Recombinant Acanthamoeba polyphaga mimivirus DNA topoisomerase 1B (TOP1E)

What is Acanthamoeba polyphaga mimivirus DNA topoisomerase 1B and how was it discovered?

Acanthamoeba polyphaga mimivirus DNA topoisomerase IB (MimiTopIB) is a functional enzyme encoded by the largest known DNA virus, mimivirus, which infects Acanthamoeba polyphaga amoebae. It belongs to the type IB family of topoisomerases that catalyze the relaxation of supercoiled DNA through a transient single-strand break mechanism. MimiTopIB was identified following the discovery and genomic analysis of mimivirus, which revealed its extraordinarily large genome (1.2 Mb) and extensive protein-coding capacity .

The enzyme displays primary structure similarity to poxvirus topoisomerases and, to an even greater extent, to the bacterial poxvirus-like TopIB enzyme family. MimiTopIB consists of 336 amino acids, comparable in size to poxvirus TopIB (314 aa) and bacterial TopIB homologs. Notably, it shares the critical "catalytic pentad" (Arg-Lys-Arg-(His/Asn)-Tyr) responsible for the DNA transesterification reaction that is characteristic of type IB topoisomerases .

How does mimivirus TOP1B structurally and functionally compare to other viral and cellular topoisomerases?

Mimivirus TOP1B represents a structural and functional homolog of poxvirus TOP1B and the poxvirus-like topoisomerases found in certain bacteria. Comparative analysis reveals several important similarities and differences:

Despite its greater primary structure similarity to bacterial topoisomerases, functional analyses suggest that mimivirus TopIB is functionally more akin to poxvirus TopIB. Both share the ability to incise duplex DNA at the specific 5'-CCCTT cleavage site, a characteristic recognition sequence for poxvirus topoisomerases .

What is the predicted biological role of TOP1B in the mimivirus replication cycle?

The presence of TopIB in mimivirus suggests important parallels with poxviruses and their replication strategies. In poxviruses, which also replicate in the cytoplasm of host cells, TopIB is encapsidated within the viral particle and plays a crucial role in the synthesis of viral early mRNAs .

Mimivirus, like poxviruses, encodes its own transcriptional machinery, including a multisubunit RNA polymerase and RNA capping enzymes, suggesting a poxvirus-like gene expression strategy. The presence of TopIB in this context strongly indicates its involvement in early viral transcription or DNA replication processes that occur in the cytoplasm of the amoeba host cell .

The enzyme likely participates in resolving DNA topological problems that arise during viral DNA replication or transcription in the cytoplasmic environment, where access to host nuclear topoisomerases would be limited. This function would be essential for the successful completion of the viral life cycle within Acanthamoeba polyphaga.

What expression systems are optimal for producing recombinant mimivirus TOP1B?

Recombinant expression of mimivirus TopIB can be achieved using several expression systems, each with advantages and limitations. Based on successful topoisomerase expression strategies, the following approaches are recommended:

• Bacterial Expression (E. coli): The most commonly used system employs BL21(DE3) or similar strains with pET-based vectors containing an N-terminal His-tag or GST-tag for purification. Optimal expression typically occurs at lower temperatures (16-18°C) after induction with reduced IPTG concentrations (0.1-0.5 mM) to enhance solubility.

• Insect Cell Expression: Baculovirus-infected Sf9 or Hi5 cells provide a eukaryotic environment potentially beneficial for proper folding and post-translational modifications. This system may yield higher amounts of soluble, active enzyme compared to bacterial expression.

• Yeast Expression: Pichia pastoris or Saccharomyces cerevisiae systems offer another eukaryotic alternative that balances yield and proper protein processing.

For optimal results, codon optimization for the chosen expression host should be considered, as viral proteins often display codon bias different from the expression host. Additionally, expressing the protein with solubility-enhancing tags (MBP, SUMO) can significantly improve yield and activity of the recombinant enzyme.

What purification protocol yields the highest activity for recombinant mimivirus TOP1B?

A multi-step purification protocol is recommended for obtaining highly active mimivirus TopIB:

• Initial Capture: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a gradient elution (50-250 mM imidazole) in buffer containing 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% glycerol, and 1 mM DTT.

• Ion Exchange Chromatography: The IMAC-purified fraction should be dialyzed against low-salt buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 10% glycerol, 1 mM DTT) and applied to a heparin column, with elution using a 50-1000 mM NaCl gradient.

• Size Exclusion Chromatography: Final polishing using Superdex 75 or 200 in storage buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 10% glycerol, 1 mM EDTA, 5 mM DTT).

Throughout purification, it is critical to maintain reducing conditions (1-5 mM DTT or 0.5-2 mM TCEP) and include protease inhibitors in the initial lysis steps. Purified protein should be stored in aliquots at -80°C with glycerol (25-50%) to maintain activity during freeze-thaw cycles.

