[show abstract][hide abstract] ABSTRACT: Simple, spherical RNA viruses have well-understood, symmetric protein capsids, but little structural information is available for their asymmetric components, such as minor proteins and their genomes, which are vital for infection. Here, we report an asymmetric structure of bacteriophage MS2, attached to its receptor, the F-pilus. Cryo-electron tomography and subtomographic averaging of such complexes result in a structure containing clear density for the packaged genome, implying that the conformation of the genome is the same in each virus particle. The data also suggest that the single-copy viral maturation protein breaks the symmetry of the capsid, occupying a position that would be filled by a coat protein dimer in an icosahedral shell. This capsomere can thus fulfill its known biological roles in receptor and genome binding and suggests an exit route for the genome during infection.
[show abstract][hide abstract] ABSTRACT: All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nu-cleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å , respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply seques-tered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetra-mers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.
Nucleic Acids Research 04/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N- and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N- and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision.
Journal of Virology 08/2012; 86(20):10914-23. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viral proteins can have multiple effects on host cell biology. Human respiratory syncytial virus (HRSV) nonstructural protein 1 (NS1) is a good example of this. During the virus life cycle, NS1 can act as an antagonist of host type I and III interferon production and signaling, inhibit apoptosis, suppress dendritic cell maturation, control protein stability, and regulate transcription of host cell mRNAs, among other functions. It is likely that NS1 performs these different roles through interactions with multiple host cell proteins. To investigate this and identify cellular proteins that could interact with NS1, we used quantitative proteomics in combination with green fluorescent protein (GFP)-trap immunoprecipitation and bioinformatic analysis. This analysis identified 221 proteins that were potentially part of complexes that could interact with NS1, with many of these associated with transcriptional regulation as part of the mediator complex, cell cycle regulation, and other functions previously assigned to NS1. Specific immunoprecipitation using the GFP trap was used to confirm the ability of selected cellular proteins to interact individually with NS1. Infection of A549 cells with recombinant viruses deficient in the expression of NS1 and overexpression analysis both demonstrated that NS1 was necessary and sufficient for the enrichment of cells in the G(1) phase of the cell cycle.
Journal of Virology 05/2012; 86(15):7777-89. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Influenza A virus is one of the world's major uncontrolled pathogens, causing seasonal epidemics as well as global pandemics. This was evidenced by the recent emergence and now prevalence of the 2009 swine origin pandemic H1N1 influenza A virus. In this study, quantitative proteomics using stable isotope labelling with amino acids in cell culture was used to investigate the changes in the host cell proteome in cells infected with pandemic H1N1 influenza A virus. The study was conducted in A549 cells that retain properties similar to alveolar cells. Several global pathways were affected, including cell cycle regulation and lipid metabolism, and these could be correlated with recent microarray analyses of cells infected with influenza A virus. Taken together, both quantitative proteomics and transcriptomic approaches can be used to identify potential cellular proteins whose functions in the virus life cycle could be targeted for chemotherapeutic intervention.
[show abstract][hide abstract] ABSTRACT: Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Nairovirus genus within the Bunyaviridae family of segmented negative-sense RNA viruses. This paper describes the expression, purification and crystallization of full-length CCHFV nucleocapsid (N) protein and the collection of a 2.1 Å resolution X-ray diffraction data set using synchrotron radiation. Crystals of the CCHFV N protein belonged to space group C2, with unit-cell parameters a = 150.38, b = 72.06, c = 101.23 Å, β = 110.70° and two molecules in the asymmetric unit. Circular-dichroism analysis provided insight into the secondary structure, whilst gel-filtration analysis revealed possible oligomeric states of the N protein. Structural determination is ongoing.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 05/2012; 68(Pt 5):569-73. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viruses continue to pose some of the greatest threats to human and animal health, and food security worldwide. Therefore, new approaches are required to increase our understanding of virus-host cell interactions and subsequently design more effective therapeutic countermeasures. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture (SILAC), coupled to LC-MS/MS and bioinformatic analysis, is providing an excellent resource for studying host cell proteomes and can readily be applied for the study of virus infection. Here, we review this approach and discuss how virus-host cell interactions can best be studied, what is realistically feasible, and the potential limitations. For example, sub-cellular fractionation can reduce sample complexity for LC-MS/MS, increase data return and provide information regarding protein trafficking between different cellular compartments. The key to successful quantitative proteomics combines good experimental design and appropriate sample preparation with statistical analysis and validation of the MS data through the use of independent techniques and functional analysis. The annotation of the human genome and the increasing availability of biological reagents such as antibodies, provide the optimum parameters for studying viruses that infect humans, in human cell lines. SILAC-based quantitative proteomics can also be used to study the interactome of viral proteins with the host cell. Coupling proteomic studies with global transcriptomic and RNA depletion experiments will provide great insights into the complexity of the infection process, and potentially reveal new antiviral targets.
