Recombinant Vaccinia virus Virion membrane protein A16 (TA17L)

What is the A16 protein and what is its role in Vaccinia virus infection?

The A16 protein is a 378-amino-acid protein encoded by the A16L open reading frame in Vaccinia virus. It contains a predicted C-terminal transmembrane domain and 20 invariant cysteine residues that are conserved across all sequenced members of the poxvirus family. The protein is expressed late during infection and gets incorporated into intracellular virus particles with its N-terminal segment exposed on the viral surface. A16 plays a critical role in viral entry, specifically in the penetration of viral cores into the cytoplasm after the initial binding of virions to host cells. Additionally, it is involved in low-pH-triggered syncytium formation (cell-cell fusion). Unsuccessful attempts to isolate deletion mutants suggest that A16 is essential for virus replication, highlighting its fundamental importance in the viral life cycle .

How is the A16 protein structurally characterized?

The A16 protein has several conserved structural features that are important for its function. It has a molecular weight of approximately 43 kDa as determined by SDS-PAGE analysis. The protein contains 20 invariant cysteine residues that form disulfide bonds via the poxvirus cytoplasmic redox system, which likely contributes to its structural stability and functionality. It possesses a C-terminal hydrophobic domain that serves as a transmembrane anchor, integrating the protein into the viral membrane. Additionally, A16 has a penultimate N-terminal glycine that is consistent with evidence of myristylation, a post-translational modification that typically facilitates membrane association and protein-protein interactions. The N-terminal segment of the protein is exposed on the surface of the virus particle, making it accessible for interactions with host cell components during the entry process .

What other viral proteins work together with A16 in virus entry?

A16 functions as part of a larger protein complex involved in poxvirus entry. Research has identified at least five conserved viral membrane proteins that are required for entry of poxviruses into cells and for cell-cell fusion: A16, A21, A28, H2, and L5. When the synthesis of any of these proteins is repressed, a similar phenotype is observed—virions can bind to cells, but their cores cannot penetrate into the cytoplasm. This indicates that these proteins likely form a functional complex or act in the same pathway to facilitate membrane fusion and core entry. The coordinated action of these proteins is essential for the virus to overcome the barrier of the host cell membrane and deliver its genetic material into the cytoplasm to initiate infection. Understanding this protein complex is crucial for developing comprehensive models of poxvirus entry mechanisms .

How do the disulfide bonds in A16 contribute to its function in virus entry?

The 20 invariant cysteine residues in A16 form disulfide bonds via the poxvirus cytoplasmic redox system, creating a complex network that likely stabilizes the protein's tertiary structure. These disulfide bonds are particularly significant because they form in the reducing environment of the cytoplasm, which is unusual for most cellular proteins with disulfide bonds. The conservation of these cysteines across all poxviruses suggests they play a critical structural or functional role. For researchers investigating this aspect, site-directed mutagenesis of specific cysteine residues would be an effective approach to determine which disulfide bonds are essential for A16 function. Experiments comparing the entry efficiency of virions containing wild-type A16 versus cysteine-mutated variants could reveal how these bonds contribute to the membrane fusion process. The integrity of these disulfide bonds may be necessary for proper interaction with other entry complex proteins (A21, A28, H2, L5) or for conformational changes that occur during the fusion process .

What is the mechanistic role of A16 in the penetration of viral cores into the cytoplasm?

A16-deficient virions can bind to host cells but fail to deliver their cores into the cytoplasm, indicating that A16 functions after attachment but before core penetration. The precise mechanism involves a complex interplay between viral and cellular components. Current evidence suggests that A16, together with other entry proteins, may undergo conformational changes triggered by low pH in endosomes, leading to membrane fusion. To investigate this mechanism, researchers could employ techniques such as cryo-electron microscopy to visualize structural changes in A16 under different pH conditions, or use fluorescently labeled virions to track the entry process in real-time with high-resolution microscopy. Additionally, studying the interaction partners of A16 during different stages of entry using crosslinking and mass spectrometry would provide insights into the dynamic protein complexes formed during this process. Understanding this mechanism is crucial for developing interventions that block poxvirus entry .

How does myristylation of A16 impact its localization and function?

The conserved penultimate N-terminal glycine in A16 is consistent with evidence that the protein undergoes myristylation, a post-translational modification where a myristoyl group (a 14-carbon saturated fatty acid) is covalently attached to the glycine residue. This modification typically enhances the protein's hydrophobicity and facilitates its association with membranes. For A16, myristylation likely contributes to its correct incorporation into the viral membrane and may influence its interaction with other proteins in the entry complex. Researchers interested in this aspect could generate recombinant viruses expressing A16 with mutations at the myristylation site and assess the impact on protein localization, virus assembly, and infectivity. Biochemical fractionation experiments comparing wild-type and mutation-bearing virions would reveal differences in membrane association. Additionally, structural studies using techniques like nuclear magnetic resonance (NMR) spectroscopy could elucidate how myristylation affects the conformation and dynamics of the N-terminal region of A16, providing insights into its functional significance .