Typical yields range from 2-5 mg of purified protein per liter of bacterial culture or 5-10 mg per liter of insect cell culture, with specific activity (measured as DNA relaxation units per mg protein) often 2-3 fold higher from insect cell-derived material.

What assays are available for measuring mimivirus TOP1B activity in vitro?

Several complementary assays can be employed to measure mimivirus TopIB activity:

• DNA Relaxation Assay: The standard method measures conversion of supercoiled plasmid DNA to relaxed forms using agarose gel electrophoresis. Reaction mixtures (typically 20 μL) contain 0.2-0.5 μg supercoiled plasmid DNA, buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM DTT), and varying amounts of enzyme, incubated at 37°C for 5-30 minutes. Products are analyzed on 1% agarose gels run with or without ethidium bromide.

• DNA Cleavage Assay: This assay detects the formation of the covalent TopIB-DNA complex by using radiolabeled or fluorescently labeled oligonucleotides containing the 5'-CCCTT recognition sequence. Cleavage products are separated by denaturing polyacrylamide gel electrophoresis.

• Suicide Cleavage Substrate Assay: Using a modified oligonucleotide that traps the enzyme in the covalent complex allows quantitative measurement of the cleavage rate independent of religation.

• Fluorescence-Based Relaxation Assay: Real-time monitoring of topoisomerase activity using fluorescent intercalators or FRET-based supercoiled DNA substrates enables continuous measurement and kinetic analysis.

How does the 5'-CCCTT DNA sequence specificity of mimivirus TOP1B influence its applications in research?

The 5'-CCCTT sequence specificity of mimivirus TopIB, which it shares with poxvirus topoisomerases, has significant implications for research applications :

• Mechanistic Studies: This defined sequence specificity makes mimivirus TopIB valuable for studying the molecular basis of DNA sequence recognition by type IB topoisomerases.

• DNA Structure Analysis: The enzyme can serve as a probe for accessibility of this sequence in different DNA conformations or chromatin structures.

• Recombination Applications: The site-specific cleavage capability can be exploited for targeted DNA manipulation, similar to how vaccinia topoisomerase has been used in molecular cloning strategies.

• Inhibitor Development: The sequence specificity provides a structural basis for developing sequence-selective inhibitors that could have applications against related viral topoisomerases.

• Evolutionary Studies: The shared sequence specificity with poxvirus enzymes provides evidence for evolutionary relationships despite primary sequence differences, suggesting functional conservation that supports theories about viral topoisomerase evolution and horizontal gene transfer events .

What are the key catalytic residues in mimivirus TOP1B and how do mutations affect enzyme activity?

Mimivirus TopIB contains the conserved catalytic pentad (Arg-Lys-Arg-His-Tyr) that is essential for the transesterification reaction characteristic of type IB topoisomerases . Mutation studies reveal the following effects:

These catalytic residues are conserved and essential in mimivirus TopIB, as demonstrated by site-directed mutagenesis studies. The substitution of the active site tyrosine with phenylalanine (Y→F) abolishes all catalytic activity while preserving DNA binding capability, creating a useful catalytically inactive control for experimental studies .

What structural features distinguish mimivirus TOP1B from other type IB topoisomerases?

Mimivirus TopIB shares the core structural features of type IB topoisomerases while possessing unique characteristics:

• Domain Organization: Unlike eukaryotic TopIB enzymes that have separate core and C-terminal domains connected by a linker, mimivirus TopIB has a compact, single-domain structure similar to poxvirus and bacterial poxvirus-like topoisomerases.

• DNA Recognition Elements: The enzyme possesses specific residues that interact with the 5'-CCCTT recognition sequence, forming contacts primarily in the major groove of DNA.

• Active Site Architecture: The active site contains the catalytic pentad (Arg-Lys-Arg-His-Tyr) arranged to position the active site tyrosine for nucleophilic attack on the DNA phosphodiester bond.

• C-terminal Region: This region contains elements important for DNA binding and recognition, with structural differences from poxvirus TopIB that may account for subtle differences in catalytic properties.

• N-terminal Region: This region is involved in DNA binding and contributes to the preferential interaction with specific DNA sequences.

Structural comparison with other TopIB enzymes reveals that mimivirus TopIB appears to be a structural intermediate between bacterial and poxvirus TopIB enzymes, consistent with theories about horizontal gene transfer events potentially involving amoebae as a permissive host for this evolutionary exchange .

What crystallization conditions are recommended for structural studies of mimivirus TOP1B?

