Recombinant Vaccinia virus Protein I2 (I2L)

When during the viral life cycle is I2L expressed?

I2L is expressed at late times of the vaccinia virus infection cycle, following viral DNA replication . This timing of expression is consistent with its role in virion assembly and maturation. As a late gene product, I2L becomes incorporated into developing virions during the assembly process and remains tightly associated with the viral membranes of mature virions . The expression pattern aligns with other structural proteins that are needed during the final stages of virion morphogenesis rather than early proteins that typically function in viral DNA replication or host interaction .

How is the I2L protein localized within infected cells and virions?

Fluorescence microscopy experiments have demonstrated that I2L co-localizes with major membrane proteins of both immature and mature virions . Researchers have used GFP-tagged I2L fusion proteins to track its localization, confirming that the protein is found primarily associated with viral membrane structures within the cytoplasm of infected cells . In mature virions, I2L is tightly associated with membranes and becomes encapsidated in the final viral particles . This membrane association is critical for its function in both virion morphogenesis and entry processes .

What is the specific role of I2L in vaccinia virus morphogenesis?

The I2L protein plays a critical role in the transition from immature virions (IVs) to mature virions (MVs). Studies with I2L deletion mutants have revealed that in the absence of I2L, morphogenesis is interrupted after immature virion formation, resulting in the accumulation of dense spherical particles instead of the characteristic brick-shaped mature virions with well-defined core structures .

These abnormal particles exhibit two key defects:

• Retention of the D13 scaffold protein that normally dissociates during the IV-to-MV transition

• Severe deficiency in the transmembrane proteins that comprise the entry fusion complex (EFC)

Additionally, these particles contain increased amounts of unprocessed membrane and core proteins . This suggests that I2L functions as a regulator of the morphological transition, potentially triggering the release of the D13 scaffold and facilitating the subsequent incorporation of EFC proteins into the viral membranes . The precise molecular mechanism by which I2L mediates these changes remains under investigation, but its conservation across poxviruses underscores its essential nature in this process.

How does I2L deficiency affect the entry fusion complex (EFC) proteins?

I2L deficiency leads to a significant reduction in EFC proteins both in purified virions and in total lysates of infected cells . This reduction is attributed to protein instability caused by:

• The inherent hydrophobicity of EFC proteins

• Failure of EFC proteins to be properly inserted into viral membranes

The relationship between I2L and EFC stability appears to be indirect. When I2L is absent, viral morphogenesis is arrested at a stage where the D13 scaffold remains associated with viral membranes. This morphological block appears to prevent the proper integration of EFC proteins into these membranes, leading to their degradation due to exposed hydrophobic domains .

Interestingly, similar instability of EFC proteins has been observed with other unrelated mutants that are blocked earlier in morphogenesis and also accumulate viral membranes retaining the D13 scaffold. This suggests that proper scaffold removal, which requires I2L, is a prerequisite for successful EFC integration .

What is the relationship between I2L's roles in morphogenesis and virion entry?

The dual roles ascribed to I2L—in both morphogenesis and virion entry—appear to be interconnected. Initial studies using tetracycline-inducible I2L mutants reported that I2-deficient virions showed an approximately 400-fold reduction in specific infectivity due to an inability to enter target cells . Later studies with I2L deletion mutants revealed that the primary defect is actually in virion morphogenesis .

This apparent discrepancy can be explained by examining the different experimental approaches:

• Tetracycline-inducible systems may allow for low-level expression sufficient for partial morphogenesis but insufficient for entry function

• Complete deletion mutants reveal the earlier morphogenesis block that prevents proper formation of infectious particles

The current understanding suggests a unified model where I2L's primary function is in morphogenesis, particularly in the transition from immature to mature virions and the shedding of the D13 scaffold. This transition is required for the proper incorporation of EFC proteins, which are essential for virion entry into target cells . Thus, defects in entry observed in I2-deficient virions are likely secondary consequences of improper morphogenesis and EFC assembly.

How can researchers generate and characterize I2L mutants?

