Recombinant Vaccinia virus Myristoylated protein G9 (TG10R)
Description
Introduction to Recombinant Vaccinia Virus Myristoylated Protein G9 (TG10R)
Recombinant Vaccinia virus Myristoylated protein G9 (TG10R) is a viral protein of 340 amino acids encoded by the G9R gene (VACWR087) in the Vaccinia virus genome. The protein derives its name from the post-translational myristoylation modification at its N-terminus, which contributes significantly to its functional properties . TG10R specifically refers to this protein as expressed in the Tian Tan strain of Vaccinia virus, which serves as an important research model in poxvirus studies .
G9 functions as an integral component of the viral entry-fusion complex (EFC), a multiprotein assembly responsible for mediating fusion between viral and cellular membranes during the infection process. Previous research has demonstrated that G9 is one of eight proteins associated with this complex, highlighting its importance in viral penetration into host cells .
The significance of G9 in viral replication is underscored by unsuccessful attempts to isolate viable viral mutants lacking the G9R gene, strongly suggesting that this protein plays an essential role in the viral life cycle . This characteristic makes G9 particularly interesting from both a basic research perspective and as a potential target for antiviral interventions.
Molecular Properties
The G9 protein has a molecular weight of approximately 38,758 Da according to sequence analysis . The protein contains several conserved structural elements that contribute to its specialized function in the viral envelope. These conserved features appear throughout the poxvirus family, indicating their functional importance in viral biology .
| Property | Characteristic |
|---|---|
| Molecular Weight | 38,758 Da |
| Amino Acid Length | 340 amino acids |
| UniProt Accession | Q9JFC1 |
| Viral Strain | Vaccinia virus (strain Tian Tan) |
| Gene Name | TG10R (G9R, VACWR087) |
Conserved Domains and Post-translational Modifications
The G9 protein contains several key structural elements that are preserved across all poxviruses, demonstrating their essential role in protein function :
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N-terminal myristoylation site: This post-translational modification involves the covalent attachment of a myristate group to the N-terminal glycine residue, facilitating membrane association and proper protein function.
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Fourteen conserved cysteine residues: These amino acids likely participate in disulfide bond formation, contributing to the tertiary structure and stability of the protein.
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C-terminal transmembrane domain: This hydrophobic region anchors the protein in the viral membrane, with portions of the protein exposed on the virion surface.
These structural features collectively enable the protein to associate with membranes and participate in the complex process of virus-cell fusion during infection .
Comparative Analysis with Related Proteins
Within the poxvirus family, G9 shares structural similarities with other viral proteins, notably A16 and J5, despite having relatively low sequence homology (approximately 30%) . Both G9 and A16 undergo myristoylation, suggesting a common functional requirement for this modification in their roles within the viral replication cycle.
No non-poxvirus homologs of G9 have been detected in comprehensive position-specific iterative BLAST searches, highlighting the unique nature of this protein to the poxvirus family .
Association with the Entry-Fusion Complex
Research has established that G9 functions as a critical component of the viral entry-fusion complex, which mediates membrane fusion during infection. Experimental evidence confirms that G9 is enriched in mature virions (MVs) compared to whole-cell extracts, though at lower levels than some other fusion complex proteins such as A16 .
Studies have shown that G9 is enriched more than eightfold in MVs compared to whole-cell extracts, while A16, another component of the entry-fusion complex, is enriched more than 16-fold. This differential incorporation may result from differences in expression timing and levels when using inducible promoter systems for study .
Essential Nature for Virus Replication
The critical importance of G9 in viral replication has been demonstrated through unsuccessful attempts to isolate Vaccinia virus mutants lacking the G9R gene. When researchers constructed a recombinant virus with regulated G9 expression, they found that omission of the inducer reduced infectious virus yield by approximately 1.5 logs .
Interestingly, despite this reduction in viral infectivity, the morphological stages of virus development appeared to proceed normally in the absence of G9, with extracellular virions still present on the cell surface. This observation suggests that G9's essential function may be specifically in the infection process rather than in virion assembly .
Surface Exposure on Mature Virions
Experimental evidence indicates that G9 is exposed on the surface of mature virions. Researchers demonstrated this by successfully labeling G9 with membrane-impermeant biotinylation reagents, which can only react with proteins accessible from outside the virion membrane . This surface localization is consistent with G9's proposed role in mediating interactions with host cell membranes during the entry process.
In addition to its role in virus-cell fusion, G9 also functions in cell-cell fusion (syncytium formation), contributing to viral spread within infected tissues through the formation of multinucleated cells . This activity further supports its role in membrane fusion events during the viral replication cycle.