Based on successful crystallization of related type IB topoisomerases, the following conditions are recommended for crystallographic studies of mimivirus TopIB:

• Protein Preparation:

  • Highly purified protein (>95% by SDS-PAGE) at 5-15 mg/mL

  • Buffer composition: 20 mM Tris-HCl pH 7.5-8.0, 150-200 mM NaCl, 1 mM EDTA, 5 mM DTT

  • Fresh protein preparation, avoiding multiple freeze-thaw cycles

• Crystallization Screening:

  • Initial sparse matrix screening at both 4°C and 18°C

  • Vapor diffusion methods (hanging or sitting drop) with 1:1 protein:reservoir ratios

  • PEG-based conditions (PEG 3350, PEG 4000, PEG 8000) at 10-25% concentration

  • pH range of 6.0-8.5 with various buffers (Tris, HEPES, MES)

  • Addition of divalent cations (Mg²⁺, Mn²⁺, Ca²⁺) at 5-10 mM

• Co-crystallization with DNA:

  • Short oligonucleotides (10-14 bp) containing the 5'-CCCTT recognition sequence

  • Pre-incubation of protein with DNA at 1:1.2 molar ratio

  • Addition of non-hydrolyzable ATP analogs for stabilization

  • Use of catalytically inactive mutant (Y→F) to capture the pre-cleavage complex

• Crystal Optimization:

  • Microseeding techniques to improve crystal quality

  • Additive screening with small molecules (glycerol, alcohols, detergents)

  • Controlled dehydration to improve diffraction quality

  • Cryoprotection with 15-25% glycerol, ethylene glycol, or PEG 400

What inhibitors are effective against mimivirus TOP1B and how do they compare to other topoisomerase inhibitors?

Several classes of inhibitors show activity against type IB topoisomerases, with varying effectiveness against mimivirus TopIB:

Structure-based drug design approaches targeting the unique features of the mimivirus TopIB active site present opportunities for developing selective inhibitors. Molecular docking studies suggest that compounds targeting the interface between the enzyme and its specific DNA recognition sequence (5'-CCCTT) may offer the highest selectivity.

How can mimivirus TOP1B be used to study horizontal gene transfer mechanisms in amoebae?

The presence of TopIB in mimivirus provides a unique model for investigating horizontal gene transfer (HGT) events involving amoebae. Research applications include:

• Phylogenetic Analysis: Comprehensive sequence comparison of mimivirus TopIB with bacterial and viral homologs can trace potential evolutionary paths and identify signatures of HGT events.

• Co-infection Studies: Experimental co-infection of Acanthamoeba with mimivirus and bacteria containing TopIB genes can be monitored for genetic exchange events.

• Experimental Evolution: Long-term culturing of mimivirus in Acanthamoeba under selective pressure can reveal adaptive changes in the TopIB gene.

• Functional Complementation: Testing whether mimivirus TopIB can functionally replace topoisomerases in other systems provides evidence for functional conservation despite evolutionary divergence.

• Metagenomics Surveys: Analysis of environmental amoeba samples for the presence of diverse TopIB genes can map the distribution and diversity of these enzymes in natural settings.

The current evidence suggests that the ancestral bacterial/viral TopIB was likely disseminated by horizontal gene transfer within amoebae, which serve as permissive hosts for both intracellular bacteria and large DNA viruses . This makes mimivirus TopIB an excellent model for studying the molecular mechanisms of HGT in microbial communities.

What role does TOP1B play in mimivirus infection of Acanthamoeba polyphaga?

The role of TopIB in mimivirus infection can be investigated through several experimental approaches:

• Temporal Expression Analysis: RT-qPCR and proteomics studies demonstrate that TopIB is expressed early during infection, consistent with a role in early viral transcription or DNA replication.

• Virion Association: Proteomics analysis of purified virions confirms the presence of TopIB in the virus particle, suggesting it is packaged for immediate use upon infection.

• DNA Transfection Studies: Experiments involving transfection of mimivirus DNA into Acanthamoeba castellanii show that viral DNA with associated proteins (including potential TopIB) can generate infectious virions, while proteinase K treatment to remove these proteins prevents successful infection .

• Inhibitor Studies: Treatment with TopIB inhibitors during early infection reduces viral gene expression and progeny production, supporting a critical role in the viral life cycle.

• Interaction with Host Encystment: Mimivirus prevents Acanthamoeba encystment, potentially through mechanisms involving TopIB activity on host DNA or chromatin structure .

These lines of evidence collectively suggest that TopIB plays a crucial role in establishing productive infection, likely by supporting viral DNA replication and transcription in the cytoplasmic environment of the host cell.

How can CRISPR-Cas9 technology be applied to study mimivirus TOP1B function in vivo?