Researchers have employed several complementary approaches to generate and characterize I2L mutants:

Tetracycline-Inducible System:

• Replace the natural I2L promoter with a tetracycline-responsive promoter

• Control I2L expression by adding/removing tetracycline from culture medium

• Allows for conditional expression and study of partial defects

I2L Deletion Mutant Generation:

• Create a complementing cell line expressing I2L (essential for propagating deletion mutants)

  • Use lentiviral vectors carrying codon-optimized I2L gene with epitope tags (e.g., HA tag)

  • Introduce into permissive cells (e.g., RK-13 rabbit kidney cells)

  • Select transduced cells with appropriate antibiotics

  • Verify expression by Western blotting and immunofluorescence

• Delete I2L gene from the viral genome

  • Design recombination vectors with selection markers flanked by I2L-adjacent sequences

  • Transfect complementing cells with the recombination vector and infect with wild-type virus

  • Select recombinant viruses based on marker expression

  • Confirm deletion by PCR and sequencing

• Characterization methods:

  • Plaque assays on complementing vs. non-complementing cells to verify essentiality

  • Electron microscopy to examine viral morphogenesis

  • Western blotting to analyze viral protein expression and processing

  • Immunofluorescence to determine protein localization

  • Entry assays to assess virion infectivity

These approaches allow for comprehensive analysis of I2L function and the consequences of its absence in the viral life cycle.

What methods are effective for recombinant expression and purification of I2L protein?

Recombinant expression and purification of I2L protein presents challenges due to its small size (8.4 kDa) and hydrophobic transmembrane domain. Based on available commercial products and research protocols, the following methods have proven effective:

Expression System:

• E. coli-based expression systems have successfully produced full-length I2L protein (1-73 aa)

• Use of N-terminal His-tags facilitates purification while preserving function

• Expression vectors with strong inducible promoters optimize protein yield

Purification Protocol:

• Cell lysis: Standard methods (sonication, French press, or chemical lysis)

• Affinity chromatography: Ni-NTA resins for His-tagged proteins

• Buffer optimization: Critical for membrane protein stability

  • Tris/PBS-based buffers

  • Addition of 6% Trehalose for stability

  • pH 8.0 appears optimal

• Storage: Lyophilization or storage in glycerol (5-50%)

Quality Control:

• SDS-PAGE to verify purity (>90% purity achievable)

• Avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• Long-term storage at -20°C/-80°C with glycerol

For functional studies, reconstitution of purified I2L into liposomes or nanodiscs may preserve native conformation and activity, though specific protocols for I2L reconstitution are not detailed in the available literature.

How can researchers study I2L localization and interactions in infected cells?

Several complementary approaches can be used to study I2L localization and interactions:

Fluorescent Protein Fusions:

• Generate recombinant viruses expressing GFP-I2L fusion proteins

• Maintain the natural I2L promoter to preserve proper expression timing

• Verify functionality of fusion proteins by complementation assays

• Use live-cell imaging or fixed-cell microscopy to track localization

Epitope Tagging:

• Introduce small epitope tags (HA, FLAG, etc.) to minimize functional disruption

• Create stable cell lines expressing tagged I2L for complementation studies

• Use immunofluorescence with tag-specific antibodies for localization studies

• Apply Western blotting to detect protein expression and processing

Co-localization Studies:

• Perform dual-labeling with antibodies against known viral markers

• Examples include D13 (scaffold protein), A17 (membrane protein), or EFC components

• Assess temporal and spatial relationships during infection

• Quantify co-localization using appropriate image analysis software

Protein-Protein Interaction Analysis:

• Co-immunoprecipitation to identify binding partners

• Proximity ligation assays for in situ detection of protein interactions

• Chemical crosslinking followed by mass spectrometry for interaction mapping

• Yeast two-hybrid or mammalian two-hybrid screens for potential interactors

These methods have been successfully applied to characterize I2L localization with major membrane proteins of immature and mature virions, confirming its association with viral membranes throughout morphogenesis .

How should researchers interpret phenotypic differences between inducible and deletion I2L mutants?

