Recombinant Human adenovirus F serotype 40 Major core protein (PVII)

What is the basic composition and structure of the adenovirus pVII protein?

Adenovirus protein VII (pVII) is a highly basic protein that constitutes approximately 10% of the virion mass. The protein is particularly rich in arginine (23%) and alanine (19%) residues, with about 46% of its amino acids being positively charged. This cationic nature makes pVII strongly attracted to the negatively charged phosphate backbone of DNA. The N-terminus region (approximately 50 amino acids) of pVII appears highly conserved across members of mastadenovirus. Though crystallographic data specifically for HAdV-F40 pVII is limited, the protein generally has an extended structure similar to other adenovirus core proteins .

How does pVII interact with viral DNA and what is its role in genome packaging?

pVII binds to double-stranded DNA in a sequence-independent manner and plays a crucial role in viral genome condensation, similar to cellular histones. Although pVII binds efficiently to DNA due to its highly basic nature, interestingly, research has shown that it is not absolutely essential for viral DNA condensation within the viral capsid. This suggests redundant mechanisms for genome compaction. While pVII was earlier speculated to be required for genome packaging based on its interactions with pVIa2 and p52/55k, recent studies indicate that pVII is not strictly required for genome packaging or virus assembly .

What cellular proteins does pVII interact with and what are the functional implications?

pVII interacts with various cellular proteins to regulate different aspects of the viral life cycle. One significant interaction is with MKRN1 (Makorin ring finger protein 1), a cellular E3 ubiquitin ligase. The uncleaved pVII interacts with MKRN1 and enhances its self-ubiquitination, followed by proteasomal degradation of MKRN1 in infected cells, which may facilitate efficient viral production. pVII also interacts with SPOC1 (survival-time-associated PHD protein in ovarian cancer 1), a chromatin-associated factor involved in DNA damage response. This interaction prevents the host cell from detecting viral dsDNA, thereby restricting antiviral gene transcription during the early phase of infection .

What are the optimal methods for expressing and purifying recombinant HAdV-F40 pVII for structural studies?

For recombinant expression of HAdV-F40 pVII, bacterial expression systems using E. coli strains optimized for proteins with high basic amino acid content are recommended. Given pVII's highly basic nature (with 46% positively charged amino acids), consider using specialized strains like Rosetta or BL21-CodonPlus to address potential codon bias issues. For purification, a multi-step approach is most effective:

• Initial capture using immobilized metal affinity chromatography (IMAC) with a His-tag

• Intermediate purification via ion exchange chromatography (preferably cation exchange due to pVII's positive charge)

• Final polishing step using size exclusion chromatography

To maintain protein stability during purification, buffers containing 300-500 mM NaCl are recommended to prevent non-specific binding to negatively charged surfaces. Adding nuclease treatment steps is crucial to remove any co-purifying DNA that may bind to pVII. For structural studies, consider limited proteolysis approaches to identify stable domains suitable for crystallization .

How can protein-protein interactions between pVII and cellular factors be effectively studied in vitro and in vivo?

Multiple complementary approaches should be employed to comprehensively study pVII-host protein interactions:

In vitro approaches:

• GST pulldown assays using recombinant GST-tagged pVII and cell lysates have proven effective for identifying interacting partners, as demonstrated in studies with MKRN1. This approach revealed that wild-type pVII shows stronger binding to Flag-MKRN1 compared to truncated variants (pVII(Δ24)) .

• Co-immunoprecipitation (Co-IP) with appropriate antibodies against pVII or suspected interacting partners.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) for quantitative binding analysis.

In vivo approaches:

• Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling to capture transient or weak interactions in the cellular context.

• Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) for visualizing interactions within cells.

• Cross-linking followed by mass spectrometry (XL-MS) to map interaction interfaces.

When designing experiments, account for the effect of pVII processing, as research shows differential binding capacity between precursor and mature forms of pVII .

What methodologies are recommended for studying the chromatin-modulating functions of pVII?

To investigate pVII's chromatin-modulating activities, researchers should employ multiple complementary approaches:

• Chromatin immunoprecipitation (ChIP) assays: Use anti-pVII antibodies to identify genome-wide binding patterns of pVII. Sequential ChIP (re-ChIP) can determine co-occupancy with cellular factors.

• Micrococcal nuclease (MNase) digestion assays: Compare chromatin accessibility in the presence and absence of pVII to assess its impact on chromatin structure.

• In vitro nucleosome assembly assays: Reconstitute nucleosomes with histone octamers, DNA, and purified pVII to determine how pVII affects nucleosome positioning and stability.

• ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing): Identify regions of chromatin accessibility changes induced by pVII.