CRISPR-Cas9 technology offers several innovative approaches for studying mimivirus TopIB function:

• Gene Editing of Mimivirus Genome:

  • Creation of TopIB-null mutants through targeted disruption

  • Introduction of point mutations to modify catalytic residues

  • Generation of tagged variants for localization studies

  • Engineering chimeric TopIB enzymes with domains from other viral or bacterial sources

• Host Cell Modification:

  • Creation of Acanthamoeba lines with fluorescent reporters for monitoring infection dynamics

  • Engineering amoeba cells expressing mimivirus TopIB to assess effects on host DNA metabolism

  • Knockout of host factors that potentially interact with viral TopIB

• High-throughput Functional Screening:

  • CRISPR library screening to identify host factors affecting TopIB function

  • Creation of mimivirus TopIB variant libraries to analyze structure-function relationships

• Live Cell Imaging Applications:

  • Integration of fluorescent tags to visualize TopIB localization during infection

  • FRET-based approaches to monitor TopIB-DNA interactions in living cells

While technical challenges exist in applying CRISPR to large DNA viruses and amoeba hosts, recent advances in transfection methods for Acanthamoeba and the development of CRISPR systems optimized for diverse organisms make these approaches increasingly feasible for advanced mimivirus research.

What are common problems encountered when working with recombinant mimivirus TOP1B and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant mimivirus TopIB:

• Low Solubility:

  • Solution: Express at lower temperatures (16-18°C), use solubility-enhancing tags (MBP, SUMO), include 5-10% glycerol and 0.1% non-ionic detergents in purification buffers.

• Proteolytic Degradation:

  • Solution: Include protease inhibitor cocktail during lysis, minimize purification time, use fresh preparations, consider removing flexible regions prone to proteolysis.

• Low Specific Activity:

  • Solution: Maintain reducing conditions throughout purification, include Mg²⁺ in storage buffers, avoid multiple freeze-thaw cycles, verify DNA substrate quality.

• DNA Contamination:

  • Solution: Include DNase treatment during lysis, incorporate polyethyleneimine precipitation step, use heparin chromatography, verify final preparation purity by absorbance ratio (A260/A280).

• Inconsistent Assay Results:

  • Solution: Standardize DNA substrates, control reaction temperature precisely, use internal controls, verify buffer composition (particularly salt concentration and pH).

• Storage Stability Issues:

  • Solution: Store in small aliquots with 30-50% glycerol at -80°C, avoid repeated freeze-thaw cycles, consider lyophilization for long-term storage.

How can researchers optimize transfection of mimivirus DNA into Acanthamoeba for functional studies?

Successful transfection of mimivirus DNA into Acanthamoeba requires careful optimization of several parameters:

• Microinjection Approach:

  • Research indicates successful generation of infectious mimivirus through microinjection of viral DNA into Acanthamoeba castellanii .

  • Key factors include maintaining the association of specific proteins with the viral DNA, as proteinase K treatment prevents successful virus production .

• Transfection Protocol Optimization:

  • Cell Preparation: Use actively growing trophozoites at 70-80% confluence.

  • DNA Quality: Freshly extracted viral DNA without additional proteinase K treatment yields best results .

  • Microinjection Technique: Fluorescent markers (dextran) can verify successful injection .

  • Recovery Period: Allow 24 hours post-microinjection before assessing viability and infection progress .

• Critical Protein Factors:

  • Proteins identified as potentially important for successful transfection include uncharacterized proteins L442, L724, L829, R387, and putative GMC-type oxidoreductase R135 .

  • Of these, L442 appears to play a particularly significant role in DNA-protein interactions necessary for infection .

• Monitoring Techniques:

  • Successful transfection can be confirmed by observing cytopathic effects 5-7 days post-transfection .

  • Virus production can be verified through scanning electron microscopy showing viral particles with characteristic morphology .

  • Flow cytometry with SYBR green DNA staining provides quantitative assessment of viral production .

What quality control measures are essential for ensuring reliable results in mimivirus TOP1B research?

To ensure reliable and reproducible results in mimivirus TopIB research, several quality control measures should be implemented:

• Protein Quality Assessment:

  • Purity verification by SDS-PAGE (>95% homogeneity)

  • Mass spectrometry confirmation of protein identity

  • Activity measurement using standardized relaxation assays

  • Circular dichroism to verify proper folding

  • Dynamic light scattering to assess aggregation state

• DNA Substrate Quality:

  • Supercoiled plasmid preparation with >90% supercoiled form

  • Defined sequence oligonucleotides for cleavage assays

  • Verification of oligonucleotide integrity by denaturing PAGE

  • Consistent substrate concentration determination

• Reaction Conditions Standardization:

  • Precise temperature control (±0.5°C)

  • Calibrated pH measurements for buffers

  • Use of internal controls for comparative experiments

  • Multiple technical and biological replicates

• Equipment Validation:

  • Regular calibration of pipettes and thermal cyclers

  • Validation of gel imaging systems for linear response

  • Temperature monitoring of incubators and storage units

• Data Analysis Rigor:

  • Blinded quantification of enzyme activity where possible

  • Statistical analysis with appropriate tests

  • Clear reporting of replicate numbers and variation

  • Standardized methods for calculating enzyme kinetic parameters