Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry

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, 2). It is the most predominant pathogen within the family Caliciviridae, which also includes Sapovirus, Vesivirus, and Lagovirus (3). 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, 5). VP1 forms homodimers, and the mature Norwalk virus capsids (T = 3) are composed of 90 VP1 dimers (6, 7) and possibly a few copies of VP2 that are thought to stabilize the icosahedral structure as well as affect the expression of VP1 (7, 8). 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),¹ which are spontaneously assembled during the expression of recombinant VP1 and VP2 in insect cells (5). Importantly, these empty noninfectious particles have been demonstrated to be morphologically and antigenically similar to the genuine virion (9).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, 9–11). 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, 10), 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 (12–15). Native mass spectrometry exploits the gentle ionization conditions afforded by electrospray ionization to transfer intact non-covalently bound protein assemblies into the gas phase. Determining the mass of these complexes with high accuracy allows the oligomeric stoichiometry to be unambiguously deduced. Traditionally challenging targets for structural biology, including complexes in the megadalton range (15–17), heterogeneous or polydisperse assemblies (18, 19), and membrane-bound protein assemblies (20) can now be interrogated in this manner. Furthermore, selective dissociation of these assemblies in both the gas and solution phases allows the designation of subcomplexes, non-covalently bound species smaller than the original protein complex. Combining the knowledge obtained from such data provides information regarding subunit organization at both the architecture and subarchitecture level, allowing the generation of low resolution maps of the overall three-dimensional structure of protein complexes (21–23). Additional information can also be obtained through the combination of tandem MS techniques. CID, for example, can be used to selectively dissociate specific protein assemblies and thus provide information regarding the stability and aid in the assignment of stoichiometry for a given complex. Another tandem MS approach, ion mobility MS (IMMS), provides additional information regarding the shape of gaseous protein complexes. In IMMS, in addition to separation based on their mass-to-charge ratio, ions are also passed through an ion mobility cell with a counterflow of neutral background gas where they are separated based on their size and shape (13, 24).The ability to perform mass measurements of intact viruses has been exploited by several groups but is often limited by mass resolution, which is impeded by the incomplete desolvation of the large protein assemblies during the ionization process. Siuzdak et al. (25) and Robinson and co-workers (26) pioneered the analyses of viruses using mass spectrometry. More recently Uetrecht et al. (15, 17) 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, 17). 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).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.     

Quantitative Proteomic Analysis of Host-virus Interactions Reveals a Role for Golgi Brefeldin A Resistance Factor 1 (GBF1) in Dengue Infection

