Recombinant Fowlpox virus Protein O1 homolog (FPV091)

What is FPV091 and what is its role in Fowlpox virus?

FPV091 is a protein O1 homolog encoded by the Fowlpox virus (FPV) genome. It is classified as a hypothetical protein with a full length of 656 amino acids . While the specific function of FPV091 within the virus lifecycle is not fully characterized, it belongs to the protein family that may be involved in viral replication and host interaction mechanisms. Fowlpox virus itself is a member of the Avipoxvirus genus that primarily infects birds, particularly poultry, and has been extensively studied for its potential as a vaccine vector due to its large genome capacity and safety profile .

Research exploring FPV091's function typically involves comparative genomic analysis with other poxvirus homologs, knockout studies to observe phenotypic changes, or protein-protein interaction studies to identify binding partners within host cells or the viral proteome.

What expression systems are available for producing recombinant FPV091?

Recombinant FPV091 can be expressed using multiple heterologous expression systems, each with specific advantages depending on research requirements:

Commercial providers offer the protein with ≥85% purity as determined by SDS-PAGE . When selecting an expression system, researchers should consider the intended application, especially whether post-translational modifications are critical for functional studies. For structural studies, bacterial expression may be sufficient, while interaction studies might benefit from mammalian expression systems that better replicate the natural processing environment.

How can I verify the identity and purity of recombinant FPV091?

Verification of recombinant FPV091 identity and purity involves multiple complementary approaches:

• SDS-PAGE Analysis: Standard for purity assessment, with commercially available recombinant FPV091 typically showing ≥85% purity . Run alongside molecular weight markers to confirm the expected size (approximately 74 kDa for the full-length protein plus tag).

• Western Blotting: Using antibodies specific to the protein tag (e.g., anti-His antibody) or to FPV091 itself if available.

• Mass Spectrometry: For definitive protein identification and to detect potential post-translational modifications.

• Peptide Mapping: Digestion with specific proteases followed by HPLC analysis of the resulting peptide fragments.

• N-terminal Sequencing: To confirm the intact N-terminus and proper processing of signal peptides if present.

Researchers should implement at least two orthogonal methods to ensure both identity and purity verification, with mass spectrometry being particularly valuable for definitive characterization.

What approaches can be used to study FPV091 interactions with host proteins?

Several methodologies can elucidate FPV091 interactions with host proteins:

• Co-immunoprecipitation (Co-IP): Involves using antibodies against FPV091 or its tag to pull down the protein complex from cell lysates, followed by Western blotting or mass spectrometry to identify binding partners.

• Yeast Two-Hybrid (Y2H) Screening: Allows systematic screening of potential protein interactions, though it may yield false positives requiring validation.

• Proximity Labeling: BioID or APEX2 fused to FPV091 can label proximal proteins in living cells for subsequent identification by mass spectrometry.

• Surface Plasmon Resonance (SPR): Provides quantitative binding kinetics between purified FPV091 and candidate interacting proteins.

• Crosslinking Mass Spectrometry: Identifies interaction interfaces at amino acid resolution.

When studying viral-host protein interactions, validation across multiple systems is crucial, as interactions observed in vitro may not reflect the complexity of the infection environment. Researchers should also compare results in relevant avian cell lines, as FPV primarily infects avian hosts .

How can FPV091 be incorporated into recombinant fowlpox virus vectors for vaccine development?

Incorporating FPV091 or using it as a backbone for heterologous antigen expression in vaccine development involves several key steps:

• Vector Construction: Create a transfer plasmid containing FPV091 and the gene of interest under a strong poxvirus promoter. Two commonly used promoters are the vaccinia virus 7.5 kDa polypeptide gene promoter and synthetic poxvirus promoters, with synthetic promoters generally showing higher expression efficiency .

• Homologous Recombination: Co-transfect the transfer plasmid with infectious FPV into permissive cells (typically chicken embryo fibroblasts) to allow recombination.

• Selection: Identify recombinant viruses through plaque purification, often using fluorescent markers or drug resistance genes.