Expression Systems
Recombinant G9 protein can be produced using various expression systems, each with distinct advantages for different research applications. The available expression platforms include:
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Bacterial expression (Escherichia coli): Provides high yield but may lack some post-translational modifications
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Yeast expression systems: Offers eukaryotic processing with moderate yield
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Baculovirus-mediated expression in insect cells: Provides improved eukaryotic post-translational modifications
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Mammalian cell expression systems: Offers the most authentic post-translational modifications, particularly for myristoylation
The choice of expression system depends on specific research requirements, including the need for proper post-translational modifications (particularly myristoylation), correct protein folding, and compatibility with downstream applications.
Purification Methods and Quality Control
Recombinant G9 protein is typically purified to a high standard, with preparations achieving greater than or equal to 85% purity as determined by SDS-PAGE analysis . The purification process may involve affinity chromatography facilitated by epitope tags incorporated into the recombinant protein construct.
These tags may be positioned at either the N-terminus or C-terminus of the protein, with tag types determined based on various factors including tag-protein stability considerations . Quality control measures typically include verification of protein identity, purity assessment through SDS-PAGE, and functional validation where applicable.
| Specification | Details |
|---|---|
| Purity | ≥85% as determined by SDS-PAGE |
| Formulation | Lyophilized or liquid (determined during manufacturing) |
| Tags | N-terminal tag and potentially C-terminal tag |
| Sterility Options | Sterile filtration available upon request |
| Endotoxin Level | Low endotoxin preparations available upon request |
Role in Understanding Poxvirus Biology
The study of G9 protein contributes significantly to our understanding of poxvirus entry mechanisms and host-pathogen interactions. As an essential component of the entry-fusion complex, investigations into G9 structure and function provide insights into the molecular details of viral penetration into host cells.
Research involving recombinant G9 protein enables detailed analysis of protein-protein interactions within the entry-fusion complex and between viral and cellular components during infection. These studies help elucidate the coordinated processes that facilitate successful viral entry and subsequent replication.
Function in Viral Entry and Membrane Fusion
According to UniProt annotations, G9 functions as an envelope protein that forms part of the entry-fusion complex responsible for virus membrane fusion with host cell membranes during virus entry . This critical function places G9 at the center of the infection process, making it an important subject for research into viral pathogenesis.
The protein also plays a documented role in cell-cell fusion, contributing to the formation of syncytia (multinucleated cells) . This activity may facilitate viral spread within infected tissues by allowing the virus to bypass the extracellular environment and move directly from cell to cell.
Potential as a Therapeutic Target
Given its essential role in viral replication and surface exposure on mature virions, G9 represents a potential target for antiviral interventions. The high conservation of G9 across the poxvirus family suggests that interventions targeting this protein might have broad applicability against multiple poxvirus species.
As a surface-exposed protein essential for viral entry, G9 could potentially serve as a target for neutralizing antibodies or small-molecule inhibitors designed to block virus-cell fusion. The development of such interventions could contribute to strategies for addressing poxvirus infections, including potential emerging threats.
Application Guidelines
When utilizing recombinant G9 protein in research applications, several factors should be considered:
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The presence of epitope tags may influence certain assays or interactions
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Expression system choice may affect post-translational modifications, particularly the critical N-terminal myristoylation
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Buffer compatibility should be evaluated for specific experimental systems
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Protein activity should be verified under the conditions of intended use
Researchers are typically advised to avoid repeated freeze-thaw cycles and to prepare working aliquots to maintain protein integrity and activity .
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please specify them in your order notes, and we will accommodate your request.
Note: All of our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What are the key structural features of the vaccinia virus G9 protein?
The vaccinia virus G9R gene (VACWR087) encodes a 340 amino acid protein (38.7 kDa) with several highly conserved structural features across all poxviruses. These include a site for N-terminal glycine myristoylation, 14 conserved cysteine residues, and a C-terminal transmembrane domain . G9 is structurally related to two other vaccinia virus proteins, A16 and J5, although sequence similarity between them is relatively low . Both G9 and A16 have been experimentally confirmed to undergo myristoylation .
What is the cellular localization and function of G9 in vaccinia virus?
G9 is predominantly associated with the mature virion (MV) surface membrane. Experiments with HA epitope-tagged G9 have demonstrated that the protein is enriched approximately eightfold in MVs compared to whole-cell extracts . The protein can be labeled with membrane-impermeant biotinylation reagents, confirming its exposure on the virion surface . Functionally, G9 is one of eight proteins that form a putative entry-fusion complex essential for virus entry into host cells .
Is G9 essential for vaccinia virus replication?
