Recombinant Vaccinia virus 25 kDa core protein A12L (VACWR131)
Description
Characteristics of Vaccinia Virus Protein VP8
Vaccinia virus protein VP8, the 25 kDa product of the L4R gene, has the following characteristics:
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It can bind to both single-stranded and double-stranded DNA, with a preference for single-stranded DNA at high salt concentrations .
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The dissociation constant (Kd) for a 45-mer oligonucleotide is approximately 2 nM .
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Multiple rL4R molecules can bind to a single 45-mer oligonucleotide, with one rL4R molecule binding approximately every 17 nucleotides .
Recombinant forms
The recombinant form of the 25 kDa L4R protein (rL4R) is utilized in research to study its DNA-binding properties and to elucidate its potential roles in viral replication .
Use as an Expression Vector
Vaccinia virus is used as an expression vector for protein synthesis, immunology research, vaccines, and therapeutics . Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels .
Tables and Figures
Tables are used to organize data that is too detailed or complicated to be described adequately in the text, allowing the reader to quickly see the results . When constructing tables for research findings, the following guidelines can be considered :
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The number of tables should align with journal recommendations .
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Tables should be easily understood, allowing readers to form opinions without reading the text .
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Data in tables should match the text, with accurate totals, defined units, and indicated sample sizes .
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Values should be expressed with standard error, range, or confidence interval, and significance levels should be indicated .
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Avoid abbreviations, but if necessary, define them in footnotes .
Table 1: Histopathological Analysis of Specimens
| Histopathological diagnosis | Men n (%) | Women n (%) | Total n (%) |
|---|---|---|---|
| Adrenal cortical adenoma | 5 (31.3) | 6 (37.6) | 11 (68.8) |
| Pheochromocytoma | 1 (6.2) | 1 (6.2) | 2 (12.6) |
| Ganglioneuroma | 1 (6.2) | - | 1 (6.2) |
| Myelolipoma | - | 1 (6.2) | 1 (6.2) |
| Adrenal carcinoma | - | 1 (6.2) | 1 (6.2) |
| Total | 7 (43.7) | 9 (56.2) | 16 (100) |
Product Specs
Note: We will prioritize shipping the format currently in stock. If you require a specific format, please specify this in your order notes.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping is available upon request and incurs an additional fee. Please contact us in advance to arrange this.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
A component of the virion core that undergoes proteolytic processing during the immature virion (IV) to mature virion (MV) transition. It is essential for the formation of a structurally sound core.
- We concluded that not only A12L protein but also its cleavage processing plays an essential role in virus morphogenic transition. PMID: 17625005
KEGG: vg:3707529
Q&A
What is the A12L protein and what is its fundamental role in vaccinia virus?
The A12L protein (VACWR131) is a 25 kDa late gene product of vaccinia virus that undergoes proteolytic processing. It is initially synthesized as a precursor (p17K) that is cleaved at an N-terminal AG/A site to yield a 17 kDa product (17K) . Unlike most core protein precursors where only processed forms appear in mature virions, both the A12L precursor and its cleavage product are found in the core of mature virions, suggesting distinct roles for both forms in virus assembly . The protein appears to participate in multiple stages of viral morphogenesis through interactions with various viral core and membrane proteins .
How does A12L proteolytic processing differ from other vaccinia virus core proteins?
The A12L protein exhibits unique proteolytic processing compared to other vaccinia virus core proteins:
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While major core protein precursors (p4a, p4b, p25K) are processed at the conserved Ala-Gly-X motif to yield single cleavage products, A12L undergoes multiple cleavage events .
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Immunoblot analysis with A12L antisera reveals not only the expected 25 kDa precursor and 17 kDa product but also five additional peptides with apparent molecular weights of 21, 18, 15, 13, and 11 kDa .
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Unlike other core proteins where only processed forms appear in mature virions, both the A12L precursor and its cleavage products are present in mature virus particles .
