Recombinant Vaccinia virus Virion membrane protein A9 (VACWR128)

What is Vaccinia Virus Virion Membrane Protein A9 (VACWR128) and what is its significance in the viral structure?

Vaccinia virus virion membrane protein A9 (VACWR128) is a structural protein encoded by the A9L gene that forms part of the viral membrane architecture. It is a relatively small protein, with the mature form spanning approximately amino acids 23-108 of the full-length protein . A9 plays a critical role in the viral envelope structure and is found in the virion membrane. Its significance lies in its contribution to viral stability, morphogenesis, and potentially in host cell interaction. As a membrane component, it helps maintain the structural integrity of the viral particle, which is essential for vaccinia virus's infectious capability and environmental resilience.

What expression systems are most effective for producing recombinant VACWR128 protein?

For studies requiring authentic post-translational modifications, mammalian or insect cell expression systems may be preferable. Vaccinia virus's natural ability to replicate in mammalian cells makes them suitable hosts for expressing viral proteins with native modifications . Baculovirus expression systems using insect cells like Sf9 have also been employed for poxvirus proteins, offering a balance between yield and post-translational processing capabilities .

What methods are most effective for studying VACWR128 protein-protein interactions within the viral particle?

To effectively study VACWR128 protein-protein interactions within viral particles, several complementary approaches should be considered:

• Co-immunoprecipitation (Co-IP) represents a foundational method for detecting protein-protein interactions in near-native conditions. Using antibodies specific to VACWR128 or its interaction partners, researchers can isolate protein complexes from infected cell lysates or purified virions.

• Yeast two-hybrid screening can identify potential interaction partners when VACWR128 is used as bait. This approach is particularly useful for discovering novel interactions but requires validation with other methods.

• Proximity labeling techniques like BioID or APEX, where VACWR128 is fused with a proximity-dependent labeling enzyme, allow identification of proteins that come into close proximity with VACWR128 in living cells during viral assembly.

• Cryo-electron microscopy of purified virions can provide structural insights into how VACWR128 integrates into the viral membrane and interfaces with other viral components.

• Cross-linking mass spectrometry (XL-MS) can capture transient or stable interactions by chemically linking proteins in close proximity before identification by mass spectrometry.

The data from these complementary approaches should be compiled in interaction networks to understand VACWR128's role within the complex architecture of vaccinia virions.

How does the membrane topology of VACWR128 influence its function in virion assembly?

The membrane topology of VACWR128 (A9) is critical to its function in virion assembly. As a virion membrane protein, A9 contains hydrophobic domains that facilitate its integration into the viral membrane . The protein's orientation within the membrane likely determines its ability to interact with other viral structural proteins during morphogenesis.

Experimental approaches to study this topology include protease protection assays, where susceptibility to proteolytic cleavage indicates exposed protein regions, and fluorescence microscopy with tagged variants of VACWR128 to visualize its localization during different stages of virion assembly. Understanding this topology is essential because it directly impacts how A9 contributes to the structural organization of the viral envelope and potentially influences the incorporation of other viral proteins during virion maturation.

Researchers have observed that proper membrane insertion of virion proteins like A9 is essential for the transition from immature to mature virion forms during the vaccinia virus replication cycle. Disruption of this process through mutations in membrane proteins can lead to morphogenesis defects, highlighting the importance of A9's correct topological arrangement in viral assembly.

What are the key considerations when designing experiments to study VACWR128 function through gene modification?

When designing experiments to study VACWR128 function through gene modification, researchers should consider several critical factors:

What purification methods yield the highest purity recombinant VACWR128 protein for structural studies?

For structural studies requiring high-purity recombinant VACWR128 protein, a multi-step purification strategy is recommended:

• Expression system selection: While E. coli systems can produce substantial quantities of recombinant protein , insect cell expression systems may provide better folding and post-translational modifications for membrane proteins like VACWR128 .

• Affinity chromatography: His-tagged VACWR128 can be effectively purified using nickel or cobalt affinity resins . For membrane proteins, purification should be performed in the presence of appropriate detergents to maintain solubility.

• Size exclusion chromatography: This secondary purification step separates the target protein from aggregates and differentially sized contaminants, providing more homogeneous preparations.

• Ion exchange chromatography: As a polishing step, this technique can remove remaining contaminants based on charge differences.

• Detergent optimization: For membrane proteins like VACWR128, screening different detergents (e.g., DDM, LMNG, or amphipols) is crucial for maintaining native conformation during purification.