• Verification: Confirm correct insertion by PCR, sequencing, and expression analysis using immunofluorescence or Western blotting .

• Propagation and Purification: Grow the recombinant virus to high titers and purify for vaccine formulation.

The insertion site within the FPV genome can affect expression levels, though studies have shown that multiple non-essential regions can be used with only slight influence on expression . The direction of transcription relative to flanking FPV sequences has minimal impact on expression efficiency.

What methods are available for assessing immune responses to FPV091-based vaccines?

Comprehensive evaluation of immune responses to FPV091-based vaccines requires assessment of both humoral and cell-mediated immunity:

• Antibody Response Measurement:

  • ELISA to quantify antigen-specific antibodies in serum

  • Virus neutralization assays to evaluate functional antibody activity

  • Western blotting to determine which epitopes are recognized

• Cell-Mediated Immunity Assessment:

  • ELISpot assays to enumerate antigen-specific T cells

  • Intracellular cytokine staining to identify T cell subsets and their cytokine profiles

  • Cytotoxicity assays to measure killing of target cells by CD8+ T cells

• Cytokine Profile Analysis:

  • qPCR for cytokine gene expression in tissues

  • Multiplex assays for cytokine protein levels in serum or tissue culture supernatants

Studies have shown that FPV-vectored vaccines can induce early expression of Toll-like receptors (TLR3, TLR7), type I interferons, and proinflammatory cytokines, followed by adaptive immune responses including IFN-γ and IL-10 expression . When evaluating FPV091-based vaccines, researchers should examine responses at multiple time points, as the kinetics differ between innate and adaptive components.

How can structural biology approaches be applied to understand FPV091 function?

Structural characterization of FPV091 can provide crucial insights into its function and interactions:

• X-ray Crystallography: Requires high-purity protein crystals, typically using truncated or domain-specific constructs to improve crystallization prospects. Molecular replacement with homologous structures may facilitate structure determination.

• Cryo-Electron Microscopy (Cryo-EM): Particularly valuable for examining FPV091 in complex with binding partners or as part of larger assemblies. Recent advances allow near-atomic resolution for proteins >100 kDa.

• Nuclear Magnetic Resonance (NMR): Best suited for smaller domains of FPV091 (<25 kDa), providing dynamic information not available from static structures.

• Small-Angle X-ray Scattering (SAXS): Yields low-resolution envelope information and is useful for studying conformational changes upon ligand binding.

• Computational Structure Prediction: Tools like AlphaFold2 can provide initial structural models, especially valuable when experimental approaches face challenges.

Structural information should be integrated with functional assays to test hypotheses about interaction interfaces or enzymatic mechanisms. For vaccine development purposes, structural data can guide the design of immunologically optimized constructs that better present critical epitopes.

What are the challenges in studying FPV091 expression kinetics during infection?

Investigating FPV091 expression during infection presents several methodological challenges:

• Temporal Resolution: FPV gene expression follows a cascade pattern typical of poxviruses, with early, intermediate, and late genes. Studies using recombinant FPV have shown that peak antigen expression occurs 12-24 hours post-infection, with no active viral gene expression detected after 96 hours . Time-course experiments with sampling at multiple points are essential.

• Cell Type Specificity: FPV primarily infects avian cells, particularly chicken embryo fibroblasts. Research should use relevant cell types, as expression kinetics may vary between permissive and non-permissive cells.

• Detection Methods:

  • Real-time qPCR for mRNA expression

  • Western blotting for protein levels

  • Reporter gene fusions (e.g., GFP, mCherry) for live-cell imaging

  • In vivo imaging systems (IVIS) for whole-organism studies

• Promoter Analysis: Understanding the native FPV091 promoter is crucial for interpreting expression patterns. Comparative analysis with known early, intermediate, and late poxvirus promoters can provide insights.

• Host Response Interference: Host antiviral responses can modify viral gene expression. Studies have shown that FPV infection induces early increases in type I interferons and Toll-like receptors, followed by decreased expression of these genes at later time points .

