Recombinant Vaccinia virus Protein I2 (VACWR071)

What is the basic structure of Vaccinia virus Protein I2 (VACWR071)?

Vaccinia virus Protein I2 is a small, essential viral protein consisting of 72-73 amino acids with a molecular weight of approximately 8.4 kDa. It features a characteristic C-terminal transmembrane domain. The complete amino acid sequence is: MDKLYAAIFGVFMGSPEDDLTDFIEIVKSVLSDEKTVTSTNNTGCWGWYWLIIIFFIVLILLLLIYLYLKVVW. The protein is encoded by the I2L open reading frame in the Vaccinia virus genome and is synthesized following viral DNA replication .

Where is Protein I2 localized during viral infection?

Fluorescence microscopy experiments have demonstrated that Protein I2 colocalizes with major membrane proteins of both immature and mature virions. Studies using GFP-I2 fusion proteins (with GFP fused to the N-terminus of I2) have confirmed this localization pattern. The protein is primarily found in cytoplasmic viral factories during infection and is incorporated into the viral particles during assembly .

What is the established function of Protein I2 in the viral life cycle?

Protein I2 serves dual critical functions in the Vaccinia virus life cycle. First, it plays an essential role in virion morphogenesis, particularly in the transition from immature virions (IVs) to mature virions (MVs). Second, it is required for virion entry into host cells. Deletion mutant studies have conclusively shown that without functional I2 protein, viral replication is completely abrogated, confirming its status as an essential viral factor .

What expression systems are recommended for producing recombinant Protein I2?

Recombinant Protein I2 has been successfully expressed in E. coli expression systems with N-terminal tags (such as His-tag) to facilitate purification. The full-length protein (amino acids 1-73) can be produced and purified to >90% purity as determined by SDS-PAGE analysis. To maintain protein stability, lyophilization in Tris/PBS-based buffer with 6% trehalose (pH 8.0) is recommended .

What are the optimal storage conditions for recombinant Protein I2?

For long-term storage, recombinant Protein I2 should be stored at -20°C or preferably -80°C. After reconstitution in deionized sterile water (to a concentration of 0.1-1.0 mg/mL), it is recommended to add glycerol to a final concentration of 50% and then aliquot for storage at -20°C/-80°C. For working solutions, store aliquots at 4°C for up to one week. Repeated freeze-thaw cycles should be strictly avoided as they significantly degrade protein quality and activity .

How can complementing cell lines be developed for studying Protein I2 function?

To study the function of essential viral proteins like I2, complementing cell lines are invaluable tools. The established methodology involves:

• Creating a eukaryotic codon-optimized version of the I2L gene with an N-terminal tag (e.g., hemagglutinin/HA tag)

• Incorporating this construct into a lentiviral vector for stable gene delivery

• Transducing permissive cells (such as rabbit kidney RK-13 cells)

• Selecting transduced cells with appropriate antibiotics

• Isolating and clonally purifying the resulting cell lines

• Confirming expression by Western blotting and fluorescence microscopy

This approach enables the creation of deletion mutants for stringent functional studies by providing the essential protein in trans .

How can deletion mutants of Protein I2 be constructed and characterized?

Construction of I2L deletion mutants involves a systematic approach:

• Create a cell line expressing HA-tagged I2 protein (as described in 2.3)

• Design a deletion construct with GFP regulated by the VACV late P11 promoter, flanked by sequences upstream and downstream of I2L

• Transfect this construct into cells expressing HA-I2 that are infected with wild-type Vaccinia virus

• Allow homologous recombination to occur

• Identify mutant viruses by green fluorescent plaque formation

• Clonally purify the mutants through repeated plaque picking

• Confirm deletion of the I2L ORF by sequencing

• Verify the mutant's inability to replicate in cells not expressing I2 protein

• Analyze phenotypic characteristics through electron microscopy, immunoblotting, and infectivity assays

This approach has revealed that I2-deficient viruses produce abnormal, spherical, dense particles instead of typical brick-shaped mature virions .