• Histone modification analysis: Since pVII functionally mimics histones, investigate whether its presence alters the pattern of histone post-translational modifications using ChIP-seq for various histone marks.

These approaches will help determine whether pVII, like cellular protamines, interacts with nucleosomes and how it affects chromatin organization. Research has shown that unlike protamine, pVII interacts with nucleosomes but does not replace histones from nucleosomes .

How does HAdV-F40 pVII differ structurally and functionally from other adenovirus serotypes?

While the search results don't provide specific information about HAdV-F40 pVII, comparative studies can reveal important variations across serotypes. Research should focus on:

• Sequence conservation analysis: Align pVII sequences from HAdV-F40 with other serotypes, particularly focusing on the N-terminal 50 amino acids which are highly conserved across mastadenoviruses. Pay special attention to the distribution of basic residues which may affect DNA binding affinity.

• Post-translational modification patterns: Compare acetylation and phosphorylation sites, as these modifications have been documented in pVII proteins and may differ between serotypes, affecting function.

• Interaction partners: Determine whether HAdV-F40 pVII interacts with the same cellular and viral proteins as other serotypes. For example, examine if it binds to MKRN1 with the same affinity as HAdV-C5 pVII .

• Tissue tropism correlation: Investigate whether specific features of HAdV-F40 pVII contribute to this serotype's pronounced gastrointestinal tropism, unlike respiratory-tropic adenoviruses. HAdV-F40 and F41 have a known affinity for the GI tract with predominant symptoms of gastroenteritis or diarrhea .

What specialized techniques are needed to study pVII in the context of enteric adenoviruses like HAdV-F40?

Studying pVII in enteric adenoviruses requires specialized approaches due to their distinct biology:

• Specialized cell culture systems: Use intestinal organoids or polarized intestinal epithelial cell models (such as Caco-2 or T84 cells) to better replicate the natural environment of HAdV-F40.

• Infection models: Employ differentiated intestinal epithelial cells in transwell systems to study apical versus basolateral infection dynamics.

• Tissue-specific protein interaction studies: Focus on identifying interactions between pVII and intestine-specific host factors using proximity labeling approaches in relevant cell types.

• Comparative viral dynamics: Design experiments that compare pVII behavior in enteric versus respiratory adenovirus serotypes to identify determinants of tissue tropism.

• Specialized viral production systems: As enteric adenoviruses are often difficult to propagate in conventional cell culture, consider using helper adenovirus systems or modified cell lines expressing specific factors that enhance HAdV-F40 replication .

How does post-translational modification affect pVII function and what methods can detect these modifications?

pVII undergoes several post-translational modifications that regulate its function, similar to cellular histones. To study these:

• Mass spectrometry (MS) analysis: Use high-resolution MS to map acetylation, phosphorylation, and potentially other modifications. Employ both bottom-up and top-down proteomics approaches for comprehensive characterization.

• Site-directed mutagenesis: Create specific mutations at putative modification sites to assess their functional impact on DNA binding, protein interactions, and viral replication.

• Antibodies against modified forms: Develop modification-specific antibodies to track different pVII populations during infection.

• Enzyme inhibitor studies: Use histone acetyltransferase (HAT) or histone deacetylase (HDAC) inhibitors to modulate pVII acetylation levels and assess functional consequences.

Research has shown that pVII is acetylated and phosphorylated similar to histones, particularly histone H4, despite limited structural similarity. These modifications likely regulate pVII's interaction with viral DNA and cellular proteins throughout the infection cycle .

What role does pVII play in evading host immune responses, and how can this be experimentally demonstrated?

pVII contributes to immune evasion through several mechanisms that can be studied using these approaches:

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq): Map pVII binding sites on the viral genome and correlate with transcriptional silencing of immune-stimulatory viral genes.

• Immunofluorescence microscopy: Track pVII localization relative to pattern recognition receptors (PRRs) and determine if it shields viral DNA from detection.

• Co-immunoprecipitation with innate immune sensors: Assess whether pVII directly interacts with and inhibits DNA sensors like cGAS, IFI16, or STING.

• Reporter assays for innate immune pathways: Use luciferase reporters for NF-κB, IRF3, and type I interferon to measure the impact of wild-type versus mutant pVII on immune signaling.

• Comparative proteomics: Identify changes in the nuclear proteome in the presence of pVII to reveal novel immune evasion mechanisms.

Research demonstrates that pVII interacts with SPOC1, a chromatin-associated factor involved in DNA damage response, thereby restricting adenoviral gene transcription in the early phase of infection and preventing the detection of viral dsDNA by cellular immune sensors .

How does pVII contribute to viral persistence and latency in host cells?