Dengue virus is considered to be the most important mosquito-borne virus worldwide and poses formidable economic and health care burdens on many tropical and subtropical countries. Dengue infection induces drastic rearrangement of host endoplasmic reticulum membranes into complex membranous structures housing replication complexes; the contribution(s) of host proteins and pathways to this process is poorly understood but is likely to be mediated by protein-protein interactions. We have developed an approach for obtaining high confidence protein-protein interaction data by employing affinity tags and quantitative proteomics, in the context of viral infection, followed by robust statistical analysis. Using this approach, we identified high confidence interactors of NS5, the viral polymerase, and NS3, the helicase/protease. Quantitative proteomics allowed us to exclude a large number of presumably nonspecific interactors from our data sets and imparted a high level of confidence to our resulting data sets. We identified 53 host proteins reproducibly associated with NS5 and 41 with NS3, with 13 of these candidates present in both data sets. The host factors identified have diverse functions, including retrograde Golgi-to-endoplasmic reticulum transport, biosynthesis of long-chain fatty-acyl-coenzyme As, and in the unfolded protein response. We selected GBF1, a guanine nucleotide exchange factor responsible for ARF activation, from the NS5 data set for follow up and functional validation. We show that GBF1 plays a critical role early in dengue infection that is independent of its role in the maintenance of Golgi structure. Importantly, the approach described here can be applied to virtually any organism/system as a tool for better understanding its molecular interactions.Viruses modify the intracellular environment of infected host cells in a number of important ways, including subverting the antiviral response, reorganizing host membranes, and manipulating host signaling pathways to create an environment more favorable for infection. For example, some viral proteins co-opt host proteins to degrade host interferon signaling components, thus antagonizing the antiviral response (1, 2); other viral proteins recruit metabolic enzymes that are potentially involved in the biogenesis of replication complexes (RCs)¹ (3); and some viral proteins interact with host regulatory proteins to block the cellular stress response (4). These examples illustrate only a few of the ways in which viral-host protein-protein interactions (PPIs) enable the viral life cycle and drive pathogenicity. Because of the limited coding capacity of many viral genomes, in particular RNA virus genomes, viral-host PPIs generally occur between a remarkably small number of viral proteins and a much larger number of host proteins (5). The study of these extensive interactions necessitates comprehensive and quantitative approaches, the development and validation of which will potentially contribute to: 1) our understanding of the mechanisms by which viruses subvert cellular pathways to their own advantage; 2) our understanding of fundamental cell biology; 3) the choice of potential drug targets and the rational design of such drugs; and 4) our understanding of the host response to infection.Dengue virus (DENV) is a positive-sense, single stranded RNA virus in the family Flaviviridae that is transmitted by the bite of an infected Aedes mosquito (6). DENV is an important emerging pathogen that is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, diseases which cumulatively pose formidable economic and health care burdens in many tropical and subtropical countries worldwide (7). Recent estimates of the global burden of DENV infection have revealed that DENV infection is ∼threefold more prevalent than previously estimated, with ∼400 million annual incidences worldwide (8). Moreover, development of an anti-DENV vaccine has been hindered by the existence of four antigenically distinct DENV serotypes (DENV-1, -2, -3, and -4), each of which is capable of producing the full spectrum of DENV-induced disease (9). DENV is also related to other flaviviruses that cause significant human disease, including yellow fever virus, West Nile virus, and Japanese encephalitis virus (10). Thus, insights into DENV biology may be applicable to other flaviviruses of medical importance.The flavivirus genome encodes only three structural (C, pr/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), and is translated as a single polyprotein, which is later cleaved into the mature viral proteins (6). The three structural proteins, capsid (C), membrane (M), and envelope (E) comprise the virion, whereas the NS proteins are mainly responsible for carrying out genome replication in infected cells. Among the seven NS proteins, NS5 and NS3 are the two largest and most highly conserved proteins (11); moreover, each possesses multiple enzymatic activities. NS5 contains an RNA-dependent RNA polymerase domain as well as a nucleoside-2′-O-methyltransferase domain; both of these activities are essential for replication (12, 13). NS3, on the other hand, possesses an N-terminal serine protease domain, which is responsible for cleaving the viral polyprotein at several sites (along with its cofactor, NS2B) (14). The C-terminal domain of NS3 has 5′ RNA triphosphatase, nucleoside triphosphatase, and helicase activities (15–17). NS5 and NS3 have been shown to interact in infected cells (18), most likely in the RC. The precise composition and biogenesis mechanisms of RCs are poorly understood, but likely involve host proteins as well as viral proteins. As with other viruses, DENV-host PPIs have been interrogated by a number of high-throughput yeast two-hybrid assays (19–31) and approaches coupling either affinity purification (AP), immunoprecipitation, or immunoaffinity purification (IP) with MS (32–35). These approaches have yielded a number of putative DENV-host PPIs; however, considering the large repertoire of interactions undertaken by other viruses (36–41), our knowledge of DENV-host PPIs is likely incomplete. One advantage of IP/MS approaches is their potential to comprehensively reveal bona fide time-resolved interactions from the environment of an infected cell; however, the extremely high sensitivity of modern mass spectrometers highlights the need to develop IP/MS workflows capable of reliably discriminating between genuine interactors and nonspecific contaminants (42). Here, we present a workflow incorporating immunoaffinity purification and quantitative proteomics from infected cells, followed by robust statistical analysis to identify high confidence interactors of virtually any protein of interest, and apply this workflow to DENV NS5 and NS3.     

Protein Analysis of Purified Respiratory Syncytial Virus Particles Reveals an Important Role for Heat Shock Protein 90 in Virus Particle Assembly

In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of β-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy.Respiratory syncytial virus (RSV)¹ belongs to the paramyxovirus group of viruses, and it is the most important respiratory virus causing lower respiratory tract infection in young children and neonates. The mature RSV particle comprises a ribonucleoparticle (RNP) core formed by the interaction between the viral genomic RNA (vRNA), the nucleocapsid (N) protein (42 kDa), the phospho (P) protein (35 kDa), and the large (L) protein (250 kDa). The RNP core is visualized by electron microscopy as a strand of repeating N protein subunits that form a herringbone-like structure of ∼10–20 nm in diameter (1). Although the minimal functional polymerase activity requires an association between the N, P, and L proteins and the virus genome vRNA (2–4), additional viral proteins called the M2-1 protein (22 kDa), M2-2 protein, and M protein (28 kDa) regulate the activity of the polymerase (5–8). The virus is surrounded by a lipid envelope that is formed from the host cell during the budding process in which the three virus membrane proteins are inserted. The G protein (90 kDa) mediates attachment of the virus to the cell during virus entry (9), and the fusion (F) protein (10) mediates the fusion of the virus and host cell membranes during virus entry, whereas the role of the SH protein is currently unknown. In addition, two non-structural proteins called NS1 and NS2, which are thought not to be present in the virus particle but play a role in countering the host innate immune response (11), are expressed.During virus infection two distinct virus structures are formed, virus filaments and inclusion bodies. The virus filaments are membrane-bound structures that are ∼150–200 nm thick and can be up to 6 μm in length (1, 12–16); they form at the sites of virus assembly and are the progeny viruses. The inclusion bodies form in the cytoplasm and can be several μm in diameter, consisting of accumulations of RNP cores (17–19). Inclusion bodies are found in all RSV-infected tissue culture cells, and they have also been observed in biopsy material isolated from RSV-infected patients (20) suggesting a clinical relevance. Although the cellular processes that lead to assembly of the mature virus filaments are still poorly understood, the involvement of lipid raft microdomains and the cortical cytoskeleton network appear to play an important role in this process (16, 21–25). For example, rhoA kinase is a raft-associated signaling molecule that is involved in regulating actin structure (26), and it has been implicated in virus filament formation (27, 28). Virus filament formation also requires phosphoinositide 3-kinase (PI3K) activity (25, 29, 30); PI3K is a raft-associated kinase activated by rhoA kinase (31). The identification of cellular proteins that interact with the virus particles should further facilitate the identification of the cellular pathways that are involved in RSV maturation. In this study, we examined virus-host cell interactions during RSV assembly using a combination of advanced imaging techniques and analyzed the protein content of purified virus particles by proteomics technology. Our analysis provides evidence that cellular proteins that regulate actin structures in the cell may also play an important role in formation of infectious RSV particles, and that the HSP90 protein plays an important role in the virus assembly process.     

Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections

The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 10³ 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction. Also, our results suggest that VP4 may be involved in, but is not exclusively responsible for, host range restriction in the rabbit model. The replication efficiency of rotavirus in rabbits also is not controlled by the product of gene 5 (NSP1) alone, since a reassortant rotavirus with ALA gene 5 and all other genes from SA11 was more severely replication restricted than either parental rotavirus strain.Rotaviruses are the leading cause of acute viral gastroenteritis in humans and animals throughout the world. Rotaviruses belong to the Reoviridae family and are characterized by a genome consisting of 11 segments of double-stranded RNA (dsRNA), enclosed in a triple-layered protein capsid (28). Serotype designations are based on independent neutralization determinants on the two outer capsid proteins VP4 (P serotypes, for protease-sensitive protein) and VP7 (G serotypes, for glycoprotein) (28). Serotype specificity determined by cross-neutralization assays using hyperimmune sera against the whole virus is mainly defined by VP7, and 14 G serotypes have been identified (28). Recently, antisera or monoclonal antibodies raised to VP4 and sequence analysis of VP4 identified 12 P serotypes and 20 P genotypes, respectively (28, 39). Rotavirus VP4 protein is responsible for a number of important biological functions, such as the enhancement of infectivity by proteolytic cleavage of VP4 into VP8* and VP5*, hemagglutination, restricted growth in cell culture, virulence, initial virus attachment to cells, and protease sensitivity associated with plaque formation (1, 4, 25, 34, 40, 51).The use of animal models, including the rabbit and mouse models, has been essential to the understanding of rotavirus infection, pathology, disease, immunity, and testing of prospective vaccines in children (21). The limitations of the rabbit and adult mouse models of rotavirus infection for vaccine testing are as follows: (i) human rotavirus (HRV) strains do not efficiently replicate in either animal, (ii) clinical disease is not observed, and (iii) only homologous virus strains (isolated from the same species) replicate efficiently and spread horizontally to uninoculated control animals, whereas heterologous virus strains (isolated from a different species) do not (6, 15, 16, 29, 31, 35, 37, 44, 50, 55). We and others developed a rabbit model of rotavirus infection that is useful for defining basic parameters of active immunity, immunogenicity, and protective efficacy of vaccines (12, 15–21, 36, 61). Rabbits are productively infected with homologous lapine rotavirus strains up to at least the age of 5 years, which allows examination of active and long-term immunity for vaccine studies (13, 15–17, 36, 61). Group A lapine rotavirus strains have been isolated in Canada, Japan, Italy, and the United States, and those that have been characterized are serotype G3 (8, 11, 15, 53, 56, 61). Recently, the P type of four different strains was identified as genotype P[14] (11). Previously, limited infection of rabbits with a heterologous strain had been obtained only with SA11 Cl3 (P[2], G3) (15).Attempts to identify host range and virulence determinants for rotavirus have implicated different constellations of genes, including genes 2, 3, 4, 5, 8, 9, 10, and 11 (5, 23, 30, 33, 37, 38, 41, 43, 44, 60, 62, 65). Although host range restriction and virulence may be multigenic, two genes, 4 and 5, are of interest because they cluster according to species of origin, suggesting a role in host range restriction. The finding that genome segment 5 (NSP1) sequences cluster according to species of origin (24, 39, 65) and that, in the mouse model, gene 5 segregates with transmission of virus among littermates (5), led to the hypothesis that NSP1 is involved in host range restriction. VP4 sequence analyses of rotavirus strains isolated from different species revealed that specific VP4 types also generally correlate with the species of origin of each rotavirus strain (43, 60). Therefore, once we identified the P type of four lapine rotaviruses as P[14], we tested two P[14] HRV strains, PA169 (G6) and HAL1166 (G8) (32) to determine if VP4 is involved in host range restriction. We also tested several other HRV strains, live attenuated reassortant candidate vaccine strains [rhesus rotavirus (RRV) × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4)], and animal rotavirus strains of different P and G types to determine if they could productively infect rabbits. In addition, to evaluate whether the single rotavirus gene 5 is responsible for replication efficiency in rabbits, rabbits were inoculated with a reassortant rotavirus with the lapine ALA gene 5 and all the other genes from the simian rotavirus SA11 Cl3 strain.