[show abstract][hide abstract] ABSTRACT: Human respiratory syncytial virus (HRSV) is a member of the family Paramyxoviridae, and is responsible for serious respiratory illness in infants, the elderly and the immunocompromised. HRSV exists as two distinct lineages known as subgroups A and B, which represent two lines of divergent evolution with extensive genetic and serologic differences. While both subgroup A and B viruses contribute to overall HRSV disease, subgroup A isolates are associated with both increased frequency and morbidity of infections, and reasons for this are unclear. HRSV disease is characterized by virus-mediated cell destruction in combination with extensive inflammatory and immune modulatory responses, and for HRSV subgroup A isolates, several of these signaling pathways are regulated through activation of the transcription factor NF-κB. In contrast, the NF-κB activation characteristics of HRSV subgroup B infection remain untested. Here, we performed a quantitative and comparative analysis of NF-κB activation in response to infection of both continuous and primary cell cultures with HRSV subgroup A and B isolates. Our results showed the model HRSV subgroup A isolate consistently induced increased NF-κB activation compared to its HRSV subgroup B counterpart. The differential NF-κB activation characteristics of HRSV subgroup A and B viruses may contribute to differences in their pathogenesis.
[show abstract][hide abstract] ABSTRACT: The family Bunyaviridae of segmented, negative-stranded RNA viruses includes over 350 members that infect a bewildering variety of animals and plants. Many of these bunyaviruses are the causative agents of serious disease in their respective hosts, and are classified as emerging viruses because of their increased incidence in new populations and geographical locations throughout the world. Emerging bunyaviruses, such as Crimean-Congo hemorrhagic fever virus, tomato spotted wilt virus and Rift Valley fever virus, are currently attracting great interest due to migration of their arthropod vectors, a situation possibly linked to climate change. These and other examples of continued emergence suggest that bunyaviruses will probably continue to pose a sustained global threat to agricultural productivity, animal welfare and human health. The threat of emergence is particularly acute in light of the lack of effective preventative or therapeutic treatments for any of these viruses, making their study an important priority. This review presents recent advances in the understanding of the bunyavirus life cycle, including aspects of their molecular, cellular and structural biology. Whilst special emphasis is placed upon the emerging bunyaviruses, we also describe the extensive body of work involving model bunyaviruses, which have been the subject of major contributions to our overall understanding of this important group of viruses.
Journal of General Virology 08/2011; 92(Pt 11):2467-84. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viruses can modify conditions inside cells to make them more favorable for replication and progeny virus production. One way of doing this is through manipulation of the cell cycle, a process that describes the ordered growth and division of cells. Analysis of model cell lines, such as A549 cells and primary airway epithelial cells, infected with human respiratory syncytial virus (HRSV) has shown alteration of the cell cycle during infection, although the signaling events were not clearly understood. In this study, targeted transcriptomic analysis of HRSV-infected primary airway epithelial cells revealed alterations in the abundances of many mRNAs encoding cell cycle-regulatory molecules, including decreases in the D-type cyclins and corresponding cyclin-dependent kinases (CDK4 and CDK6 [CDK4/6]). These alterations were reflected in changes in protein abundance and/or relocalization in HRSV-infected cells; taken together, they were predicted to result in G(0)/G(1) phase arrest. In contrast, there was no change in the abundances of D-type cyclins in A549 cells infected with HRSV. However, the abundance of the G(1)/S phase progression inhibitor p21(WAF1/CIP1) was increased over that in mock-treated cells, and this, again, was predicted to result in G(0)/G(1) phase arrest. The G(0)/G(1) phase arrest in both HRSV-infected primary cells and A549 cells was confirmed using dual-label flow cytometry that accurately measured the different stages of the cell cycle. Comparison of progeny virus production in primary and A549 cells enriched in G(0)/G(1) using a specific CDK4/6 kinase inhibitor with asynchronously replicating cells indicated that this phase of the cell cycle was more efficient for virus production.