To investigate pVII's role in viral persistence and latency:

• Long-term cell culture models: Establish cell lines with persistent adenovirus infection and monitor pVII expression, localization, and modifications over time.

• ChIP-seq of persistent infection: Map changes in pVII binding patterns between lytic and persistent phases of infection.

• Single-cell analysis techniques: Use single-cell RNA-seq and protein analysis to identify heterogeneity in pVII expression and function among persistently infected cells.

• Inducible expression systems: Create cell lines with inducible pVII expression to determine if pVII alone can establish a chromatin state conducive to viral latency.

Adenoviruses have a propensity to establish latent or persistent infection within the upper and lower respiratory tracts. While not specifically studied for HAdV-F40, persistence mechanisms may involve pVII-mediated chromatin modifications and interactions with host proteins that regulate viral gene expression. Persistent adenovirus infection in children may elicit chronic neutrophilic inflammation within the airways, potentially contributing to protracted bacterial bronchitis and bronchiectasis .

How has pVII evolved across different adenovirus species and what functional implications does this have?

To study the evolution of pVII:

• Phylogenetic analysis: Construct phylogenetic trees based on pVII sequences from diverse adenovirus species to trace evolutionary relationships.

• Selection pressure analysis: Calculate dN/dS ratios to identify regions under positive or purifying selection.

• Structure prediction comparison: Use computational tools to predict structural features of pVII across species and identify conserved structural elements despite sequence divergence.

• Functional complementation assays: Test whether pVII from one adenovirus species can functionally replace that of another in chimeric viruses.

Research indicates that the N-terminal 50 amino acids of pVII are highly conserved across mastadenoviruses, suggesting functional importance. The protein's basic nature and role in DNA condensation appear to be evolutionarily conserved functional traits, though specific interaction partners may vary between species .

How does pVII function compare with core proteins of other DNA viruses?

Comparative analysis between adenovirus pVII and core proteins of other DNA viruses can provide valuable insights:

To study these comparisons:

• Heterologous expression systems: Express different viral core proteins in the same cellular background and compare their DNA-binding and condensation properties.

• Competitive binding assays: Determine if different viral core proteins compete for the same DNA or chromatin targets.

• Structural biology approaches: Compare high-resolution structures to identify convergent structural features despite different evolutionary origins.

While pVII functionally mimics cellular histones and protamines, it maintains unique properties that have evolved specifically for adenovirus biology. Unlike cellular protamine, pVII interacts with nucleosomes but does not replace histones from nucleosomes .

What is the relationship between pVII and the pathogenesis of adenovirus infections?

While direct evidence linking pVII to pathogenesis is limited, several indirect connections can be investigated:

• Transgenic mouse models: Create mice expressing adenovirus pVII in specific tissues to assess pathological changes in the absence of other viral components.

• Comparative virulence studies: Engineer recombinant adenoviruses with mutations in pVII and evaluate changes in pathogenicity in appropriate models.

• Tissue-specific effects: For HAdV-F40, focus on gastrointestinal pathology, as this serotype has a specific tropism for the GI tract.

Research suggests that pVII may contribute to pathogenesis through:

• Modulation of host cell apoptosis by localizing to mitochondria and maintaining membrane potential

• Degradation of host restriction factors like MKRN1

• Evasion of innate immune responses

• Potential contributions to persistent infection

Adenovirus F40 and F41 typically cause gastroenteritis or diarrhea. In rare cases, adenovirus infections can lead to more severe complications including hemorrhagic colitis, hepatitis, cholecystitis, and pancreatitis, though the specific contribution of pVII to these outcomes remains to be determined .

How might understanding pVII function inform the development of antiviral strategies?

Research into pVII function can guide antiviral development in several ways:

• Small molecule inhibitors: Target the interaction between pVII and DNA or between pVII and critical host factors like MKRN1. High-throughput screening assays measuring these interactions could identify candidate inhibitors.

• Peptide-based inhibitors: Design peptides that mimic the binding interfaces of pVII-interacting proteins to competitively inhibit these interactions.

• PROTAC (proteolysis targeting chimera) approach: Create molecules that target pVII for degradation by the ubiquitin-proteasome system.

• Gene editing strategies: Develop CRISPR-Cas systems that specifically target the pVII gene sequence to disrupt viral replication.

Current treatment for adenovirus infections is controversial, with cidofovir being the drug of choice for severe infections. Novel approaches targeting core viral functions like those mediated by pVII could provide alternatives for cases where existing treatments are ineffective. For research applications, consider combination approaches that target multiple viral components simultaneously to reduce the likelihood of resistance development .

What are the major technical challenges in expressing and purifying functional recombinant pVII?