How can advanced immunological techniques be used to characterize T cell responses to FPV091?

Comprehensive characterization of T cell responses to FPV091 requires sophisticated immunological approaches:

• Epitope Mapping:

  • Overlapping peptide libraries spanning FPV091 sequence

  • MHC-binding prediction algorithms to identify potential T cell epitopes

  • In vitro restimulation assays to confirm epitope immunogenicity

• Single-Cell Technologies:

  • Multiparameter flow cytometry for phenotypic characterization

  • Single-cell RNA sequencing to identify transcriptional profiles

  • Mass cytometry (CyTOF) for high-dimensional analysis of cell populations

• In Vivo Tracking:

  • Adoptive transfer of labeled antigen-specific T cells

  • MHC tetramer staining to quantify epitope-specific T cells

  • Intravital microscopy to visualize T cell behavior in tissues

• Functional Assessment:

  • Multiplexed cytokine profiling

  • Cytotoxicity assays against target cells expressing FPV091

  • T cell receptor sequencing to analyze clonal expansion

Studies with TROVAC-AIV H5 (an FPV-vectored vaccine) have demonstrated that these vaccines can elicit cell-mediated responses against expressed heterologous antigens . When investigating T cell responses to FPV091, researchers should examine both CD4+ and CD8+ compartments and consider tissue-specific responses, as FPV primarily infects the initial vaccination site (lung and nasal cavity) without disseminating to distal sites .

What controls should be included when studying recombinant FPV091 activity?

Rigorous experimental design for FPV091 research requires appropriate controls:

• Protein Controls:

  • Empty vector expression product (tag-only protein)

  • Heat-denatured FPV091 to control for non-specific effects

  • Related viral proteins to assess specificity

  • Truncated variants to map functional domains

• Cell Culture Controls:

  • Uninfected cells treated with culture medium only

  • Cells infected with parental (non-recombinant) FPV

  • Cells expressing irrelevant proteins of similar size/structure

• In Vivo Controls:

  • Mock-vaccinated animals

  • Animals receiving vector without FPV091 insert

  • Historical controls from previous vaccine studies

• Technical Controls:

  • Standard curves for quantitative assays

  • Isotype controls for antibody-based methods

  • Internal reference genes for gene expression studies

When analyzing host responses, researchers should be particularly attentive to baseline variations between individuals and establish clear criteria for defining positive responses .

How can researchers overcome challenges in expressing functional FPV091?

Expression of functional viral proteins like FPV091 can present specific challenges:

• Solubility Issues:

  • Test multiple expression systems (bacterial, yeast, insect, mammalian)

  • Optimize cultivation conditions (temperature, induction time)

  • Use solubility-enhancing tags (MBP, SUMO)

  • Express individual domains separately

  • Screen various buffer compositions for purification

• Post-translational Modifications:

  • Use eukaryotic expression systems for authentic modifications

  • Analyze glycosylation, phosphorylation, or other modifications

  • Consider in vitro modification approaches when necessary

• Cytotoxicity:

  • Implement inducible expression systems

  • Use less toxic truncated variants

  • Optimize cell density and expression duration

• Functional Verification:

  • Design activity assays based on predicted function

  • Compare activities across expression systems

  • Ensure proper folding using circular dichroism or thermal shift assays

Expression systems should be selected based on downstream applications. For structural studies, high yields from E. coli may be preferred, while functional studies might require mammalian expression to ensure proper folding and modifications .

What approaches can address data inconsistencies in FPV091 research?