Journal of Virology 07/2011; 85(19):10300-9. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human respiratory syncytial virus (HRSV) is a leading cause of serious lower respiratory tract infections in infants. The virus has two subgroups A and B, which differ in prevalence and (nucleotide) sequence. The interaction of subgroup A viruses with the host cell is relatively well characterized, whereas for subgroup B viruses it is not. Therefore quantitative proteomics was used to investigate the interaction of subgroup B viruses with A549 cells, a respiratory cell line. Changes in the cellular proteome and potential canonical pathways were determined using SILAC coupled to LC-MS/MS and Ingenuity Pathway Analysis. To reduce sample complexity and investigate potential trafficking both nuclear and cytoplasmic fractions were analyzed. A total of 904 cellular and six viral proteins were identified and quantified, of which 112 cellular proteins showed a twofold or more change in HRSV-infected cells. Data sets were validated using indirect immunofluorescence confocal microscopy on independent samples. Major changes were observed in constituents of mitochondria including components of the electron transport chain complexes and channels, as well as increases in the abundance of the products of interferon-stimulated genes. This is the first quantitative proteomic analysis of cells infected with HRSV-subgroup B.
[show abstract][hide abstract] ABSTRACT: Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.
[show abstract][hide abstract] ABSTRACT: The genome of Bunyamwera virus (BUNV) comprises three RNA segments that are encapsidated by the virus-encoded nucleocapsid (N) protein to form ribonucleoprotein (RNP) complexes. These RNPs are the functional templates for RNA synthesis by the virus-encoded RNA-dependent RNA polymerase (RdRp). We investigated the roles of conserved positively charged N-protein amino acids in RNA binding, in oligomerization to form model RNPs and in generating RNP templates active for both RNA replication and mRNA transcription. We identified several residues that performed important roles in RNA binding, and furthermore showed that a single amino acid change can differentially affect the ability of the resulting RNP templates to regulate the transcription and replication activities of the RdRp. These results indicate that the BUNV N protein possesses functions outside of its primary role of RNA encapsidation.
Journal of General Virology 10/2010; 92(Pt 1):80-4. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract disease in infants. The HRSV small hydrophobic (SH) protein plays an important role in HRSV pathogenesis, although its mode of action is unclear. Analysis of the ability of SH protein to induce membrane permeability and form homo-oligomers suggests it acts as a viroporin. For the first time, we directly observed functional SH protein using electron microscopy, which revealed SH forms multimeric ring-like objects with a prominent central stained region. Based on current and existing functional data, we propose this region represents the channel that mediates membrane permeability.
[show abstract][hide abstract] ABSTRACT: The genomic termini of RNA viruses contain essential cis-acting signals for such diverse functions as packaging, genome translation, mRNA transcription, and RNA replication, and thus preservation of their sequence integrity is critical for virus viability. Sequence alteration can arise due to cellular mechanisms that add or remove nucleotides from terminal regions, or, alternatively, from introduction of sequence errors through nucleotide misincorporation by the error-prone viral RNA-dependent RNA polymerase (RdRp). To preserve template function, many RNA viruses utilize repair mechanisms to prevent accumulation of terminal alterations. Here we show that Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family of segmented negative-sense RNA viruses, also can repair its genomic termini. When an intact nontranslated region (NTR) was added to the anti-genomic 3' end, it was precisely removed, to restore both length and RNA synthesis function of the wild-type template. Furthermore, when nucleotides were removed from the anti-genome 3' end, and replaced with a duplicate and intact NTR, both the external NTR were removed, and the missing nucleotides were restored, thus, indicating that the BUNV RdRp can both remove and add nucleotides to the template. We show that the mechanism for repair of terminal extensions is likely that of internal entry of the viral RdRp during genome synthesis. Possible mechanisms for repair of terminal deletions are discussed.
[show abstract][hide abstract] ABSTRACT: RNA genomes are vulnerable to corruption by a range of activities, including inaccurate replication by the error-prone replicase, damage from environmental factors, and attack by nucleases and other RNA-modifying enzymes that comprise the cellular intrinsic or innate immune response. Damage to coding regions and loss of critical cis-acting signals inevitably impair genome fitness; as a consequence, RNA viruses have evolved a variety of mechanisms to protect their genome integrity. These include mechanisms to promote replicase fidelity, recombination activities that allow exchange of sequences between different RNA templates, and mechanisms to repair the genome termini. In this article, we review examples of these processes from a range of RNA viruses to showcase the diverse approaches that viruses have evolved to maintain their genome sequence integrity, focusing first on mechanisms that viruses use to protect their entire genome, and then concentrating on mechanisms that allow protection of the genome termini, which are especially vulnerable. In addition, we discuss examples in which it might be beneficial for a virus to 'lose' its genomic termini and reduce its replication efficiency.