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While the I7L proteinase is involved in A12L processing, the cleavage occurs with slower kinetics and remains incomplete, with additional cleavages occurring at sites other than the expected AG/X motifs .
When is A12L expressed during the viral replication cycle?
A12L is synthesized at late times during infection, consistent with its classification as a viral late gene product . This timing aligns with its role in viral assembly and morphogenesis, which occur in the later stages of the viral life cycle. Like other structural proteins, its expression follows viral DNA replication and is part of the coordinated expression program that ensures viral components are available at the appropriate time for virion assembly .
How is A12L proteolysis regulated during infection?
The proteolytic processing of A12L is regulated by rifampicin, similar to other vaccinia virus core proteins. Research using rifampicin-reversibility experiments demonstrates:
| Experimental Condition | Observed Effect on A12L Processing |
|---|---|
| No rifampicin | Complete expression of A12L precursor and all cleavage products |
| Rifampicin (100-200 μg/ml) | Expression of A12L precursor but suppression of cleavage products |
| Rifampicin (>200 μg/ml) | Inhibition of both precursor and cleavage product expression |
| Rifampicin removal after treatment | Resumption of A12L proteolytic processing |
When rifampicin is added at 5 hours post-infection (allowing sufficient A12L precursor accumulation) and then removed, the A12L-derived multiple cleavage products reappear, confirming the regulated nature of this process . This rifampicin-regulated proteolysis suggests that A12L processing is linked to virus morphogenesis, similar to other core proteins, yet displays distinctive characteristics in terms of processing kinetics and completeness .
What protein interactions does A12L engage in during viral assembly?
Immunoprecipitation experiments followed by N-terminal sequencing and mass spectrometry reveal that A12L associates with multiple vaccinia virus proteins involved in different aspects of virion assembly:
| Associated Protein | Molecular Weight | Function in Virus Assembly |
|---|---|---|
| A4L | 39 kDa | Core protein; associates with A10L; stimulates IV to IMV progression |
| A10L | Unknown | Core protein precursor; forms complexes with A4L |
| L4R | 28 kDa | DNA-binding protein; involved in genome packaging and transcription |
| F17R | 11 kDa | DNA-binding phosphoprotein; involved in genome packaging |
| A17L | 21 kDa | Membrane protein; interacts with A14L; initiates membrane formation |
| A14L | 15 kDa | Phosphorylated membrane protein; works with A17L |
| A27L | 13 kDa | Envelope protein; incorporates with A17L; involved in IMV envelopment |
These interactions suggest that A12L and its cleavage products participate in multiple stages of virion morphogenesis, from the organization of viral core components to the development of mature infectious particles .
What experimental evidence demonstrates the importance of A12L in viral replication?
The critical role of A12L in viral replication has been demonstrated through the construction of a conditional lethal mutant virus (vvtetOA12L) that regulates A12L expression based on the presence or absence of tetracycline. Key findings include:
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In the absence of tetracycline (which suppresses A12L expression), viral replication was inhibited by 80% .
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This inhibition could be overcome by transient expression of the wild-type copy of the A12L gene, confirming that the replication defect was specifically due to A12L depletion .
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The requirement for both the precursor and processed forms in mature virions suggests that proper A12L proteolysis is crucial for its function in viral assembly .
This evidence collectively indicates that A12L plays an essential role in the vaccinia virus replication cycle that cannot be compensated for by other viral proteins .
What is the significance of the multiple cleavage products of A12L?
The presence of multiple A12L cleavage products (with apparent molecular weights of 21, 18, 17, 15, 13, and 11 kDa) is a unique feature that distinguishes it from other vaccinia virus core proteins . These multiple processing events suggest:
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A12L may have multiple functional domains that play different roles when separated through proteolysis.
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The different cleavage products may interact with different viral proteins at various stages of virus assembly.
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This complex processing pattern may represent a regulatory mechanism that coordinates the participation of A12L in different aspects of virion morphogenesis.