Yes, G9 appears to be essential for efficient vaccinia virus replication. Attempts to isolate a deletion mutant lacking the G9R gene have been unsuccessful . When using an inducible system to regulate G9 expression, omission of the inducer reduced infectious virus yield by approximately 1.5 logs . Furthermore, rescue experiments using a bacterial artificial chromosome (BAC) system confirmed that the G9R gene is required for viable virus production, as VAC-BACΔG9 (with G9R replaced by an ampicillin resistance gene) could only be rescued when complemented with intact G9R DNA .
What approaches have been used to study G9 function given its essential nature?
Due to G9's essential role in viral replication, researchers have developed conditional expression systems rather than complete gene deletion. A key approach involves constructing recombinant viruses (such as vG9i) where G9R expression is regulated by an inducer like IPTG . This system places the G9R open reading frame under control of the bacteriophage T7 RNA polymerase promoter and the E. coli lac operator . Additionally, researchers have attempted to use the vaccinia virus bacterial artificial chromosome (VAC-BAC) system to study G9, though complete deletion mutants were not viable without complementation .
How can researchers distinguish between G9 and other vaccinia virus membrane proteins in experimental analyses?
Since highly specific antibodies to G9 are not always available, researchers often use epitope-tagged versions of G9 (such as HA-tagged G9) for detection and analysis . Western blotting can be used to analyze both whole-cell extracts and purified virions, with relative enrichment in virions serving as an indicator of incorporation into viral particles . When studying multiple vaccinia proteins simultaneously, comparative enrichment analysis can help distinguish between different membrane proteins - for example, A16 was found to be enriched more than 16-fold in virions compared to the eightfold enrichment observed for G9 .
What control systems are recommended when constructing conditional G9 expression systems?
When constructing conditional expression systems for G9, it is advisable to include a fluorescent marker gene (such as enhanced GFP) regulated by a constitutive viral promoter like the VACV P11 late promoter . This allows for visual confirmation of viral infection and recombinant virus selection. Additionally, to prevent RNA polymerase read-through from neighboring genes, the inducible G9R open reading frame should be inserted in its natural genomic site but in the opposite orientation . Verification of the construction should include PCR amplification and sequencing of relevant genomic segments .
How does G9 differ functionally from other myristoylated vaccinia virus proteins?
Although both G9 and A16 undergo myristoylation and share some structural features, they likely play distinct roles in the viral life cycle. Both proteins are components of the entry-fusion complex, but their precise individual contributions to membrane fusion events remain an area of ongoing research . The myristoylation of G9 is believed to facilitate membrane association and potential protein-protein interactions, but direct comparative studies examining the specific functional consequences of myristoylation between G9 and other myristoylated vaccinia proteins (like A16) would provide valuable insights into their unique roles.
What is the relationship between G9 and vaccinia virus decapping enzymes like D9 and D10?
G9 appears to function independently from the vaccinia virus decapping enzymes D9 and D10. While G9 is a structural protein associated with the viral entry-fusion complex , D9 and D10 are enzymes involved in mRNA degradation by removing the protective 5' caps of transcripts . D9 primarily targets viral transcripts, while D10 has broader activity against host cell mRNAs . Unlike G9, which is essential for virus replication, functional studies with D9 and D10 mutants show that viruses can still replicate when these enzymes are inactivated, although with altered host and viral transcript profiles .
How might the conserved cysteine residues in G9 contribute to protein function?
The 14 conserved cysteine residues in G9 likely play critical roles in protein folding and stability through disulfide bond formation. These residues could participate in intramolecular bonds that maintain proper protein conformation or intermolecular bonds that facilitate interactions with other components of the entry-fusion complex . The high conservation of these cysteines across poxviruses suggests they are functionally significant. Research approaches to investigate their importance might include site-directed mutagenesis studies replacing specific cysteine residues and analyzing the effects on protein localization, virion incorporation, and virus infectivity.
What are the key considerations when designing experiments to study G9 interactions with other entry-fusion complex proteins?
When investigating G9 interactions with other components of the entry-fusion complex, researchers should consider both in vivo and in vitro approaches. Co-immunoprecipitation studies using epitope-tagged G9 can identify direct binding partners . For studying membrane topology and surface exposure, biotinylation assays with membrane-impermeant reagents have proven effective . When designing experiments, it's important to account for the essential nature of G9 - using conditional expression systems rather than complete knockouts allows for controlled reduction of G9 levels without eliminating viral replication entirely. Additionally, researchers should consider the timing of G9 expression and complex formation during the viral life cycle.
What challenges might researchers encounter when expressing recombinant G9 protein, and how can these be addressed?