Journal of General Virology 03/2010; 91(Pt 6):1373-87. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bunyamwera virus (BUNV) is the prototypic member of both the Orthobunyavirus genus and the Bunyaviridae family of negative stranded RNA viruses. In common with all negative stranded RNA viruses, the BUNV genomic and anti-genomic strands are not naked RNAs, but instead are encapsidated along their entire lengths with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex. This association is critical for the negative strand RNA virus life cycle because only RNPs are active for productive RNA synthesis and RNA packaging. We are interested in understanding the molecular details of how N and RNA components associate within the bunyavirus RNP, and what governs the apparently selective encapsidation of viral replication products. Toward this goal, we recently devised a protocol that allowed generation of native BUNV N protein that maintained solubility under physiological conditions and allowed formation of crystals that yielded high-resolution x-ray diffraction data. Here we extend this work to show that this soluble N protein is able to oligomerize and bind RNA to form a highly uniform RNP complex, which exhibits characteristics in common with the viral RNP. By extracting and sequencing RNAs bound to these model RNPs, we determined the stoichiometry of N-RNA association to be approximately 12 nucleotides per N monomer. In addition, we defined the minimal sequence requirement for BUNV RNA replication. By comparing this minimal sequence to those bound to our model RNP, we conclude that N protein does not obligatorily require a sequence or structure for RNA encapsidation.
[show abstract][hide abstract] ABSTRACT: The heptauridine tract at each gene end and intergenic region (IGR) at the gene junctions of vesicular stomatitis virus (VSV) have effects on synthesis of the downstream mRNA, independent of their respective roles in termination of the upstream mRNA. To investigate the role of the U tract and the IGR in downstream gene transcription, we altered the N/P gene junction of infectious VSV such that transcription levels would be affected and result in altered molar ratios of the N and P proteins, which are critical for optimal viral RNA replication. The changes included extended IGRs between the N and P genes and shortening the length of the heptauridine tract upstream of the P gene start. Viruses having various combinations of these changes were recovered from cDNA and selective pressure for efficient viral replication was applied by sequential passage in cell culture. The replicative ability and sequence at the altered intergenic junctions were monitored throughout the passages to compare the effects of the changes at the IGR and U tract. VSV variants with wild-type U tracts upstream of the P gene replicated to levels similar to wt VSV. Variants with shortened U tracts were reduced in their ability to replicate. With passage, populations emerged that replicated to higher levels. Sequence analysis revealed that mutations had been selected for in these populations that increased the length of the U tract. This correlated with an increase in abundance of P mRNA and protein to provide improved N:P protein molar ratios. Extended IGRs resulted in decreased downstream transcription but the effect was not as extensive as that caused by shortened U tracts. Extended IGRs were not selected against in 5 passages. Our results indicate that the size of the upstream gene end U tract is an important determinant of efficient downstream gene transcription in infectious virus.
[show abstract][hide abstract] ABSTRACT: Transcription by the vesicular stomatitis virus (VSV) polymerase has been characterized as obligatorily sequential with transcription of each downstream gene dependent on termination of the gene immediately upstream. In studies described here we investigated the ability of the VSV RNA-dependent RNA polymerase (RdRp) to access mRNA initiation sites located at increasing distances either downstream or upstream of a transcription termination signal. Bi-cistronic subgenomic replicons were constructed containing progressively extended intergenic regions preceding the initiation site of a downstream gene. The ability of the RdRp to access the downstream sites was progressively reduced as the length of the intergenic region increased. Alternatively, bi-cistronic replicons were constructed containing an mRNA start signal located at increasing distances upstream of a termination site. Analysis of transcription of these "overlapped" genes showed that for an upstream mRNA start site to be recognized it had to contain not only the canonical 3'-UUGUCnnUAG-5' gene start signal, but that signal needed also to be preceded by a U7 tract. Access of these upstream mRNA initiation sites by the VSV RdRp was proportionately reduced with increasing distance between the termination site and the overlapped initiation signal. Possible mechanisms for how the RdRp accesses these upstream start sites are discussed.
[show abstract][hide abstract] ABSTRACT: Messenger RNA transcription by Bunyaviridae family members is unique within the group of negative-strand RNA viruses as it requires on-going protein synthesis. The long-standing model explaining this phenomenon proposes that the translational requirement is not for a protein product, but instead is for ribosomal translocation along nascent mRNAs. This movement is proposed to disrupt spurious transcription termination signals that otherwise cause premature mRNA truncation leading to a fatal loss of gene expression. This model was tested by introducing translational stop codons into model RNA genomes of Bunyamwera virus, the prototypic member of the Bunyaviridae family. This directly showed that translation of nascent mRNAs prevents transcription termination. While such coupling of transcription and translation is common in prokaryotic systems, these results represent the first report of such obligatory coupling in a eukaryotic cell environment. The results also provide insight into the bunyavirus termination mechanism and suggest it is mechanistically similar to prokaryotic intrinsic termination.