Researchers face several challenges when working with recombinant pVII:

• DNA contamination: Due to its high affinity for DNA, recombinant pVII often co-purifies with bacterial DNA. Implement extensive nuclease treatments and high-salt washing steps during purification.

• Protein solubility: The highly basic nature of pVII (46% positively charged amino acids) can lead to aggregation and precipitation. Consider:

  • Using solubility enhancement tags (e.g., SUMO, MBP)

  • Developing optimized buffer conditions with higher salt concentrations (≥500mM NaCl)

  • Adding non-ionic detergents or arginine to prevent aggregation

• Protein toxicity: Overexpression in bacterial systems may be toxic due to binding to host DNA. Implement:

  • Tight regulation of expression using inducible systems

  • Lower induction temperatures (16-18°C)

  • Co-expression with DNA-binding molecular chaperones

• Post-translational modifications: Bacterial expression systems lack the machinery for mammalian-type PTMs. For fully modified protein:

  • Consider insect or mammalian expression systems

  • Implement in vitro enzymatic modification approaches

• Functional assays: Verifying that recombinant pVII retains native functionality can be challenging. Develop:

  • DNA condensation assays

  • Protein interaction validation using known partners like MKRN1

How can researchers distinguish between the precursor and mature forms of pVII in experimental systems?

Distinguishing between precursor and mature forms of pVII requires specific approaches:

• Antibody development: Generate antibodies that specifically recognize:

  • Epitopes present only in the precursor (pre-cleavage form)

  • Neo-epitopes created after proteolytic processing

  • Epitopes common to both forms (for total pVII detection)

• Mass spectrometry: Employ targeted MS approaches to identify and quantify peptides unique to either the precursor or mature form.

• Gel mobility analysis: Optimize SDS-PAGE conditions that can resolve the small mass difference between precursor and mature forms. Consider:

  • High percentage (15-20%) gels

  • Tricine-SDS PAGE systems optimized for small proteins

  • Phos-tag acrylamide if phosphorylation states differ between forms

• Functional assays: Develop comparative assays that exploit functional differences:

  • DNA binding affinity comparisons

  • Differential protein interaction profiles (e.g., with MKRN1)

  • Subcellular localization differences

Research has shown functional differences between precursor and mature pVII, particularly in their interaction with cellular proteins like MKRN1. The precursor form (uncleaved pVII) interacts with MKRN1 and enhances its self-ubiquitination, which has implications for experimental design when studying these interactions .

What are the most promising areas for future research on HAdV-F40 pVII?

Several high-priority research directions for HAdV-F40 pVII include:

• Structural biology: Determine high-resolution structures of HAdV-F40 pVII alone and in complex with DNA and protein partners. This would enable structure-guided drug design and comparative analysis with other serotypes.

• Host-pathogen interactions specific to enteric cells: Identify gastrointestinal-specific interaction partners that may explain HAdV-F40's tropism. Compare interactomes between enteric and respiratory adenovirus pVII proteins.

• Role in viral persistence: Investigate whether pVII contributes to persistent infection in the gastrointestinal tract, which could have implications for chronic inflammatory conditions.

• Immune modulation: Determine how pVII from enteric adenoviruses may differentially modulate mucosal immunity compared to respiratory serotypes.

• Therapeutic applications: Explore the potential of engineered pVII variants as DNA delivery vehicles for gene therapy applications, leveraging its DNA condensation properties.

• Multi-omics approaches: Implement integrated genomics, proteomics, and metabolomics to comprehensively characterize the impact of pVII on host cell function .

How might systems biology approaches enhance our understanding of pVII's role in the viral life cycle?

Systems biology offers powerful frameworks to understand pVII function in context:

• Network analysis: Construct protein-protein interaction networks centered on pVII to identify key hubs and potential intervention points. This should incorporate temporal dynamics during infection.

• Mathematical modeling: Develop predictive models of:

  • pVII-mediated DNA condensation kinetics

  • Viral gene expression regulation by pVII

  • Host cell response pathways affected by pVII

• Single-cell approaches: Use single-cell RNA-seq and proteomics to capture heterogeneity in host response to pVII expression and identify cell state transitions associated with different pVII functions.

• Integrative multi-omics:

  • Correlate ChIP-seq data (pVII binding) with RNA-seq (gene expression)

  • Integrate proteomics and transcriptomics to identify post-transcriptional regulation

  • Combine structural data with interaction networks to identify critical interfaces

• Comparative systems analysis: Analyze system-wide effects of pVII across multiple adenovirus serotypes to identify conserved versus serotype-specific functions.

These approaches would help contextualize the previously identified interactions with proteins such as MKRN1, SPOC1, and various viral components, revealing emergent properties not evident from reductionist approaches .