• Quality control assessment: The final preparation should be evaluated through:

  • SDS-PAGE and Western blotting

  • Dynamic light scattering to assess homogeneity

  • Mass spectrometry for identity confirmation

  • Circular dichroism to verify secondary structure

Typical yields for recombinant VACWR128 from bacterial systems range from 2-5 mg/L of culture, with purity exceeding 95% after optimized purification protocols. For structural studies using techniques like X-ray crystallography or cryo-EM, protein purity should be at least 98%.

What are the most reliable methods for detecting VACWR128 expression in infected cells?

Detection of VACWR128 expression in infected cells can be accomplished through several complementary techniques:

• Western blot analysis using specific antibodies against VACWR128 or epitope tags if using recombinant viruses with tagged A9 protein . This approach provides quantitative information about expression levels and protein processing.

• Immunofluorescence microscopy to visualize the subcellular localization of VACWR128 during different stages of infection, providing insights into its trafficking and incorporation into viral particles.

• Quantitative RT-PCR to measure VACWR128 mRNA levels, which is particularly useful for temporal expression studies during the viral life cycle.

• Mass spectrometry-based proteomics for unbiased detection and quantification of VACWR128 along with other viral and cellular proteins.

• Flow cytometry if using fluorescently tagged VACWR128 constructs, enabling quantitative single-cell analysis of expression levels across a population of infected cells.

For temporal studies, researchers should collect samples at multiple time points post-infection (e.g., 0, 2, 4, 8, 12, and 24 hours) to track the expression dynamics of VACWR128 throughout the vaccinia virus replication cycle.

How does modification of VACWR128 affect the immunogenicity of recombinant vaccinia virus vaccines?

Modification of VACWR128 (A9) can significantly impact the immunogenicity of recombinant vaccinia virus vaccines through several mechanisms:

The A9 protein, as a virion membrane component, contributes to the structural integrity of the viral particle, which in turn affects how the virus interacts with host cells and the immune system. Alterations to VACWR128 may modify viral infectivity, stability, or the presentation of other immunogenic viral epitopes on the virion surface.

Research has demonstrated that viral membrane proteins like A9 can influence the balance between intracellular mature virion (IMV) and extracellular enveloped virion (EEV) forms . This balance is critical because these different virion forms elicit distinct immune responses. While IMVs typically induce strong antibody responses, EEVs may be important for cell-to-cell spread and can elicit complementary immune responses targeting different viral epitopes.

When considering VACWR128 modifications in vaccine design, researchers should evaluate:

• Effects on virion stability and infectivity in different cell types

• Changes in the kinetics of antigen presentation

• Alterations in the magnitude and quality of antibody responses

• Modifications to T-cell epitope presentation and subsequent cellular immunity

Systematic immunogenicity studies comparing wild-type and VACWR128-modified vaccinia viruses should include:

• Antibody titer measurements

• Neutralization assays against different virion forms

• T-cell response analysis (CD4+ and CD8+)

• Challenge studies in appropriate animal models to assess protective efficacy

These comprehensive evaluations will determine whether VACWR128 modifications enhance or diminish vaccine efficacy, guiding rational design of next-generation recombinant vaccinia vaccines.

What insertion sites near VACWR128 are optimal for foreign gene expression in recombinant vaccinia vectors?

When considering insertion sites near VACWR128 for foreign gene expression in recombinant vaccinia vectors, researchers should evaluate several factors:

The thymidine kinase (TK) locus (J2R, VACV-WR94) has been extensively used as an insertion site for foreign genes in vaccinia virus and provides a reliable location that typically doesn't interfere with VACWR128 function . This site allows for stable insertion of foreign DNA and can accommodate relatively large inserts (up to 25 kb) .

When selecting insertion sites near VACWR128, researchers should consider:

• Genomic context: Avoid disrupting essential genes or regulatory elements that might affect VACWR128 expression or function.

• Promoter selection: The synthetic early/late promoter has been successfully used to drive strong expression of foreign genes in vaccinia vectors . This promoter ensures expression throughout the viral replication cycle.

• Reporter systems: Including reporter genes like GFP or luciferase facilitates identification and isolation of recombinant viruses . The Luc-2A-GFP dual reporter system has proven effective for this purpose.

• Selection method: Homologous recombination with flanking sequences upstream and downstream of the insertion site is the standard approach, followed by plaque purification to isolate recombinant viruses .

For optimal expression and minimal interference with viral functions, researchers have successfully used the following approach:

The final recombinant viruses should be verified through PCR, sequencing, and expression analysis to confirm proper insertion and functionality of the foreign gene.

How does VACWR128 contribute to the differential behavior of intracellular mature virions (IMV) versus extracellular enveloped virions (EEV)?

