Recombinant Variola virus Virion membrane protein A16 (A16L)

What is the structure and function of Variola virus A16L protein?

A16L is an essential component of the Entry/Fusion Complex (EFC) in poxviruses. This 377-amino acid membrane protein (UniProt P33841) contains a conserved N-terminal MG motif that serves as the site for N-myristoylation . The protein plays a critical role in virus entry by facilitating fusion of the viral membrane with host cell membranes.

The A16L protein functions within a multiprotein complex alongside other proteins, including G9, to mediate the delivery of viral cores into the cytoplasm. Research with vaccinia virus (VACV), which shares high homology with variola virus, demonstrates that A16L is indispensable for viral infection, making it a potential target for antiviral development .

How conserved is A16L across the poxvirus family?

The A16L protein demonstrates remarkable conservation across all members of the Poxviridae family. Research indicates that the N-terminal MG N-myristoylation motif is conserved in all poxviral A16 proteins, suggesting an important evolutionary role in poxvirus biology .

What post-translational modifications occur in A16L?

N-myristoylation represents the primary documented post-translational modification of A16L. This irreversible lipid modification occurs at the N-terminal glycine residue (position 2) following removal of the initiating methionine. The 14-carbon saturated fatty acid (myristate) attachment appears to influence protein function, particularly in viral spread .

Research demonstrates that inhibiting N-myristoylation using compounds like IMP-1088, which targets N-myristoyltransferase (NMT), reduces the incorporation of myristate analogs into A16L. While this modification contributes to viral spread, mutational studies suggest it may not be essential for initial virus infectivity .

Additionally, A16L likely undergoes disulfide bond formation, as the protein contains multiple cysteine residues. Research with related poxviral proteins indicates that N-myristoylation can influence the formation of intramolecular disulfide bonds, which may also be relevant for A16L structure and function .

How does A16L contribute to the Entry/Fusion Complex (EFC) and virus entry?

A16L functions as a core component of the Entry/Fusion Complex (EFC), a specialized multiprotein assembly that mediates fusion between viral and cellular membranes during poxvirus entry. Based on studies with vaccinia virus, A16L works in concert with other EFC proteins, including G9 and L1, to facilitate virus entry .

Experimental evidence suggests that A16L plays a distinct role from other EFC components. While N-myristoylation is critical for L1 protein function and virus infectivity, A16(G2A) mutants that lack N-myristoylation remain infectious but form smaller plaques, indicating reduced virus spread rather than compromised initial infection .

What is the impact of N-myristoylation on A16L function and viral fitness?

N-myristoylation of A16L appears to play a nuanced role in poxvirus biology. Research with vaccinia virus A16(G2A) mutants, which prevent N-myristoylation, demonstrated that these viruses retained infectivity (with unchanged genome/PFU ratios) but formed smaller plaques compared to wild-type virus . This suggests that while N-myristoylation of A16 is not strictly required for initial infection, it significantly contributes to efficient virus spread.

This finding contrasts with another EFC protein, L1, where N-myristoylation is critical for infectivity. The research data suggests that N-myristoylation of different EFC proteins may serve distinct functional roles .

Mechanistically, N-myristoylation likely promotes proper protein folding, membrane association, or protein-protein interactions within the Entry/Fusion Complex. For related proteins, it has been shown that this modification can influence the formation of intramolecular disulfide bonds, which may also apply to A16L .

What experimental approaches are optimal for studying A16L mutations?

Comprehensive mutational analysis of A16L requires several complementary approaches:

• Site-directed mutagenesis: Targeting specific residues predicted to be functionally important, such as the G2 position that prevents N-myristoylation. This approach has already demonstrated that G2A mutations in A16 affect viral spread while maintaining infectivity .

• Domain mapping: Creating truncated versions of A16L to identify functional regions. The full-length protein spans 377 amino acids, with the expression region typically used for recombinant protein production covering amino acids 2-377 .

• Cysteine modification: Given the importance of disulfide bonds in related proteins, systematically mutating cysteine residues to assess their role in A16L function and folding.

• Charge replacement: Altering charged residues at potential interaction interfaces to disrupt protein-protein contacts within the EFC.

The effects of mutations can be assessed through plaque size measurements, one-step growth curves, electron microscopy of virion morphogenesis, and specialized assays measuring membrane fusion activity. Importantly, due to variola virus research restrictions, these studies typically utilize vaccinia virus as a model system .

What are the challenges in recombinant A16L protein production and purification?