When analyzing I2L mutants, researchers should consider the following factors that may explain phenotypic differences between inducible and deletion mutants:

Expression Leakiness:

• Tetracycline-inducible systems may exhibit baseline expression even under repressive conditions

• Low-level expression might be sufficient for some but not all I2L functions

• Quantify residual expression by sensitive methods (RT-qPCR, Western blot with enhanced chemiluminescence)

Temporal Considerations:

• Inducible systems may alter the timing of I2L expression

• Complete absence (deletion) versus reduced/delayed expression (inducible) can lead to different phenotypes

• Conduct time-course experiments to assess sequential effects of I2L absence

Functional Thresholds:

• Different I2L functions may require different protein levels

• Morphogenesis may have a lower threshold requirement than entry functions

• Serial dilution of inducer can help establish dose-response relationships

Comparative Analysis Framework:

This framework helps reconcile apparently contradictory findings regarding I2L function. Initial studies with inducible mutants emphasized an entry defect , while later deletion studies revealed the primary block in morphogenesis . The current model suggests that entry defects are secondary consequences of morphogenesis failure and reduced EFC incorporation.

What contradictions exist in the I2L literature and how might they be resolved?

Several apparent contradictions exist in the literature regarding I2L function:

Entry vs. Morphogenesis Role:

• Initial studies: I2L primarily functions in virion entry

• Later studies: I2L is essential for morphogenesis, with entry defects being secondary

Resolution Strategy: These findings can be reconciled by considering the experimental approaches. The tetracycline-inducible system used initially may have allowed sufficient I2L expression for partial morphogenesis but insufficient for entry function. The deletion mutant studies revealed the earlier block in morphogenesis that naturally leads to entry defects.

Mechanism of EFC Reduction:

• Direct interaction: I2L directly stabilizes EFC proteins

• Indirect effect: I2L affects morphogenesis, indirectly impacting EFC stability

Resolution Strategy: The current evidence favors the indirect model. Multiple unrelated mutants blocked at similar morphogenesis stages show similar EFC stability issues, suggesting the common denominator is failure to properly integrate EFC proteins into membranes rather than specific interactions with I2L.

Experimental Data Integration:
Researchers should integrate multiple lines of evidence when interpreting I2L function:

• Genetic (mutant phenotypes)

• Biochemical (protein stability and processing)

• Structural (electron microscopy of virion morphology)

• Functional (entry and replication assays)

This integrated approach supports a model where I2L primarily functions in morphogenesis, specifically in the transition from immature to mature virions and the release of the D13 scaffold, which is a prerequisite for proper EFC integration and subsequent entry function .

How can structural analysis of I2L inform its mechanistic function?

While high-resolution structural data for I2L is currently limited, researchers can extract meaningful insights through several approaches:

Sequence-Based Structural Prediction:

• Secondary structure prediction suggests a C-terminal α-helical transmembrane domain

• Hydrophobicity analysis confirms membrane association

• Conserved residues across orthopoxviruses may indicate functional sites

• The N-terminal hydrophilic domain likely contains interaction motifs

Structure-Function Analysis Through Mutagenesis:
A transient complementation assay revealed key structural features:

• The C-terminal hydrophobic domain is essential for protein stability

• Several regions within the N-terminal hydrophilic domain are essential for biological function

Comparative Structural Biology:

• Identify structural homologs with known functions

• Map conserved motifs onto predicted structural models

• Consider potential interactions with known binding partners like D13 scaffold

Proposed Structural Mechanism:
Based on available data, a mechanistic model can be proposed where:

• The C-terminal transmembrane domain anchors I2L in viral membranes

• The N-terminal domain likely extends into the cytoplasm or virion interior

• This domain may interact with the D13 scaffold, potentially triggering conformational changes

• These changes could lead to scaffold release and subsequent membrane remodeling

• Remodeled membranes then become competent for EFC integration

Future structural studies using techniques like cryo-electron microscopy of I2L-containing viral particles or reconstituted systems could provide higher-resolution insights into the precise structural basis of I2L function.