VACWR128 (A9) plays a differential role in the biology of intracellular mature virions (IMV) versus extracellular enveloped virions (EEV), although its primary association is with the IMV form. Understanding this distinction is crucial for advanced vaccinia virus research:

IMVs constitute the majority of infectious particles (>99%) and are characterized by a single membrane, while EEVs have an additional outer membrane and comprise less than 1% of the viral population . The A9 protein is predominantly found in the IMV membrane, contributing to its structural integrity.

Research suggests that membrane proteins like A9 in the IMV may influence the subsequent formation of EEVs through interactions with wrapping membrane components. The balance between IMV and EEV forms affects viral dissemination strategy - IMVs are released upon cell lysis and mediate host-to-host transmission, while EEVs facilitate long-range spread within an infected host .

Experimental evidence indicates that IMVs containing A9 induce different neutralizing antibody responses compared to EEVs, which express distinct envelope proteins like B5R . This immunological difference is significant for vaccine development, as protective immunity may require responses against both virion forms.

Advanced research techniques to study A9's differential role include:

• Immunoelectron microscopy to visualize A9 localization in different virion forms

• Conditional expression systems to modulate A9 levels and observe effects on virion morphogenesis

• Correlative light and electron microscopy to track A9-tagged virions during morphogenesis

• Proteomics analysis comparing protein composition of IMV versus EEV membranes

The data suggests that while A9 primarily functions in IMV structure, its proper incorporation affects the subsequent formation and function of EEVs, highlighting its importance in the complete viral replication cycle.

What role might VACWR128 play in host immune evasion strategies of vaccinia virus?

While VACWR128 (A9) is primarily characterized as a structural protein, emerging research suggests it may contribute to vaccinia virus immune evasion strategies through several mechanisms:

As a membrane protein, A9 potentially influences how vaccinia virions interact with pattern recognition receptors (PRRs) of the innate immune system. The arrangement of viral membrane proteins could theoretically mask pathogen-associated molecular patterns (PAMPs) that would otherwise trigger antiviral responses.

Research into vaccinia immune evasion has revealed that structural proteins can sometimes perform dual functions, serving both architectural roles and specifically counteracting host defense mechanisms. Whether A9 has such dual functionality remains an active area of investigation.

Advanced research approaches to explore A9's potential immune evasion functions include:

• Proteomic analyses of A9 interaction partners during different stages of infection, focusing on components of host immune signaling pathways

• Comparative studies using wild-type versus A9-modified viruses to assess differences in:

  • Type I interferon induction

  • NF-κB pathway activation

  • Inflammasome activation

  • Antigen presentation efficiency

• Systems biology approaches integrating transcriptomics, proteomics, and functional assays to build comprehensive models of how A9 might influence host-virus interactions

Preliminary evidence suggests that proper virion membrane formation, which involves A9, affects the exposure of viral DNA to cytosolic DNA sensors, potentially influencing early innate immune detection. Additionally, the incorporation of host proteins into the viral membrane during morphogenesis, a process in which A9 participates, may contribute to immune evasion by displaying self-antigens on the viral surface.

These hypotheses require rigorous testing through genetic manipulation of A9 combined with comprehensive immunological assessments in both in vitro and in vivo systems.

How can VACWR128 modifications be leveraged for developing improved oncolytic vaccinia virus therapies?

VACWR128 (A9) modifications present several strategic opportunities for enhancing oncolytic vaccinia virus therapies:

As a structural membrane protein, A9 modifications could alter viral tropism, stability, and immunogenicity - all critical factors for oncolytic efficacy. Research into vaccinia virus-based oncolytic therapies has demonstrated that viral engineering can significantly enhance therapeutic outcomes .

Strategic approaches for leveraging A9 modifications include:

• Tropism modification: Altering A9's membrane interaction domains could potentially redirect viral tropism toward specific cancer cells by changing virion surface properties. This approach would require detailed structural information about A9's membrane topology.

• Immunomodulation enhancement: A9 could serve as a scaffold for displaying immunostimulatory molecules on the viral surface. Genetic fusion of immune-activating ligands to A9 might enhance anti-tumor immune responses without disrupting viral replication.

• Combination with therapeutic transgenes: Recombinant vaccinia viruses can accommodate large DNA inserts (up to 25 kb) , allowing for the expression of therapeutic proteins alongside potential A9 modifications. This approach enables multipronged therapeutic strategies.

• Virion stability optimization: Engineered changes to A9 might enhance viral particle stability in the tumor microenvironment, potentially improving delivery and persistence within tumors.

Experimental design for testing A9-modified oncolytic vectors should include:

When combined with other genetic modifications such as immune checkpoint inhibitors or cytokine expression, strategically engineered A9 variants could contribute to next-generation oncolytic platforms with enhanced efficacy and safety profiles .