Recombinant Vaccinia virus Protein F9 (F9L)

How is F9L protein expressed and incorporated into virions?

F9L is regulated by a viral late promoter, meaning the protein is expressed following viral DNA replication. This can be experimentally verified through time-course analysis of protein expression and by demonstrating sensitivity to DNA replication inhibitors such as AraC. For protein detection, Western blotting with anti-F9 polyclonal antibodies shows that unreduced F9 migrates more rapidly than expected from its mass due to intramolecular disulfide bonds. The protein becomes incorporated into mature virion (MV) membranes with the N-terminal domain exposed on the virion surface. This topological orientation can be confirmed through surface biotinylation experiments using membrane-nonpermeating reagents like sulfo-NHS-SS-biotin that selectively label exposed primary amines of lysine residues. Following biotinylation, the labeled proteins can be captured using immobilized NeutrAvidin and analyzed by Western blotting. The presence of biotinylated F9 in this fraction confirms its surface exposure on virions .

What is the functional difference between F9 and the structurally similar L1 protein?

Despite their structural similarity, F9 and L1 perform distinct functions in the viral life cycle:

The key functional difference is that F9 is critical for virus entry but not for morphogenesis, while L1 is essential for a late stage of morphogenesis. When F9 expression is repressed, virions form normally but are 150-fold less infectious than wild-type virions, as F9 is needed specifically for the virus penetration step after cell binding .

How can researchers generate conditional F9L mutants for functional studies?

To generate conditional F9L mutants, researchers can employ an inducible system that allows controlled expression of the F9L gene. A methodological approach involves:

• Constructing a recombinant virus (e.g., vF9Li) in which F9L expression is controlled by an inducible promoter system, such as the lac operator/repressor system.

• Reversing the orientation of the F9L gene to prevent read-through transcription from neighboring genes.

• Placing the F9L gene under the control of a T7 promoter and lac operator.

• Ensuring continuous expression of the E. coli lac repressor using an early-late VACV promoter.

• Incorporating a reporter gene (e.g., EGFP) to distinguish plaques containing the recombinant virus.

In this system, isopropyl-β-d-thiogalactopyranoside (IPTG) controls F9L expression. Without IPTG, the lac repressor binds to the lac operator upstream of both the T7 RNA polymerase gene and the F9L gene, repressing transcription. With IPTG present, the lac repressor is inactivated, allowing T7 RNA polymerase expression and subsequent F9L transcription. Optimal IPTG concentration can be determined by measuring virus yields at different IPTG levels (typically plateauing at around 100 μM) .

What methods can be used to analyze F9L incorporation into virions?

Several complementary approaches can be employed to analyze F9L incorporation into virions:

• Virion Purification: Mature virions (MVs) can be purified by sedimentation through two consecutive 36% sucrose cushions followed by a 25-40% continuous sucrose gradient.

• Western Blotting: Purified virions can be analyzed by SDS-PAGE and Western blotting using anti-F9 polyclonal antibodies. Comparison of migration patterns under reducing and non-reducing conditions can verify the presence of intramolecular disulfide bonds.

• Surface Biotinylation: To confirm membrane localization and topology, purified MVs can be incubated with sulfo-NHS-SS-biotin (a membrane-nonpermeating reagent). After removing excess biotin, virions are solubilized with SDS without reducing agent. Biotinylated proteins are captured using immobilized NeutrAvidin and eluted by incubation with DTT. Analysis of bound and unbound fractions by Western blotting can confirm F9's presence on the virion surface.

• Immunofluorescence: For cellular localization studies, infected cells can be fixed, permeabilized, and incubated with anti-F9 antibodies along with antibodies against known viral membrane proteins. Visualization with fluorescent secondary antibodies and confocal microscopy can determine co-localization patterns.

• Detergent Extraction: Membrane association properties can be further characterized by extracting virions with non-ionic detergents (e.g., NP-40) with or without reducing agents (DTT) .

How can researchers evaluate the role of F9L in virus entry and penetration?