When facing conflicting or inconsistent data in FPV091 research, systematic troubleshooting approaches are essential:

• Methodological Variation:

  • Standardize protocols across experiments

  • Document all experimental variables

  • Implement quality control checkpoints

  • Utilize multiple detection methods for key findings

• Biological Variability Management:

  • Increase biological replicates

  • Account for genetic background variations

  • Consider age, sex, and health status of experimental animals

  • Standardize cell line passages and growth conditions

• Statistical Approaches:

  • Perform power analysis to ensure adequate sample sizes

  • Apply appropriate statistical tests for data type

  • Consider non-parametric methods for non-normally distributed data

  • Implement mixed-effects models to account for repeated measures

• Collaboration and Validation:

  • Reproduce key findings in independent laboratories

  • Share materials and detailed protocols

  • Combine complementary expertise for comprehensive analysis

Participatory theme elicitation (PTE) methods, which encourage involvement from people with varying research experience, can be valuable for analyzing complex datasets with potential inconsistencies .

How might next-generation sequencing enhance our understanding of FPV091 function?

Next-generation sequencing (NGS) technologies offer powerful approaches to elucidate FPV091 function:

• Transcriptome Analysis (RNA-Seq):

  • Compare host cell gene expression before and after exposure to FPV091

  • Identify differentially regulated pathways suggesting protein function

  • Track temporal changes in gene expression during infection

• CLIP-Seq (Cross-Linking Immunoprecipitation Sequencing):

  • If FPV091 interacts with nucleic acids, CLIP-Seq can identify binding sites

  • Reveals potential roles in transcriptional or translational regulation

• Ribosome Profiling:

  • Investigate FPV091's impact on host cell translation

  • Identify viral and cellular mRNAs affected by FPV091 expression

• ChIP-Seq (Chromatin Immunoprecipitation Sequencing):

  • If FPV091 associates with chromatin, ChIP-Seq reveals genomic binding sites

  • Maps potential epigenetic regulatory functions

• Comparative Genomics:

  • Analyze FPV091 sequence conservation across poxvirus species

  • Identify conserved domains suggesting functional importance

  • Correlate sequence variations with host specificity

These genomic approaches should be integrated with proteomic and biochemical analyses for comprehensive functional characterization.

What are the prospects for using CRISPR-Cas9 to study FPV091 function?

CRISPR-Cas9 genome editing offers transformative approaches for FPV091 research:

• FPV091 Knockout Studies:

  • Generate FPV091-deficient virus to assess its role in replication

  • Compare phenotypes between wild-type and knockout viruses

  • Identify compensatory mechanisms through transcriptomic analysis

• Domain Mutagenesis:

  • Create precise mutations in functional domains

  • Generate chimeric proteins with domains from related viral proteins

  • Introduce reporter tags at specific locations while maintaining function

• Host Factor Identification:

  • Conduct genome-wide CRISPR screens to identify host factors interacting with FPV091

  • Knockout candidate interacting partners to confirm their relevance

  • Create cell lines with tagged endogenous proteins for interaction studies

• Conditional Expression Systems:

  • Implement CRISPR-based inducible systems to control FPV091 expression

  • Study temporal requirements for FPV091 during viral replication

CRISPR approaches should consider the large size of the poxvirus genome (approximately 288 kbp for FPV) and establish efficient delivery methods for guide RNAs and Cas9 into appropriate cell types.

How can systems biology approaches integrate FPV091 into viral-host interaction networks?

Systems biology offers frameworks to understand FPV091 function within the broader context of viral-host interactions:

• Interactome Mapping:

  • High-throughput protein-protein interaction screens

  • Correlation networks from multi-omics data

  • Computational prediction of interaction partners based on structural features

• Pathway Analysis:

  • Integrate FPV091 into known host immune signaling pathways

  • Identify novel pathways affected by FPV091 expression

  • Map temporal dynamics of pathway perturbations

• Mathematical Modeling:

  • Develop predictive models of FPV091's impact on viral replication

  • Simulate the effects of FPV091 variants on host cell responses

  • Integrate models with experimental validation

• Multi-Omics Integration:

  • Combine transcriptomics, proteomics, metabolomics, and lipidomics data

  • Identify emergent properties not apparent from single-omics approaches

  • Resolve temporal relationships between molecular events

Systems approaches are particularly valuable for understanding the context-dependent functions of viral proteins like FPV091, which may have different roles depending on the stage of infection and cell type .