Producing functional recombinant A16L protein presents several technical challenges:

• Expression system selection: Bacterial systems offer high yield but lack post-translational modifications, including the critical N-myristoylation. Eukaryotic systems (insect or mammalian cells) provide better post-translational processing but with lower yields.

• N-myristoylation: To obtain properly modified protein, co-expression with N-myristoyltransferase is typically required in eukaryotic systems, or chemical coupling approaches may be employed post-purification.

• Membrane protein extraction: As a membrane protein, A16L requires careful optimization of detergent conditions to maintain native conformation during extraction from cellular membranes.

• Purification strategy: The amino acid sequence of A16L requires consideration of pH, salt concentration, and buffer composition during chromatography steps to preserve structure and function.

• Quality assessment: Confirming proper folding through biophysical methods (circular dichroism, thermal shift assays) and functional assays (protein-protein interactions with other EFC components).

For studies requiring authentic N-myristoylated A16L, an insect cell or mammalian expression system coupled with affinity purification offers the best compromise between yield and proper modification.

How can researchers study A16L within the constraints of variola virus research regulations?

Given that variola virus research is restricted to two WHO-designated facilities (CDC in Atlanta, USA, and the Russian State Centre for Research on Virology and Biotechnology in Koltsovo) , researchers have developed several alternative approaches:

• Surrogate poxvirus models: Vaccinia virus serves as the prototypic orthopoxvirus, sharing high sequence similarity with variola virus. Researchers can generate recombinant vaccinia viruses expressing variola virus A16L to study the protein in a related viral context.

• Recombinant protein approaches: Expression and purification of recombinant variola virus A16L protein (as described in search result ) allows for in vitro studies without requiring intact variola virus.

• Computational analysis: Comparative sequence analysis, structural modeling, and molecular dynamics simulations provide insights into A16L function based on homology with related proteins.

• Collaborative arrangements: Partnerships with authorized facilities like CDC can facilitate validation of findings in the context of authentic variola virus.

These approaches have successfully advanced our understanding of poxvirus biology while maintaining the security protocols essential for work with eradicated pathogens like variola virus .

What assays can measure A16L protein activity and interactions?

Several complementary methods can assess A16L functionality:

• Protein-protein interaction assays:

  • Co-immunoprecipitation to identify protein binding partners

  • Surface plasmon resonance or biolayer interferometry for quantitative binding kinetics

  • Proximity-dependent biotin labeling (BioID, TurboID) to identify proteins near A16L in living cells

• Membrane association assays:

  • Liposome binding assays to measure membrane association

  • Flotation assays to assess protein partitioning into membrane fractions

  • Microscopy-based colocalization with membrane markers

• Functional complementation:

  • Trans-complementation of A16-deficient viruses to assess functional restoration

  • Cell-cell fusion assays using A16L expressed alone or with other EFC components

• Conformational analysis:

  • Limited proteolysis to identify stable domains and flexible regions

  • Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

When evaluating N-myristoylated versus non-myristoylated A16L, comparative analysis using these assays can reveal functional differences attributable to this modification, as suggested by research showing different phenotypes between wild-type and G2A mutant viruses .

How can A16L be utilized for developing poxvirus countermeasures?

A16L presents several opportunities for antiviral development:

• Direct inhibitors: Small molecules that bind directly to A16L could disrupt its function in the Entry/Fusion Complex. The search results indicate that inhibiting N-myristoylation with compounds like IMP-1088 affects viral infection by preventing proper modification of proteins including A16L .

• Protein-protein interaction disruptors: Compounds that interfere with A16L interactions with other EFC components could prevent complex assembly and inhibit viral entry.

• Diagnostic applications: Recombinant A16L protein can serve as an antigen in ELISA-based tests for detecting anti-poxvirus antibodies or as a component in protein microarrays .

• Vaccine development: Understanding A16L's role in the EFC may inform the design of next-generation vaccines with improved safety profiles. Currently, vaccine research is one of the primary goals of smallpox research as outlined by WHO .

• Structure-based drug design: Detailed structural information about A16L could guide the development of antivirals with high specificity and potency.

The development of antivirals targeting A16L aligns with the WHO's goals for smallpox research, which include finding better treatments and improving diagnostics for poxvirus infections .

What is the relationship between A16L and other Entry/Fusion Complex proteins?

A16L functions within a complex network of protein-protein interactions in the Entry/Fusion Complex (EFC). Research has identified several key relationships:

Understanding these relationships requires sophisticated approaches such as crosslinking mass spectrometry, co-immunoprecipitation under various conditions, and analysis of compensatory mutations that restore function in defective variants.