To evaluate F9L's role in virus entry and penetration, researchers can employ several sophisticated assays:

• Early Gene Transcription Assay: This highly sensitive approach exploits the presence of a complete transcription system within VACV cores that activates upon cytoplasmic entry. Cells are infected with equal numbers of F9-positive or F9-negative virions in the presence of AraC (which prevents viral DNA replication and late gene transcription). After 3 hours, total RNA is extracted and analyzed by Northern blotting using a radiolabeled probe complementary to an early viral transcript (e.g., C11R gene). Detection of the early transcript indicates successful virus entry and core activation.

• Cell Binding and Penetration Assay: This assay differentiates between binding and penetration defects, based on the principle that antibodies to core proteins cannot bind virions prior to membrane removal, even after fixation and permeabilization. The protocol involves:

  • Incubating cells with virions at 4°C for 1 hour to allow binding

  • Washing extensively to remove unbound virus

  • Shifting to 37°C for 2 hours to permit penetration

  • Fixing and permeabilizing cells

  • Staining with antibodies specific for membrane proteins (e.g., anti-L1) and core proteins (e.g., anti-A4)

  • Analyzing by immunofluorescence microscopy

Successful penetration is indicated by detection of core protein staining in the cytoplasm. F9-deficient virions show binding (indicated by membrane protein staining) but lack core protein staining after the temperature shift, demonstrating a specific defect in the penetration step .

What approaches can be used to investigate F9L interactions with other viral proteins?

Investigating F9L interactions with other viral proteins requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Cells infected with recombinant viruses expressing tagged versions of F9 (e.g., F9-V5) can be lysed and immunoprecipitated with anti-tag antibodies. Interacting proteins can be identified by Western blotting with antibodies against suspected partner proteins or by mass spectrometry for unbiased discovery.

• Proximity Ligation Assays: This technique can detect protein-protein interactions in situ with high sensitivity. It involves using primary antibodies against F9 and potential partner proteins, followed by secondary antibodies linked to oligonucleotides that can be ligated when in close proximity and subsequently amplified and detected.

• Crosslinking Studies: Chemical crosslinkers can stabilize transient protein interactions before cell lysis. After crosslinking, complexes containing F9 can be immunoprecipitated and analyzed by mass spectrometry.

• Fusion/Entry Complex Analysis: Since F9 has been implicated in the entry/fusion process, techniques to analyze its association with known components of the poxvirus entry/fusion complex can provide valuable insights. This might involve sequential immunoprecipitation steps or gradient fractionation followed by Western blotting.

• Yeast Two-Hybrid or Mammalian Two-Hybrid Screens: These can be employed for systematic screening of potential interaction partners, although they may miss interactions dependent on the membrane environment .

How do recombinant F9L-deficient viruses compare to wild-type in morphogenesis studies?

Detailed morphogenesis studies comparing F9L-deficient and wild-type viruses reveal important distinctions about F9's role in the viral life cycle:

• Core Protein Processing: In wild-type virus infection, viral precursor proteins p4a and p4b are processed to 4a and 4b during morphogenesis. This processing can be monitored by metabolic labeling with [35S]methionine and cysteine, followed by pulse-chase analysis. Unlike L1-deficient viruses, which show a block in core protein processing, F9-deficient viruses exhibit normal processing of these precursor proteins, indicating that F9 is not required for this morphogenesis step.

• Electron Microscopy: Transmission electron microscopy of cells infected with F9-deficient viruses shows the complete array of viral forms, including immature virions, mature virions (MVs), and extracellular virions (EVs), all indistinguishable from those formed by wild-type virus. This contrasts with the morphogenesis defect observed in L1-deficient viruses.

• Protein Composition Analysis: SDS-PAGE and Western blotting analysis of purified F9-deficient virions shows a protein composition identical to wild-type virions, except for the specific absence of F9 itself. This confirms that F9 is not required for the incorporation of other viral proteins into the virion.

• Infectivity Assays: Despite normal morphology and protein composition, F9-deficient virions exhibit dramatically reduced infectivity (approximately 150-fold less than wild-type), highlighting F9's specific role in the entry process rather than assembly .

What are the best practices for expressing and purifying recombinant F9L protein?

