Brian K. Dove

Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism.

ABSTRACT A recombinant infectious bronchitis virus (IBV), BeauR-M41(S), was generated using our reverse genetics system (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001), in which the ectodomain region of the spike gene from IBV M41-CK replaced the corresponding region of the IBV Beaudette genome. BeauR-M41(S) acquired the same cell tropism phenotype as IBV M41-CK in four different cell types, demonstrating that the IBV spike glycoprotein is a determinant of cell tropism.

Quantitative Proteomic Analysis of A549 Cells Infected with Human Respiratory Syncytial Virus

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.

Mass Spectroscopic Characterization of the Coronavirus Infectious Bronchitis Virus Nucleoprotein and Elucidation of the Role of Phosphorylation in RNA Binding by Using Surface Plasmon Resonance

ABSTRACT Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphosphorylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5′ end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Cell Cycle Perturbations Induced by Infection with the Coronavirus Infectious Bronchitis Virus and Their Effect on Virus Replication

ABSTRACT In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.

Using SILAC and quantitative proteomics to investigate the interactions between viral and host proteomes

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.

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.

Cell Cycle Dependent Nucleolar Localization of the Coronavirus Nucleocapsid Protein

The nucleolus is a dynamic sub-nuclear structure which is involved in ribosome subunit biogenesis, modulation of cell growth and response to cell stress. The nucleolar proteome varies particularly with regard to the cell cycle. Viral proteins can localise to the nucleolus and using the coronavirus nucleocapsid (N) protein as a model, the cell cycle dependent trafficking of viral proteins to the nucleolus was investigated. Cell synchronisation studies coupled to live cell confocal microscopy indicated that nucleolar localisation of N protein was greater in the G2/M phase of the cell cycle than at other stages. This result was reinforced when FRAP and FLIP analysis indicated that N protein was more mobile within the nucleoplasm and nucleolus in the G2/M phase of the cell cycle. The data suggested that viral nucleolar proteins can also localise to the nucleolus in a cell cycle dependent manner and this may be related to dynamic trafficking.

A quantitative proteomic analysis of lung epithelial (A549) cells infected with 2009 pandemic influenza A virus using stable isotope labelling with amino acids in cell culture

Influenza A virus is one of the worlds 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

The Cell Cycle and Virus Infection

A number of different viruses interact with the cell cycle in order to subvert host-cell function and increase the efficiency of virus replication; examples can be found from DNA, retro, and RNA viruses. The majority of studies have been conducted on DNA and retroviruses whose primary site of replication is the nucleus, but increasingly a number of researchers are demonstrating that RNA viruses, whose primary site of replication is normally the cytoplasm, also interfere with the cell cycle. Viral interference with the cell cycle can have a myriad of different effects to improve virus infection, for example to promote replication of viral DNA genomes, or to delay the cell cycle to allow sufficient time for RNA virus assembly. Although cell cycle control is fairly well characterized in terms of checkpoints and control molecules (e.g., cyclins), in recent years several researchers have demonstrated that the nucleolus is also involved in cell cycle control. The nucleolus and associated subnuclear structures can sequester cell cycle regulatory complexes, and nucleolar proteins also have a direct and indirect effect on the cycling cell. Viruses also interact with the nucleolus. In order to study the interactions between a virus and the cell cycle and vice versa we have developed and adapted a number of different approaches and strategies. These include determinations of virus yield and measurements of virus replication to flow cytometry and confocal analysis of the host cell. Increasingly we have found that proteomic approaches allow the rapid analysis of a whole plethora of cell cycle proteins that may be affected by virus infection.

Hydrogen peroxide vapour decontamination of surfaces artificially contaminated with norovirus surrogate feline calicivirus

BACKGROUND Noroviruses are a leading cause of gastrointestinal disease and are of particular concern in healthcare settings such as hospitals. As the virus is reported to be environmentally stable, effective decontamination following an outbreak is required to prevent recurrent outbreaks. AIM To investigate the use of hydrogen peroxide vapour to decontaminate a number of surfaces that had been artificially contaminated with feline calicivirus (FCV), a surrogate for norovirus. The surfaces tested were representative of those found in hospital wards. METHODS FCV was used to contaminate materials representative of a hospital setting (stainless steel, glass, vinyl flooring, ceramic tile and PVC plastic cornering). The carriers were exposed to 30% (w/w) hydrogen peroxide vapour at 5-min intervals over 20 min, after which postexposure viral titres were measured. FINDINGS Hydrogen peroxide vapour reduced the viral titre by 4 log(10) on all surfaces tested within 20 min of exposure. The reduction in viral titre took longest to achieve on stainless steel (20 min), and the quickest effect was seen on vinyl flooring (10 min). For glass, plastic and ceramic tile surfaces, the desired reduction in viral titre was seen within 15 min of exposure. Hydrogen peroxide vapour allows for large-scale decontamination of areas following outbreaks of infectious organisms. CONCLUSION Hydrogen peroxide vapour is effective against FCV and is active on a range of surfaces. Therefore, it may represent a suitable decontamination system for use following a hospital outbreak of norovirus.