The association of A12L-derived peptides with both core proteins (A4L, A10L, L4R, F17R) and membrane proteins (A17L, A14L, A27L) supports the hypothesis that these cleavage products serve as bridges between different structural components during virus assembly .
What techniques are most effective for studying A12L proteolytic processing?
Several complementary techniques have proven valuable for investigating A12L proteolysis:
| Technique | Application in A12L Research | Key Findings |
|---|---|---|
| Immunoblot analysis | Detection of precursor and cleavage products | Identified six A12L-derived peptides with apparent molecular weights of 21, 18, 17, 15, 13, and 11 kDa |
| Rifampicin-reversibility assays | Study of proteolysis regulation | Demonstrated rifampicin-regulated A12L processing similar to other core proteins |
| Immunoprecipitation | Isolation of A12L and associated proteins | Identified viral core and membrane proteins that associate with A12L |
| N-terminal sequencing | Identification of cleavage sites | Confirmed cleavage at the N-terminal AG/A site and identified additional cleavage events |
| Mass spectrometry | Identification of A12L-associated proteins | Revealed associations with A4L, L4R, A10L, A27L, A17L, A14L, and F17R |
For rifampicin-reversibility experiments, the protocol involves infecting cells with vaccinia virus, adding rifampicin (150 μg/ml) at 5 hours post-infection, and then removing the drug after a specified period to observe the resumption of proteolysis through immunoblot analysis .
How can researchers generate and purify recombinant A12L protein for functional studies?
Based on published methodologies, effective approaches for A12L expression and purification include:
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Bacterial expression system using an N-terminal 7× His-tagged full-length A12L protein .
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Affinity purification over a Ni-NTA-agarose column to obtain purified protein .
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For studying A12L in its native context, construction of a conditional lethal virus (vvtetOA12L) that regulates A12L expression based on tetracycline presence/absence .
When preparing cell lysates for A12L analysis, it is important to include protease inhibitors to prevent non-specific degradation. Research protocols have used PBS with a protease inhibitor cocktail tablet for cell lysate preparation .
What approaches can be used to study A12L protein-protein interactions?
Several approaches have proven effective for investigating A12L interactions with other viral proteins:
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Co-immunoprecipitation: Using antibodies against A12L to precipitate the protein and its interacting partners from infected cell lysates, followed by identification through immunoblotting, N-terminal sequencing, or mass spectrometry .
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Reciprocal immunoprecipitation: Confirming interactions by performing immunoprecipitation with antibodies against putative binding partners (such as A17L and F17R) and detecting A12L in the precipitates through immunoblot analysis .
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Two-dimensional electrophoresis: For better resolution of A12L-associated proteins, 2D PAGE followed by mass spectrometry has successfully identified multiple interacting partners .
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Functional complementation: Using the conditional lethal mutant virus (vvtetOA12L) with transient expression of wild-type or mutant A12L to assess which interactions are functionally important .
These methodological approaches provide a framework for detailed investigation of A12L structure, function, and interactions in the context of vaccinia virus replication and assembly.
How can the functional domains of A12L be mapped experimentally?
To map functional domains of the A12L protein, researchers could employ the following experimental strategies:
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Site-directed mutagenesis: Introducing specific mutations at the AG/A cleavage site and the two AG/K sites to determine their significance in proteolytic processing and virus replication.
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Deletion analysis: Creating a series of N-terminal and C-terminal truncations of A12L to identify regions necessary for interactions with other viral proteins and for supporting viral replication.
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Domain swap experiments: Replacing segments of A12L with corresponding regions from related poxvirus proteins to identify functionally conserved domains.
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Expression of individual cleavage products: Expressing each of the identified A12L-derived peptides independently to determine which can complement the replication defect of the vvtetOA12L conditional mutant in the absence of tetracycline.
These approaches, combined with the conditional expression system already established for A12L, would provide valuable insights into the functional organization of this multifaceted viral protein .