Expressing and purifying functional recombinant F9L protein presents several challenges due to its membrane association and disulfide bond formation requirements. Recommended approaches include:

• Expression System Selection:

  • Mammalian expression systems (e.g., HEK293 cells) are preferred for proper folding and disulfide bond formation

  • Baculovirus expression systems can provide higher yields while maintaining proper folding

  • Bacterial systems typically yield misfolded protein unless specialized strains with enhanced disulfide bond formation capabilities are used

• Construct Design:

  • Include a cleavable signal peptide for secretion or membrane targeting

  • Consider truncating the transmembrane domain for improved solubility

  • Add affinity tags (His6, FLAG, etc.) for purification, preferably at the C-terminus

  • Ensure the construct preserves all cysteine residues critical for disulfide bonding

• Purification Strategy:

  • Use mild detergents (DDM, CHAPS) for extraction from membranes

  • Employ affinity chromatography as the initial purification step

  • Include reducing agents during initial purification, followed by controlled oxidation for proper disulfide bond formation

  • Consider on-column refolding protocols for proteins expressed in inclusion bodies

  • Use size exclusion chromatography as a final polishing step

• Quality Control:

  • Verify correct folding through circular dichroism and thermal shift assays

  • Confirm disulfide bond formation through non-reducing SDS-PAGE

  • Validate biological activity through neutralization assays or cell binding studies

  • Check homogeneity by dynamic light scattering

How can researchers develop neutralizing antibodies against F9L for functional studies?

Developing neutralizing antibodies against F9L requires careful consideration of epitopes and screening methods:

• Immunization Strategies:

  • Use properly folded recombinant F9 protein with intact disulfide bonds

  • Consider DNA immunization encoding F9 for in vivo expression

  • Employ prime-boost strategies combining DNA and protein immunizations

  • Include appropriate adjuvants to enhance immune responses

• Screening for Neutralizing Activity:

  • Develop a flow cytometric virus neutralization assay using recombinant vaccinia virus encoding an easily detectable reporter (e.g., EGFP)

  • Pre-incubate potential neutralizing antibodies with purified virus

  • Infect target cells with the antibody-virus mixture

  • Analyze reporter gene expression (e.g., EGFP) by flow cytometry after an appropriate incubation period

  • Calculate neutralization based on the reduction in percentage of reporter-positive cells

• Epitope Mapping:

  • Generate a panel of overlapping peptides spanning the F9 N-terminal domain

  • Use ELISA or peptide array technologies to identify binding regions

  • Create point mutations in key residues for fine mapping

  • Consider hydrogen-deuterium exchange mass spectrometry for conformational epitope mapping

• Antibody Characterization:

  • Determine if neutralization occurs at the binding or post-binding stage

  • Test antibody efficacy against different poxvirus strains

  • Evaluate neutralization in the presence of complement

  • Assess antibody-dependent cellular cytotoxicity potential

What are the critical considerations when constructing recombinant viruses with modified F9L genes?

When constructing recombinant vaccinia viruses with modified F9L genes, researchers should consider these critical factors:

• Selection of Recombination Strategy:

  • Homologous recombination: Standard approach using flanking sequences of approximately 500 bp on each side of the F9L locus

  • CRISPR-Cas9 facilitated insertion: Can improve recombination efficiency

  • Bacterial artificial chromosome (BAC) systems: Allow manipulation of the viral genome in bacterial cells

• Promoter Selection:

  • Natural F9L promoter: Maintains authentic late expression timing

  • Synthetic early/late promoter: Extends expression throughout infection

  • Inducible promoters (e.g., T7-lac system): Enables controlled expression for functional studies

• Modification Strategies:

  • For knockout studies: Replace F9L with a reporter gene (e.g., EGFP) under a viral promoter

  • For expression of tagged proteins: Add epitope tags (V5, HA, etc.) preferably at the C-terminus to avoid interfering with N-terminal functions

  • For expression of heterologous genes: Position them in relation to the F9L locus to minimize effects on neighboring genes

• Virus Propagation and Purification:

  • For F9L-deficient constructs: Propagate in complementing cell lines or under inducing conditions if using an inducible system

  • Purify virions using sucrose cushion centrifugation followed by sucrose gradient purification

  • Determine particle counts by optical density measurements (OD260)

  • Calculate specific infectivity (particle-to-PFU ratio) to assess the impact of F9L modifications

• Verification Steps:

  • Confirm genomic modifications by PCR and sequencing

  • Verify protein expression by Western blotting

  • Check growth kinetics through single-step growth curves

  • Assess virion morphology by electron microscopy

What are the unresolved questions about F9L's mechanism of action in viral entry?

