ABSTRACT
Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious agents on host cells.
Subject(s)
Epithelial Cells/immunology , Host-Pathogen Interactions/immunology , Proteome/immunology , Respiratory Mucosa/immunology , Respiratory Syncytial Virus, Human/immunology , Cell Extracts/chemistry , Cell Line , Chromatography, High Pressure Liquid , Epithelial Cells/metabolism , Epithelial Cells/virology , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Humans , Interferons/genetics , Interferons/immunology , Interferons/metabolism , NF-kappa B p52 Subunit/genetics , NF-kappa B p52 Subunit/immunology , NF-kappa B p52 Subunit/metabolism , Peptide Fragments/analysis , Proteolysis , Proteome/genetics , Proteome/metabolism , Respiratory Mucosa/metabolism , Respiratory Mucosa/virology , Respiratory Syncytial Virus, Human/metabolism , Signal Transduction , Tandem Mass Spectrometry , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolismABSTRACT
The International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group prepares proposals on the classification and nomenclature of filoviruses to reflect current knowledge or to correct disagreements with the International Code of Virus Classification and Nomenclature (ICVCN). In recent years, filovirus taxonomy has been corrected and updated, but parts of it remain controversial, and several topics remain to be debated. This article summarizes the decisions and discussion of the currently acting ICTV Filoviridae Study Group since its inauguration in January 2012.
Subject(s)
Classification/methods , Filoviridae/classification , Terminology as Topic , HumansABSTRACT
Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratory-adapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming,
Subject(s)
Filoviridae/classification , Filoviridae/genetics , Reassortant Viruses/classification , Reassortant Viruses/genetics , Genome, ViralSubject(s)
Animals, Laboratory/virology , Filoviridae , Terminology as Topic , Animals , Filoviridae/classificationABSTRACT
Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets.
Subject(s)
Interferon Type I/metabolism , Interferon-gamma/metabolism , Respiratory Syncytial Virus, Human/physiology , Viral Nonstructural Proteins/physiology , Animals , Catalase/genetics , Catalase/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Cluster Analysis , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Interferon Type I/genetics , Interferon Type I/physiology , Interferon-gamma/genetics , Interferon-gamma/physiology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Oxidative Stress , Proteome/genetics , Proteome/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Signal Transduction , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Transcription, Genetic , Two-Dimensional Difference Gel Electrophoresis , Vero CellsABSTRACT
We previously used human parainfluenza virus type 3 (HPIV3) as a vector to express the Ebola virus (EBOV) GP glycoprotein. The resulting HPIV3/EboGP vaccine was immunogenic and protective against EBOV challenge in a non-human primate model. However, it remained unclear whether the vaccine would be effective in adults due to preexisting immunity to HPIV3. Here, the immunogenicity of HPIV3/EboGP was compared in HPIV3-naive and HPIV3-immune Rhesus monkeys. After a single dose of HPIV3/EboGP, the titers of EBOV-specific serum ELISA or neutralization antibodies were substantially less in HPIV3-immune animals compared to HPIV3-naive animals. However, after two doses, which were previously determined to be required for complete protection against EBOV challenge, the antibody titers were indistinguishable between the two groups. The vaccine virus appeared to replicate, at a reduced level, in the respiratory tract despite the preexisting immunity. This may reflect the known ability of HPIV3 to re-infect and may also reflect the presence of EBOV GP in the vector virion, which confers resistance to neutralization in vitro by HPIV3-specific antibodies. These data suggest that HPIV3/EboGP will be immunogenic in adults as well as children.