with two the two VP1 monomer species in as are three at the VP1 mass heterogeneity in the VP1 is an important for the lack of for the similar heterogeneity in VP1 monomer species has been using L.J. Hardy M.E. Estes M.K. of a smaller of recombinant Norwalk virus capsids assembled in insect Virol. PubMed Google Scholar). the mass was the minor structural protein It was that a few copies of VP2 could be capsid, VP2 was in the MS analysis or on the Its and in capsid assembly remain The stability of rNVLPs over a range of solution has been using and techniques (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). From was that the rNVLPs are stable under acidic are sensitive to alkaline treatment. with these under acidic pH, to the intact capsid were observed in the mass at pH intact rNVLPs the small VP1 oligomers, predominantly VP1 and dimers, were also upon alkaline capsid was mass of the rNVLPs in a over a pH range of are in the ion for species were for solution pH and are in the in It be that for the collected in important as VP1 and ionic were be in 2, at pH capsid was observed as VP1 dimers were in the (dis)assembly were also consisting of 4, 6, 18, and copies of with the VP160 species being the most of the VP1 oligomers at pH. smaller capsids were also in a study L.J. Hardy M.E. Estes M.K. of a smaller of recombinant Norwalk virus capsids assembled in insect Virol. PubMed Google Scholar). smaller particles a diameter of and were to be T = particles of copies of VP1. that at pH the intact capsid smaller VP1 oligomers, predominantly small VP1 oligomers to oligomers, predominantly VP160 at pH the has been that in solution the VP1 dimers in two and that the of VP1 as a to these two T = 3 icosahedral assembly 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 and the in structures of icosahedral Opin. Biol. PubMed Scopus Google Scholar). are within the VP1 dimers, the and to the formation of capsids with and of the solution pH to to the complete of the VP1180 capsid and the formation of midsize VP1 At the a of VP1 oligomers was and a small of and In the the VP160 species was in VP1 oligomers consisting of 18, 40, and 80 copies of VP1 were the VP1 oligomers the and are the most of the species. Middaugh and (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) that three forms of VP1 at alkaline pH on data to a we could these species and of the of an to study protein as is to oligomers within a the of these VP1 oligomers, to species were in the and to of the VP160 species are in by the of VP1 with at most being at the collision The of mass by VP1 monomer the of the VP180 could be using the mass for mass spectrometry as is the for ion the were analyzed with a in the collision cell to of the mass in the collision be in the of the the of the the of these mass of the two VP1 oligomers were over the experimental mass of the VP160 and VP180 at and excellent of these using for and which and icosahedral the experimental the of the VP180 species. The for the VP180 and VP160 oligomers were also a of VP1 and From and be that a of VP180 and VP160 represents the to the experimental the VP1 oligomers were in the of the the of mass be in the VP1 oligomers of the are at the It has been demonstrated for icosahedral as that capsid assembly also be by ionic A. R. M.J. of capsid assembly for an icosahedral virus.Virology. 2000; PubMed Scopus Google Scholar). investigate the effect of ionic on the assembly of these VP1 oligomers, analysis was of the intact rNVLPs into with a range of and a pH range of The ion for VP1 were over at solution and are as in the range of ionic strength, to intact rNVLPs the mass at physiological pH with a small of VP1 a effect under alkaline capsid was observed at ionic as by the formation of and VP1 at pH the intact capsid was most at an intermediate ionic of At pH, the ionic also to which VP1 oligomers are a small of and VP160 was is in to the at ionic in which VP160 was and the VP180 was the mass At ionic data small VP1 oligomers were at pH is that the of the with with a in VP160 that a ionic VP1 dimers into the VP160 species. are at pH At pH, was intact VP1180 at of the ionic was observed at pH at ionic was an to the mass VP1 oligomers, as VP160 and with a in In a ionic were oligomers as VP1 dimers were in the mass important in the formation of protein is the of protein investigate the effect of VP1 on the assembly of these novel