Despite significant progress in understanding F9L's role in vaccinia virus entry, several important questions remain unresolved:

• Interaction with Cellular Receptors: While F9 is known to be required for virus penetration, the identity of any cellular receptors it might interact with remains unknown. Future research should focus on identifying potential cellular binding partners using techniques such as:

  • Cross-linking followed by mass spectrometry

  • Proximity labeling approaches (BioID, APEX)

  • Genome-wide CRISPR screens to identify cellular factors required for F9-dependent entry

• Fusion Mechanism: The precise mechanism by which F9 contributes to membrane fusion during virus entry is not fully understood. Key questions include:

  • Does F9 undergo conformational changes during the fusion process?

  • Is F9 directly involved in membrane merger or does it play a regulatory role?

  • How do the intramolecular disulfide bonds influence the fusion activity?

• Integration with Known Entry-Fusion Complex: F9 is known to interact with components of the entry-fusion complex, but the stoichiometry and structural arrangement of these interactions remain undefined. Cryo-electron microscopy of entry complexes may help resolve these questions.

• pH-Dependent Functions: Cells infected with F9-negative virions do not fuse after brief low-pH treatment, unlike those infected with F9-positive virions. The molecular basis for this pH sensitivity and F9's role in this process requires further investigation .

How might F9L be utilized in the development of novel oncolytic vaccinia viruses?

F9L modifications present several opportunities for enhancing oncolytic vaccinia virus therapies:

• Targeted Entry Enhancement: Since F9 is critical for virus entry, modifications to its sequence or expression level could potentially enhance viral entry into specific cancer cell types while maintaining normal expression in non-target cells. This might involve:

  • Creating chimeric F9 proteins with cancer-specific targeting domains

  • Placing F9 under the control of cancer-specific promoters to enhance entry specifically in tumor cells

  • Engineering F9 to interact with receptors overexpressed on cancer cells

• Immune Evasion Modulation: By engineering F9 alongside immune regulatory proteins, researchers could potentially:

  • Reduce premature neutralization of the oncolytic virus by the host immune system

  • Enhance immune recognition of tumor cells after viral infection

  • Create a more favorable tumor microenvironment for immune cell infiltration

• Combination with Immunomodulatory Transgenes: As demonstrated in the context of oncolytic virus development, F9-modified viruses could be engineered to co-express therapeutic transgenes such as:

  • Human GM-CSF to enhance antitumor immunity

  • Complement regulatory proteins like CD55 to enhance virus survival in the presence of human complement

  • Immune checkpoint inhibitors to augment T-cell responses against tumor cells

• Optimization of Virus Production: Understanding F9's role in the viral life cycle could inform strategies to improve manufacturing and stability of oncolytic virus preparations, potentially increasing their therapeutic efficacy .

What advanced techniques are emerging for studying F9L interactions in the context of the viral entry complex?

Cutting-edge techniques are providing new insights into F9L interactions within the viral entry complex:

• Cryo-Electron Tomography: This technique allows visualization of viral entry complexes in their native environment, potentially revealing the three-dimensional arrangement of F9 in relation to other complex components during different stages of the entry process.

• Super-Resolution Microscopy: Techniques such as STORM, PALM, or STED microscopy can track the dynamics of fluorescently labeled F9 during the entry process with nanometer precision, revealing spatial and temporal aspects of its function.

• Single-Particle Tracking: By labeling individual virions and tracking their movement during cell entry, researchers can correlate F9 function with specific stages of the entry process in real-time.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This approach can identify regions of F9 that undergo conformational changes during the entry process or upon interaction with other proteins, providing insights into its molecular mechanism.

• AlphaFold and Other Structural Prediction Tools: Advanced protein structure prediction algorithms can generate models of F9 interactions with other viral and cellular proteins, generating hypotheses that can be tested experimentally.

• CRISPR Screening: Genome-wide or targeted CRISPR screens can identify cellular factors required for F9-dependent entry, complementing biochemical approaches to understanding its mechanism.

• Protein Correlation Profiling: This mass spectrometry-based technique can identify proteins that co-migrate with F9 during various steps of virus assembly and entry, potentially revealing new interaction partners .