How can researchers create conditional A16-deficient vaccinia virus for functional studies?

To generate conditional A16-deficient vaccinia virus, researchers can utilize the Escherichia coli lac operator system to regulate A16L transcription. This methodology involves several key steps: First, construct a recombinant virus (like vA16Li) containing the lac repressor gene under the control of a vaccinia virus early/late promoter and the bacteriophage T7 RNA polymerase gene adjacent to a vaccinia virus late promoter regulated by the lac operator. Next, modify the A16L gene to be driven by a T7 promoter and regulated by the lac operator. In this system, the lac repressor inhibits expression by binding to the lac operators in the absence of isopropyl-β-d-thiogalactopyranoside (IPTG), while IPTG addition inactivates the repressor to allow expression of T7 RNA polymerase and transcription of the A16L gene. Researchers should perform clonal purification of the virus in the presence of IPTG (typically 50 μM) and confirm the construct using PCR and sequencing. The resulting conditional mutant allows tight control of A16 expression—without IPTG, A16 synthesis is repressed; with IPTG, A16 is expressed at levels comparable to those under its natural promoter .

How can researchers directly visualize and quantify the entry defect of A16-deficient virions?

To directly visualize and quantify the entry defect of A16-deficient virions, researchers can employ a modified version of the entry assay developed by Vanderplasschen et al. The protocol involves purifying +A16 and -A16 virions and adsorbing them to cells for 1 hour at 4°C to allow binding but prevent internalization. Subsequently, raise the temperature to 37°C for 2 hours to permit penetration, maintaining cycloheximide in the medium to prevent cytopathic effects and core disassembly. Fix cells with paraformaldehyde and perform immunofluorescence staining using: (1) antibodies against L1 membrane protein to detect virions on cell surfaces, and (2) antibodies against A4 core protein to identify cores in the cytoplasm. Counterstain nuclei with DAPI. Analyze samples using confocal microscopy, capturing multiple fields for statistical analysis. Quantify the number of surface-bound virions (L1-positive particles) and internalized cores (A4-positive particles not co-localizing with L1) per cell. This approach provides both qualitative visual evidence and quantitative data on the entry defect, allowing researchers to precisely determine at which stage of the entry process A16-deficient virions are blocked .

How should researchers interpret differences in plaque formation between wild-type and A16-deficient viruses?

When analyzing plaque formation differences between wild-type and A16-deficient viruses, researchers should consider multiple parameters beyond simple plaque size. The conditional A16 mutant (vA16Li) forms tiny plaques in the absence of IPTG and nearly normal-size plaques in its presence, indicating impaired cell-to-cell spread when A16 is deficient. For robust interpretation, researchers should: (1) Measure both plaque diameter and plaque number across multiple dilutions and time points; (2) Quantify the reduction in plaque size using image analysis software and calculate statistical significance; (3) Perform one-step growth curves to distinguish between defects in entry, replication, assembly, or release; (4) Examine the morphology of cells at plaque edges using phase-contrast microscopy to assess cytopathic effects; and (5) Consider that residual A16 expression (due to incomplete repression) may allow limited replication, resulting in the observed tiny plaques rather than no plaques. The approximately 1.5 log unit reduction in virus yield for vA16Li without IPTG correlates with its 60-to-100-fold lower specific infectivity, suggesting that entry defects primarily account for the plaque phenotype rather than post-entry replication issues .

How can researchers differentiate between A16's role in virus entry versus other potential functions?

To definitively differentiate between A16's role in virus entry versus other potential functions, researchers should implement a multifaceted experimental approach that systematically evaluates distinct stages of the viral life cycle. First, use binding assays with labeled virions to confirm that A16-deficient virions attach to cells normally, establishing that initial receptor interaction remains intact. Second, employ the core penetration assay with immunofluorescence detection of L1 (membrane) and A4 (core) proteins to visualize the entry defect. Third, complement the entry defect through artificial delivery methods—treat A16-deficient virions with a membrane fusion agent like polyethylene glycol (PEG) that bypasses the need for virus-mediated fusion. If PEG treatment restores infectivity, this confirms the specific role in membrane fusion. Fourth, perform transmission electron microscopy of infected cells to examine morphogenesis stages and verify that A16-deficient virions assemble normally. Fifth, analyze protein composition of purified virions to ensure other structural proteins are correctly incorporated. Finally, conduct transcriptome analysis of cells infected with A16-deficient virions to determine if any viral genes beyond those involved in entry are affected. This comprehensive approach allows researchers to isolate A16's function in entry from potential roles in assembly, morphogenesis, or gene regulation .