Expression of recombinant G9 presents several challenges due to its structural features. The presence of a transmembrane domain makes G9 difficult to express in soluble form, while the N-terminal myristoylation requires eukaryotic expression systems or specialized bacterial systems with co-expression of N-myristoyltransferase . To address these challenges, researchers might:
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Express truncated versions lacking the transmembrane domain for solubility
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Use mammalian or insect cell expression systems to ensure proper myristoylation
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Consider fusion tags that enhance solubility while permitting myristoylation
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For structural studies, employ detergent-based purification methods suitable for membrane proteins
The choice of epitope tags should also be considered carefully, as they may interfere with myristoylation if placed at the N-terminus.
How can researchers distinguish between defects in G9 incorporation into virions versus functional defects in correctly incorporated G9?
This distinction requires a multi-faceted experimental approach:
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Western blot analysis of purified virions can quantify G9 incorporation levels relative to other virion proteins
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Surface biotinylation assays can determine if incorporated G9 is correctly exposed on the virion surface
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Electron microscopy with immunogold labeling can visualize G9 localization on virions
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Entry and fusion assays using virions with wild-type levels of G9 but containing specific mutations can separate incorporation from functional defects
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Single-cycle growth curves with complementation by wild-type G9 expressed in trans can help distinguish between defects in assembly versus entry functions
What controls are essential when analyzing the effects of reduced G9 expression on virus replication?
When analyzing the effects of reduced G9 expression using inducible systems, several controls are critical:
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A titration of inducer concentrations to establish dose-dependent effects on G9 expression and virus yield
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Time-course experiments to distinguish between delays in virus production versus absolute reductions
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Parallel analysis of other virion proteins to ensure observed effects are specific to G9 reduction
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Complementation studies with wild-type G9 to verify that replication defects are directly attributable to G9 deficiency
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Microscopy analysis of virus morphogenesis to determine which stage of the viral life cycle is affected
The presence of small plaques and low but detectable levels of replication even in the absence of inducer should be carefully interpreted, as this may represent either incomplete repression of G9 synthesis or indicate that extremely low levels of G9 are sufficient for minimal replication .
How should researchers interpret differences in G9 incorporation efficiency compared to other virion membrane proteins?
The observed difference in virion enrichment between G9 (eightfold) and other proteins like A16 (16-fold) may reflect biological differences in incorporation efficiency or technical aspects of the expression system . When interpreting such data, researchers should consider:
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The potential impact of using inducible promoters, which may alter the timing or level of protein expression compared to natural conditions
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Whether differences in enrichment correlate with functional importance or simply reflect varying stoichiometry of different components in the entry-fusion complex
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How incorporation efficiency might change under different infection conditions or in different cell types
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Whether protein stability or turnover rates differ between viral proteins
Quantitative proteomics approaches comparing the abundance of various viral proteins in purified virions versus infected cells can provide more comprehensive data on relative incorporation efficiencies across the viral proteome.
What are the implications of G9's essentiality for developing poxvirus-based vaccine vectors and oncolytic therapies?
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Conditional expression systems for G9 could be used to create self-limiting vaccine vectors where replication is restricted based on inducer availability
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Modifications to G9 might alter cell tropism, potentially redirecting vectors to specific tissue types
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Knowledge of entry-fusion complex function could inform the development of more efficient viral vectors with enhanced cell entry capabilities
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The high conservation of G9 across poxviruses suggests that insights might be applicable to multiple poxvirus-based platforms
Research exploring subtle modifications to G9 that maintain essential functions while altering other properties could lead to novel vector designs with improved safety or efficacy profiles.
How might comparative studies of G9 across different poxviruses inform our understanding of viral evolution and host adaptation?
The high conservation of G9 structural features across the poxvirus family offers a valuable opportunity for comparative studies . Such research could:
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Identify subtle sequence variations that correlate with host range or tissue tropism differences between poxvirus species
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Examine whether species-specific differences in G9 interact with host factors in ways that influence viral adaptation
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Use ancestral sequence reconstruction to trace the evolutionary history of G9 and the entry-fusion complex
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Investigate whether G9 is under different selective pressures compared to other viral proteins
Cross-complementation studies testing whether G9 from one poxvirus species can functionally replace G9 in another species would provide insights into the degree of functional conservation versus specialization.
What potential exists for targeting G9 in antiviral drug development strategies?
As an essential component of the viral entry machinery that is highly conserved across poxviruses, G9 presents an attractive target for broad-spectrum antipoxviral therapeutics. Potential approaches include:
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Small molecule inhibitors that disrupt G9's interactions with other entry-fusion complex components
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Compounds that interfere with G9 myristoylation or membrane association
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Peptide inhibitors derived from interaction domains of G9 binding partners
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Antibodies or antibody fragments that recognize exposed portions of G9 on the virion surface
Drug development efforts would benefit from detailed structural information about G9 and its binding interfaces, as well as high-throughput screening assays to identify compounds that specifically disrupt G9 function without affecting host cell proteins.