What techniques can be used to study the interaction of Protein I2 with other viral components?

Multiple complementary approaches can be employed:

• Co-immunoprecipitation (Co-IP): Using antibodies against tagged versions of I2 to pull down interacting partners

• Proximity labeling: Employing BioID or APEX2 fused to I2 to identify proteins in close proximity within the cellular environment

• Yeast two-hybrid screening: For detecting binary protein interactions

• Mass photometry: For analyzing protein-protein interactions at a single-molecule level, enabling detection of even transient or low-abundance complexes

• Fluorescence microscopy with dual labeling: To visualize co-localization with other viral proteins

• Cross-linking mass spectrometry: To capture and identify direct interaction partners

Mass photometry is particularly valuable for studying weak protein interactions as it rapidly provides detailed information on mass distribution at the single-molecule level, allowing detection and quantification of trace protein complexes .

What phenotypic changes are observed in virions lacking Protein I2?

In the absence of Protein I2, several critical phenotypic changes occur:

• Morphogenesis defects: Formation of spherical dense particles instead of brick-shaped mature virions

• Scaffold retention: Increased amounts of D13 scaffold protein, indicating failure to disassemble the scaffold

• Protein processing abnormalities: Increased amounts of unprocessed A3 and A17 proteins

• Entry Fusion Complex (EFC) deficiency: Significant reduction in EFC proteins in the viral membrane

• Reduced protease activity: Decreased amounts of I7 proteinase, which normally cleaves A17 and several core proteins

These observations indicate that I2 plays a crucial role in late stages of virion morphogenesis, particularly in the transition from immature to mature virions, scaffold disassembly, and the incorporation of entry fusion complex components .

How does Protein I2 contribute to viral entry mechanisms?

Studies using inducible mutants have demonstrated that I2-deficient virions show a profound reduction in infectivity due to an inability to enter host cells. While the exact mechanism remains under investigation, it appears that I2 affects the incorporation or stability of Entry Fusion Complex (EFC) proteins in the viral membrane. The EFC is essential for fusion of the viral membrane with cellular membranes during entry.

Research indicates a complex relationship between proper virion morphogenesis and entry function, as defects in morphogenesis in I2-deficient virions lead to downstream effects on entry capabilities. The dual role of I2 in both processes suggests it may serve as a structural or regulatory component that ensures proper virion architecture necessary for subsequent entry functions .

What are common challenges in expressing and purifying functional Protein I2?

Several challenges must be addressed when working with Protein I2:

• Transmembrane domain issues: The C-terminal transmembrane domain can cause aggregation and solubility problems

• Expression toxicity: Expression in bacterial systems may be toxic, requiring optimization of induction conditions

• Protein instability: The small size and hydrophobic regions can lead to instability during purification

• Functional validation: Confirming that recombinant protein retains native conformation and function

To overcome these challenges:

• Consider expressing truncated versions without the transmembrane domain for certain applications

• Use mild detergents during purification to maintain solubility

• Optimize expression at lower temperatures (16-18°C) to improve proper folding

• Validate function through complementation assays in I2-deficient systems

How can researchers distinguish between direct and indirect effects of Protein I2 deletion?

Distinguishing direct from indirect effects requires a multi-faceted approach:

• Temporal studies: Using inducible systems to track the sequence of events following I2 depletion

• Complementation analyses: Testing whether specific defects can be rescued by providing I2 at different stages

• Domain mapping: Creating point mutations or truncations to identify functional domains responsible for specific activities

• Interaction studies: Identifying direct binding partners to establish mechanistic connections

• Conditional mutants: Developing temperature-sensitive mutants to enable rapid inactivation and temporal tracking of effects

These approaches can help differentiate between primary effects directly caused by the absence of I2 and secondary consequences that arise from earlier disruptions in the viral life cycle .

What are promising approaches for targeting Protein I2 in antiviral development?