VP1 oligomers, of a VP1 solution in a were and analyzed by was found that at of VP1 the oligomers were very in and was the species in the VP1 VP160 and VP180 the of was also on VP1 concentration. At a VP1 of oligomers were with similar by at the 80 the VP180 species was the VP160 The assembly of these VP1 oligomers was also found to be a In the intact rNVLPs were into for the formation of VP160 and was into the and analyzed by From two were and into with a ionic or pH. was into a ionic at the pH prior to analysis The mass is in with the VP160 and VP180 species were because of the ionic that was into the ionic the VP1 oligomers to be with the range of species being by the solution pH and ionic The mass in was by the VP1 solution into a of the ionic at pH these conditions, intact rNVLPs are and the mass these the of the VP1 to and structures upon the solution It has been of capsid assembly that are to capsid formation as the effect of of in viral capsid a for PubMed Scopus Google Scholar). to be the for the rNVLPs and the VP1 oligomers and Norwalk virus is a and the presence of VP1 oligomers VP160 and and to into the of the VP1 were collected under the solution as used in the MS From the MS was found that at a pH VP1180 is at ionic to these were in VP1 in a at pH the of the particles. image and for of the T = 3 particles is as an Importantly, the particles were found to be spherical and to an diameter of 3 nm, which is in excellent with the of for VP1180 and X-ray crystallographic studies (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). we to of the intermediate VP1 VP160 and From the MS is that the formation of of these a pH and a ionic VP1 was analyzed by in a at pH with the MS VP1180 particles were and a of smaller particles was the particles were that these smaller VP1 oligomers are capsids of and The most of these particles was a of species these conditions, was found MS that the most VP1 was information regarding MS with the structural data by that the VP160 is a spherical capsid with T = icosahedral for is provided by as also observed a T = capsid with a diameter of L.J. Hardy M.E. Estes M.K. of a smaller of recombinant Norwalk virus capsids assembled in insect Virol. PubMed Google Scholar). is the VP180 Although most of the particles of nm, were species in the analysis to because of is to regarding the of the VP180 species. It is to that at of VP1 and at solution ionic strength, and pH, very VP180 was by as well From the MS a VP1 to the formation of It is to the analysis at VP1 because the with the of particles. Importantly, at VP1 a of particles with that is with the MS structural into these VP1 oligomers, the VP180 oligomers were using In the mobility of a is which information be the of the which be used to structural information the as K. mobility mass spectrometry of proteins and protein PubMed Scopus Google Scholar). The for the and VP160 oligomers are in mass the of oligomers a and with that these a similar in the are for the two observed for the capsids and of hepatitis B virus capsids in Chem. PubMed Scopus Google Scholar). In was demonstrated by that the capsids most a spherical upon to the and the be in the capsids the in with mass observed for the VP1 oligomers, that also observed and have also a is with the and that that the VP160 and VP1180 a spherical icosahedral the VP180 species on the that the VP180 also a as to an intermediate the to the T = 3 VP1180 capsid or a assembly. Although a structure consisting of 80 copies of VP1 with structures icosahedral is in the the P domain of VP1 with M. T. M. Jiang Jiang X. P structure, function and in PubMed Scopus Google Scholar). The stability of the rNVLPs was over a range of solution using native From these was that upon alkaline treatment the rNVLPs and the VP1 dimers that are into VP1 oligomers on the solution pH and ionic ionic and solution pH the assembly the formation of species consisting of and 80 copies of VP1. It was also demonstrated that the capsid formation of the VP1 oligomers was Structural analysis using and of the fully assembled and VP1 oligomers revealed that these particles a spherical was to be for the fully assembled rNVLPs and for the the structure of the VP180 capsid (dis)assembly was found to be a very and very sensitive to solution pH, ionic strength, and VP1 concentration. the of native to the of forms of a protein assembly into the a as viral assembly.