Recombinant Orf virus Protein F9 homolog (A3R)

What is Orf virus Protein F9 homolog (A3R) and what are its key structural features?

Orf virus Protein F9 homolog (A3R) is a membrane protein of the Orf virus (ORFV) that shares structural similarity with Vaccinia virus (VACV) F9 protein, with approximately 20% amino acid identity . The full-length protein consists of 73 amino acids with the sequence: GHAAANCALARVATALTRRVPASRHGLAEGGTPPWTLLLAVAAVTVLGVVAVSLLRRALRVRYRFARPAALRA . The protein contains a predicted transmembrane domain that facilitates its incorporation into the viral membrane .

The recombinant form typically includes an N-terminal His-tag when expressed in E. coli expression systems, which facilitates purification while maintaining the protein's structural integrity . Like its VACV counterpart, A3R contains disulfide bonds that affect its migration pattern in gel electrophoresis under non-reducing conditions .

How is the Orf virus F9 homolog protein expressed during viral infection?

The F9 protein is expressed as a late viral protein during ORFV infection. Based on studies of the related VACV F9, its expression is regulated by a viral late promoter, meaning the protein is synthesized following viral DNA replication . Experimental evidence shows that treatment with AraC, a DNA replication inhibitor, prevents F9 protein synthesis, confirming its classification as a late viral protein . This temporal expression pattern suggests that F9 plays a role in the later stages of viral replication, particularly in virion assembly and maturation rather than early viral processes.

What is the cellular and virion localization of the F9 protein?

The F9 protein is incorporated into the membrane of mature virions (MVs) . Surface biotinylation experiments with sulfo-NHS-SS-biotin (a membrane-nonpermeating reagent) demonstrated that F9 is exposed on the surface of MVs, similar to the L1 protein . The protein shows poor solubility when extracted with NP-40 or NP-40 plus DTT, a characteristic shared with components of the poxvirus entry/fusion complex . This membrane localization is consistent with its role in viral entry processes.

What are the optimal conditions for expressing and purifying recombinant Orf virus Protein F9 homolog?

The recombinant F9 homolog protein can be effectively produced using E. coli expression systems with an N-terminal His-tag to facilitate purification . Based on product information and experimental protocols, the following conditions are recommended:

For reconstitution, it's recommended to centrifuge the vial briefly before opening and reconstitute the lyophilized protein in deionized sterile water . Adding glycerol (5-50% final concentration) and aliquoting for long-term storage at -20°C/-80°C helps prevent protein degradation from repeated freeze-thaw cycles .

How does F9 protein contribute to Orf virus infectivity?

The F9 protein plays a critical role in ORFV infectivity, particularly during viral entry. Based on studies with the related VACV F9 protein, F9-deficient virions can bind to cells, but their cores cannot penetrate into the cytoplasm, indicating that F9 is essential for the entry process but not for virion assembly .

Experimental evidence from a recombinant VACV with inducible F9 expression (vF9Li) showed that virus replication and plaque formation were dependent on the inducer (IPTG) . Without F9 expression, there was only a slight increase in virus titer during infection, demonstrating that F9 is needed for the production of infectious virus .

What experimental approaches are most effective for studying F9 protein function?

Based on published research methodologies, these approaches have proven effective for studying F9 protein function:

• Inducible expression systems: Creating recombinant viruses with inducible F9 expression (e.g., IPTG-inducible systems) allows for controlled studies of F9's role in the viral life cycle .

• Surface biotinylation: Using membrane-nonpermeating reagents like sulfo-NHS-SS-biotin to selectively label exposed proteins on intact virions, followed by affinity purification and Western blotting .

• Virus neutralization assays: Using anti-F9 antibodies in flow cytometric virus neutralization assays to assess the role of F9 in virus entry .

• Single-step growth experiments: Comparing virus yields over time with and without F9 expression to quantify its impact on replication kinetics .

• Fusion assays: Examining low-pH-induced cell-cell fusion in cells infected with F9-expressing versus F9-deficient viruses to assess the protein's role in membrane fusion events .

How does the F9 protein interact with the poxvirus entry/fusion complex?

The F9 protein appears to be functionally associated with the poxvirus entry/fusion complex, despite having no structural homology to other complex components. Evidence indicates that F9-deficient virions exhibit a phenotype identical to virions lacking individual components of the previously described poxvirus entry/fusion complex: they can bind to cells, but their cores fail to penetrate into the cytoplasm .

Furthermore, experimental data shows that F9 physically interacts with proteins of the entry/fusion complex . Cells infected with F9-negative virions do not undergo fusion after brief low-pH treatment, unlike cells infected with F9-expressing virus, providing additional evidence for F9's role in the fusion process .

These findings suggest that F9 may function as an accessory protein for the entry/fusion complex, potentially stabilizing the complex or facilitating conformational changes required for fusion pore formation. Future research utilizing co-immunoprecipitation and cryo-electron microscopy could further elucidate the structural interactions between F9 and other complex components.

What is the relationship between F9 and the Type I interferon response?

While F9 itself has not been directly linked to interferon (IFN) antagonism, ORFV as a whole shows increased sensitivity to IFN-β compared to VACV . ORFV lacks homologues of the VACV IFN-resistance genes KIL and C7L, yet can still replicate in cells with constitutive expression of interferon-stimulated genes (ISGs) such as HeLa cells .

This suggests that ORFV must encode alternative mechanisms to combat the antiviral effects of ISGs like SAMD9 and SAMD9L, which are critical targets of both KIL and C7L in VACV . Given F9's role in viral entry, it may function in concert with other ORFV proteins to overcome IFN-induced restriction factors during the entry process.

Research examining the replication efficiency of F9-deficient ORFV in the presence of various concentrations of type I IFNs could provide insights into potential roles of F9 in IFN resistance.

How do F9-deficient virions differ from wild-type virions in structure and function?

F9-deficient virions exhibit several key differences from wild-type virions:

Interestingly, unlike L1-deficient virions which fail to form normal mature particles, F9-deficient virions form morphologically normal intracellular and extracellular virions . This indicates that while both L1 and F9 share structural similarities, they have distinct functions: L1 in assembly and F9 in entry .

What are the implications of F9 protein research for developing antiviral strategies against poxviruses?

Understanding the structure and function of F9 protein has significant implications for developing targeted antiviral strategies against poxviruses:

• Neutralizing antibodies: The F9 protein can induce neutralizing antibodies, suggesting it could be a potential target for vaccine development or therapeutic antibody approaches .

• Entry inhibitors: Given F9's essential role in viral entry, small molecule inhibitors that interfere with F9 function could effectively block poxvirus infection at an early stage.

• Broad-spectrum antivirals: Since F9 homologs are conserved across all sequenced poxviruses, targeting this protein could lead to broad-spectrum antipoxviral drugs .

• Understanding tropism: Research on F9 and related proteins helps explain the tissue tropism of poxviruses like ORFV, which primarily infects skin. This could inform the development of oncolytic virus therapies with specific tissue targeting .

• Comparative analysis with other viruses: The entry mechanisms of poxviruses, including the role of F9, may have parallels with other enveloped viruses, potentially leading to broader antiviral strategies.