Recombinant Acanthamoeba polyphaga mimivirus Uncharacterized protein R710 (MIMI_R710)

What is MIMI_R710 and what do we currently know about its characteristics?

MIMI_R710 is an uncharacterized protein encoded by the Acanthamoeba polyphaga mimivirus genome. According to available data, MIMI_R710 (UniProt ID: Q5UQ56) is a 194 amino acid protein with the sequence: MSWHTGSNQDNKLFPKGKLSGSYAPLDIAFENSPAMNEFENRLCHNNPIISERSMSPAVS ASYSNPEATSCGCMQTQTQPQHQTLSQHLPQTHHTDAHDQQKLSGIFYNRTTDAQNQFSE TINPPPSYTVHNTDIRIPLNRQQQYPANHLGSELLEGYNNVGTEPCMGFWEILLLIILIA VLVYGIYWLYKSEK .

It has been identified as a late virion-associated protein, suggesting its involvement in the later stages of viral replication or virion assembly . The C-terminal region of the protein appears to contain hydrophobic residues that might indicate membrane association properties. While its precise function remains undetermined, its conservation in the Mimivirus genome suggests biological significance.

How can I obtain recombinant MIMI_R710 for my research?

Recombinant MIMI_R710 can be produced using heterologous expression systems, with E. coli being the most commonly used host. Commercial sources offer the full-length protein (1-194 amino acids) fused to an N-terminal His-tag . For research purposes, the recombinant protein is typically provided as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE .

When working with the recombinant protein, it is recommended to:

• Briefly centrifuge the vial before opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol (5-50% final concentration) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• Store at -20°C/-80°C for optimal stability

What expression systems are most effective for producing recombinant MIMI_R710?

E. coli expression systems have been successfully employed for the production of recombinant MIMI_R710 . The protein has been expressed as a His-tagged fusion protein to facilitate purification using affinity chromatography. When designing an expression strategy, consider the following:

What are the optimal storage conditions for recombinant MIMI_R710?

To maintain the stability and activity of recombinant MIMI_R710, proper storage is essential. Based on available data, the recommended storage conditions are:

• Store lyophilized protein at -20°C/-80°C upon receipt

• After reconstitution, add glycerol (recommended final concentration 50%) and store at -20°C/-80°C

• Prepare working aliquots to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

The storage buffer typically used is Tris/PBS-based buffer with 6% trehalose at pH 8.0 . This formulation helps maintain protein stability during freeze-thaw cycles.

What techniques can be used to study the localization of MIMI_R710 during Mimivirus infection?

Understanding the spatiotemporal dynamics of MIMI_R710 during viral infection can provide valuable insights into its function. Several approaches can be employed:

Fluorescent protein tagging: Similar to methods used for other Mimivirus proteins, MIMI_R710 can be tagged with fluorescent markers such as EGFP or mCherry. Homologous recombination strategies can be used for in-frame fusion of fluorescent tags at the C-terminal end of the target gene . This approach allows real-time visualization of protein localization during infection.

Experimental methodology:

• Design primers to amplify the R710 gene with appropriate flanking regions for homologous recombination

• Create a construct with EGFP or mCherry fused in-frame to the C-terminus of R710

• Transfect Acanthamoeba cells with the construct and infect with Mimivirus

• Monitor fluorescence using confocal microscopy at different time points post-infection (e.g., 6h, 8h, 12h, 24h)

• Co-stain with DAPI to visualize viral and host DNA

• Compare localization patterns with other viral components such as capsid proteins (e.g., L425/gp455)

This approach has been successfully used to demonstrate that late virion-associated proteins localize to the viral factory (VF) during Mimivirus infection .

How can I determine the potential function of MIMI_R710 in the Mimivirus replication cycle?

As an uncharacterized protein, elucidating the function of MIMI_R710 requires a multifaceted approach:

1. Temporal expression analysis:
Determine when during infection MIMI_R710 is expressed using RT-qPCR or proteomics. Late virion-associated proteins like MIMI_R710 are typically expressed after viral DNA replication and are involved in virion assembly or maturation .

Interaction studies:

• Perform co-immunoprecipitation using anti-His antibodies with recombinant MIMI_R710 as bait

• Use mass spectrometry to identify viral or host proteins that interact with MIMI_R710

• Validate interactions using techniques such as proximity ligation assay (PLA) or FRET

3. Localization patterns:
The virus factory (VF) of Mimivirus shows distinct zones with specific functions:

• Inner replication center: DNA replication and possibly protein synthesis

• Intermediate assembly zone: Capsid assembly

• Peripheral zone: Fibril acquisition

Determining where MIMI_R710 localizes within these zones can provide functional clues.

Structural analysis:

• Perform secondary structure prediction to identify potential functional domains

• Use homology modeling if distant homologs with known structures exist

• Consider the hydrophobic C-terminal region which might indicate membrane association

How can I design experiments to investigate if MIMI_R710 is involved in viral factory formation?