How do inhibitors of N-myristoylation affect A16L and viral infection?

Inhibitors of N-myristoyltransferase (NMT), such as IMP-1088, provide valuable insights into A16L function and potential therapeutic strategies. Research demonstrates several important findings:

• Selective inhibition: IMP-1088 potently inhibits the N-myristoylation of multiple VACV proteins, including A16, G9, and L1, with L1 showing the strongest response to inhibition .

• Impact on viral lifecycle: Treatment with IMP-1088 potently decreased VACV infection and spread without significantly affecting viral early or late protein synthesis, suggesting no effect on DNA replication and gene expression .

• Effect on virion infectivity: Electron microscopy revealed that virus morphogenesis proceeded in the presence of IMP-1088, but the resulting viral particles exhibited reduced infectivity, defects in membrane fusion, and impaired core release into the cytoplasm .

• Protein-specific responses: While multiple proteins are affected by NMT inhibition, L1 appears to be the most critical for the antiviral effect, with A16 and G9 showing more nuanced responses .

• Therapeutic potential: The low cytotoxicity observed with IMP-1088 treatment suggests that NMT inhibitors could represent promising candidates for poxvirus treatment .

These findings highlight the importance of N-myristoylation for poxvirus biology and identify a potential vulnerability that could be exploited for antiviral development.

What is known about the evolutionary conservation of A16L across different poxvirus species?

The A16L protein exhibits remarkable conservation across poxviruses, suggesting its fundamental importance to viral biology. Several key evolutionary aspects include:

• Motif conservation: The N-terminal MG N-myristoylation motif is conserved in all poxviral A16 proteins, indicating strong selective pressure to maintain this feature throughout poxvirus evolution .

• Functional conservation: The essential role of A16L in the Entry/Fusion Complex appears to be maintained across diverse poxviruses, from the eradicated variola virus to contemporary viruses like vaccinia.

• Sequence homology: Comparison of A16L sequences across orthopoxviruses reveals high similarity, facilitating the use of surrogate models like vaccinia virus for studying variola virus A16L function.

• Host adaptation: Subtle sequence variations in A16L between poxviruses that infect different hosts may reflect adaptations to species-specific cellular factors.

This conservation has important implications for both fundamental research and applied studies, suggesting that insights gained from studying vaccinia virus A16L likely apply to variola virus and other poxviruses of concern.

How does A16L contribute to poxvirus host range and pathogenesis?

While A16L is primarily recognized for its role in viral entry, its contributions to host range determination and pathogenesis may be significant:

• Entry efficiency: As a component of the Entry/Fusion Complex, A16L influences the efficiency of viral entry into host cells, potentially affecting tissue tropism and disease progression.

• Species specificity: Unlike some poxviruses that can infect multiple species, variola virus exclusively infects humans . The EFC, including A16L, may contribute to this strict host specificity.

• Spread dynamics: Research with G2A mutants demonstrates that N-myristoylation of A16 affects viral spread , which could influence dissemination within the host and between individuals.

• Immune recognition: Surface-exposed regions of A16L may serve as targets for neutralizing antibodies, affecting viral clearance and influencing the design of next-generation vaccines.

Understanding these aspects requires integration of molecular virology with immunological and pathogenesis studies, ideally using animal models of poxvirus infection that approximate human disease.

What methodologies are most appropriate for studying the structural biology of A16L?

Elucidating the three-dimensional structure of A16L presents technical challenges but offers valuable insights into function:

• X-ray crystallography: Requires purification of properly folded, homogeneous protein. For membrane proteins like A16L, this often involves detergent solubilization or incorporation into lipidic cubic phases.

• Cryo-electron microscopy: Particularly valuable for visualizing A16L in the context of intact virions or purified Entry/Fusion Complexes, potentially revealing conformational states relevant to function.

• NMR spectroscopy: Suitable for studying dynamics and interactions of specific domains of A16L, though challenging for the full-length protein.

• Integrative structural biology: Combining low-resolution techniques (SAXS, negative stain EM) with computational modeling and crosslinking mass spectrometry to build composite structural models.

• In silico approaches: Homology modeling based on related proteins with known structures, molecular dynamics simulations to predict conformational changes.

For N-myristoylated A16L, maintaining this modification throughout purification represents a significant challenge. Specialized approaches such as chemical ligation or co-expression with N-myristoyltransferase in eukaryotic systems may be required to obtain structurally authentic protein for analysis.