Based on its essential nature and conserved sequence across poxviruses, Protein I2 represents a compelling target for antiviral development:

• Structure-based drug design: Determining the three-dimensional structure of I2 to design small molecules that disrupt its function

• Peptide inhibitors: Developing peptide mimetics that compete with I2 for binding to interaction partners

• RNA interference: Using siRNA or antisense oligonucleotides to reduce I2 expression

• CRISPR interference: Employing CRISPRi systems to repress I2L gene expression

• High-throughput screening: Identifying compounds that interrupt I2-dependent processes

The dual role of I2 in both assembly and entry provides multiple intervention points, potentially increasing the barrier to resistance development .

How might mass photometry advance our understanding of Protein I2 interactions?

Mass photometry offers several advantages for studying I2 protein interactions:

• Single-molecule sensitivity: Enables detection of rare or transient complexes that may be missed by bulk methods

• Label-free analysis: Avoids potential artifacts introduced by fluorescent tags or other modifications

• Native conditions: Allows study of interactions under physiologically relevant conditions

• Stoichiometry determination: Provides precise information about the composition of protein complexes

• Quantitative binding analysis: Permits determination of dissociation constants (KD) for protein interactions

This technique could reveal previously undetected interactions between I2 and other viral or cellular proteins, potentially uncovering new functions or regulatory mechanisms. For example, it could help clarify the relationship between I2 and components of the Entry Fusion Complex or scaffold proteins during virion assembly .

How conserved is Protein I2 across different poxviruses?

Protein I2 is highly conserved across the chordopoxvirus family, suggesting its critical importance in the viral life cycle. Homologs are found in all chordopoxviruses that have been sequenced to date. This conservation extends to both sequence similarity and predicted structural features, particularly the C-terminal transmembrane domain. The high degree of conservation makes I2 an attractive target for broad-spectrum antipoxviral strategies and implies that findings from vaccinia virus studies may be applicable to other poxviruses of medical or veterinary importance .

What experimental approaches can determine if Protein I2 interacts with the Entry Fusion Complex?

To investigate potential interactions between Protein I2 and the Entry Fusion Complex (EFC), researchers should consider:

• Co-immunoprecipitation with EFC components: Using antibodies against I2 or EFC proteins to detect physical associations

• Proximity labeling in intact virions: Employing techniques like BioID fused to I2 to identify nearby proteins

• Cross-linking mass spectrometry: To capture and identify direct interaction partners within the viral membrane

• Fluorescence resonance energy transfer (FRET): To detect close proximity between I2 and EFC proteins

• Genetic complementation studies: Testing whether mutants in I2 can be suppressed by mutations in EFC components

• Cryo-electron microscopy: To visualize the structural organization of I2 relative to EFC components in virions

While previous studies noted a reduction in EFC components in I2-deficient virions, direct physical associations have not been fully investigated. These approaches would help clarify whether I2 directly interacts with the EFC or influences its incorporation through indirect mechanisms .

What criteria should be used to validate successful expression of functional recombinant Protein I2?

Validation of successfully expressed recombinant Protein I2 should include multiple criteria:

A comprehensive validation approach incorporating these criteria ensures that the recombinant protein faithfully represents the native Protein I2 and is suitable for downstream applications .

How should researchers interpret contradictory findings regarding Protein I2 function?

When faced with contradictory findings regarding Protein I2 function, researchers should systematically:

• Compare experimental systems: Different cell types, virus strains, or expression systems may yield varying results

• Evaluate methodology sensitivity: Some approaches may detect only strong interactions, missing transient or weak associations

• Consider timing of observations: The role of I2 may differ at various stages of the viral life cycle

• Assess protein modifications: Post-translational modifications may affect function in context-dependent ways

• Examine protein levels: Different expression levels may reveal threshold effects or dose-dependent functions

• Investigate conditional requirements: Function may depend on specific environmental conditions or cellular factors