[Show abstract][Hide abstract] ABSTRACT: Objectives
The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness.Methods
Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion.Key findingsThe data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection.Conclusions
Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology.
[Show abstract][Hide abstract] ABSTRACT: Background
Ebolaviruses causes a severe and often fatal hemorrhagic fever in humans, with some species such as Ebola virus having case fatality rates approaching 90%. Currently the worst Ebola virus outbreak since the disease was discovered is occurring in West Africa. Although thought to be a zoonotic infection, a concern is that with increasing numbers of humans being infected, Ebola virus variants could be selected which are better adapted for human-to-human transmission.ResultsTo investigate whether genetic changes in Ebola virus become established in response to adaptation in a different host, a guinea pig model of infection was used. In this experimental system, guinea pigs were infected with Ebola virus (EBOV), which initially did not cause disease. To simulate transmission to uninfected individuals, the virus was serially passaged five times in naive animals. As the virus was passaged, virulence increased and clinical effects were observed in the guinea pig. An RNAseq and consensus mapping approach was then used to evaluate potential nucleotide changes in the Ebola virus genome at each passage.Conclusions
Upon passage in the guinea pig model, EBOV become more virulent, RNA editing and also coding changes in key proteins become established. The data suggest that the initial evolutionary trajectory of EBOV in a new host can lead to a gain in virulence. Given the circumstances of the sustained transmission of EBOV in the current outbreak in West Africa, increases in virulence may be associated with prolonged and uncontrolled epidemics of EBOV.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication.
Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription, with a view to providing potential targets for the development of a specific, transient therapeutic which disrupts virus biology but prevents the emergence of resistance, while maintaining cell viability. In particular, protein chaperones (heat shock proteins 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested, and this study demonstrates to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.
Journal of Virology 10/2014; 89(2). DOI:10.1128/JVI.01783-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral pathogenesis in the infected cell is a balance between anti-viral responses and subversion of host cell processes. Many viral proteins specifically interact with host cell proteins in order to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for anti-viral therapy. One such virus is Ebola that has profound consequences for human health, and causes viral haemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response, and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed thus placing confidence in the technique but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signalling. Disrupting the activity of ATP1A1 in Ebola virus infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.
Journal of Proteome Research 08/2014; 13(11). DOI:10.1021/pr500556d · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.
Proceedings of the National Academy of Sciences 01/2014; 111(4). DOI:10.1073/pnas.1317262111 · 9.67 Impact Factor
[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
nucleocapsid (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 sequestered, 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 tetramers
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; DOI:10.1093/nar/gkt268 · 9.11 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. DOI:10.1128/JVI.01555-12 · 4.44 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 G1 phase of the cell cycle.
Journal of Virology 05/2012; 86(15):7777-89. DOI:10.1128/JVI.00460-12 · 4.44 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. DOI:10.1107/S1744309112009736 · 0.53 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. DOI:10.1099/vir.0.035105-0 · 3.18 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. DOI:10.1128/JVI.05164-11 · 4.44 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. DOI:10.1099/vir.0.024240-0 · 3.18 Impact Factor