The Mimivirus replication cycle involves the formation of a distinctive virus factory (VF) with a volcano-like structure that emerges from the cell surface . To investigate MIMI_R710's potential role in VF formation:

Experimental approach:

• Gene silencing or CRISPR interference: Design guide RNAs targeting the R710 gene to reduce expression

• Dominant negative mutants: Express truncated versions of MIMI_R710 that might interfere with native protein function

• Time-course imaging: Using fluorescently tagged MIMI_R710, monitor its distribution relative to VF formation markers

• Electron microscopy: Compare ultrastructure of VFs in wild-type infections versus those with R710 perturbation

• Functional complementation: In systems with reduced R710 function, determine if providing recombinant protein rescues normal VF formation

The Mimivirus factory exhibits a three-zone structure (replication center, assembly zone, and peripheral zone) . Careful analysis of each zone's formation in the presence/absence of functional MIMI_R710 can reveal its specific role.

What approaches are recommended for studying protein-protein interactions involving MIMI_R710?

Understanding the interaction partners of MIMI_R710 is crucial for functional characterization. Several complementary methods can be employed:

In vitro approaches:

• Pull-down assays: Use His-tagged recombinant MIMI_R710 as bait with Mimivirus-infected cell lysates

• Surface Plasmon Resonance (SPR): Measure binding kinetics between purified MIMI_R710 and candidate partners

• Crosslinking Mass Spectrometry: Identify proteins in close proximity to MIMI_R710 during infection

In vivo approaches:

• Bimolecular Fluorescence Complementation (BiFC): Split fluorescent proteins fused to MIMI_R710 and potential partners

• Proximity-dependent biotin labeling (BioID or TurboID): Identify proteins in the vicinity of MIMI_R710 during infection

• Co-localization studies: Use dual-color fluorescence microscopy to visualize MIMI_R710 alongside other viral components

Data analysis considerations:

• Employ appropriate controls to distinguish specific from non-specific interactions

• Validate key interactions using multiple independent methods

• Consider the temporal dynamics of interactions during the 24h infectious lytic cycle

What are the best methods for tagging MIMI_R710 without affecting its function?

When designing tagged versions of MIMI_R710 for functional studies, several factors must be considered:

How should I analyze contradictory results regarding MIMI_R710 localization or function?

Contradictory findings are common in research on uncharacterized proteins. When encountering discrepancies in MIMI_R710 studies:

• Evaluate methodological differences:

  • Different detection methods may have varying sensitivities

  • Fixation procedures can affect protein localization

  • Antibody specificity should be rigorously validated

• Consider temporal dynamics:

  • MIMI_R710 localization may change during the 24h infectious cycle

  • Sample timing should be precisely reported and compared

• Account for experimental conditions:

  • Host cell state (age, passage number)

  • Virus-to-cell ratio (multiplicity of infection)

  • Temperature and other environmental factors

• Systematic approach to resolution:

  • Design experiments that directly test competing hypotheses

  • Use multiple independent techniques to verify key findings

  • Consider contacting authors of contradictory studies for additional details

• Context analysis techniques:

  • Methods such as those described by Alamri and Stevenson can help identify and characterize apparent contradictions in the literature

  • Normalize terms and concepts across studies to ensure valid comparisons

What structural studies could provide insights into MIMI_R710 function?

Structural biology approaches could significantly advance our understanding of MIMI_R710:

• X-ray crystallography: Requires high-purity recombinant protein and crystallization screening

• Cryo-electron microscopy: Particularly useful if MIMI_R710 forms part of a larger complex

• NMR spectroscopy: Suitable for analyzing structure and dynamics if protein size permits

When combined with computational approaches such as molecular dynamics simulations, these methods could reveal:

• Potential binding sites for DNA, RNA, or other proteins

• Structural homology to proteins of known function

• Conformational changes that might occur during virus assembly

How might studying MIMI_R710 contribute to our understanding of giant virus evolution?

Characterizing MIMI_R710 could provide insights into the evolutionary history of Mimiviridae:

• Comparative genomics: Identify homologs in other giant viruses and trace evolutionary relationships

• Structural conservation: Determine if MIMI_R710 shares structural features with proteins from other viral families or cellular organisms

• Functional parallels: Compare the role of MIMI_R710 with functionally analogous proteins in other large DNA viruses

This research could address fundamental questions about the origins of giant viruses and their relationship to cellular life forms, contributing to ongoing debates about the position of Mimiviridae in the tree of life.

What emerging technologies could advance our understanding of MIMI_R710?

Several cutting-edge approaches hold promise for MIMI_R710 research:

• CryoET (Cryo-electron tomography): Could reveal the precise location of MIMI_R710 within the virus particle or factory

• AlphaFold2 and other AI protein structure prediction tools: May provide structural insights even without experimental structures

• Single-particle tracking: Could monitor the dynamic behavior of MIMI_R710 molecules during infection

• Microfluidics-based single-cell analysis: May reveal cell-to-cell variability in MIMI_R710 function

• CRISPR-based viral genome engineering: Could facilitate the creation of targeted mutations to test MIMI_R710 function