Recombinant Acanthamoeba polyphaga mimivirus Uncharacterized protein R213 (MIMI_R213)

What is Acanthamoeba polyphaga mimivirus and why is it significant in virology?

Acanthamoeba polyphaga mimivirus (APMV) represents one of the largest known viruses, discovered in 2003. It belongs to the nucleocytoplasmic large DNA viruses (NCLDVs) group and infects Acanthamoeba species. APMV gained significant attention due to its exceptionally large genome (approximately 1.2 million base pairs) and virion size (approximately 750 nm in diameter), which challenged traditional definitions of viruses .

The virus establishes distinct viral factories within infected amoeba cells where genome replication and virion assembly occur. These factories are critical sites where the virus produces hundreds of copies of its genome that must be properly segregated and packaged into new virions . APMV also features a unique two-portal system for genome packaging and delivery, representing a novel mechanism among viruses .

What are the basic structural and functional characteristics of MIMI_R213?

MIMI_R213 is an uncharacterized protein encoded by the APMV genome. Based on available information, it consists of 142 amino acids, as indicated by the recombinant protein description (Q5UQ28, 1-142aa) . As an uncharacterized protein, its precise function within the viral life cycle remains undetermined.

While the specific function of R213 has not been established, it may be involved in viral processes similar to other characterized mimivirus proteins. These processes could include virion structure, DNA replication, transcription, genome packaging, or host interaction. The protein's relatively small size (142 amino acids) suggests it might function as part of a larger protein complex rather than independently.

How does MIMI_R213 fit into the context of mimivirus proteome organization?

Mimivirus encodes numerous proteins, many of which remain uncharacterized like R213. The proteome can be broadly categorized into structural proteins, those involved in genome replication and transcription, and those that interact with the host. The "R" in R213 indicates that the gene is located on the right strand of the viral genome, while "213" denotes its sequential position among right-strand genes.

Research has demonstrated that mimivirus contains many proteins and RNAs within the virion, which play roles in the early stages of infection . Some mimivirus proteins, like L442, L724, L829, R387, and R135, have been shown to be critical for virus production after DNA transfection into host cells . MIMI_R213 may have similar importance, though its specific function requires further investigation.

What expression systems are recommended for producing recombinant MIMI_R213?

For optimal expression of recombinant MIMI_R213, researchers should consider several expression systems:

Based on available information about mimivirus proteins, many recombinant mimivirus proteins including uncharacterized ones are successfully produced with N-terminal His-tags to facilitate purification . This approach is likely suitable for MIMI_R213 as well.

What purification strategies yield the highest purity and activity for recombinant MIMI_R213?

A multi-step purification protocol is recommended for obtaining high-purity MIMI_R213:

• Initial capture using immobilized metal affinity chromatography (IMAC):

  • Use Ni-NTA resin with His-tagged MIMI_R213

  • Equilibrate column with 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Apply cleared lysate, wash with 20-30 mM imidazole

  • Elute with 250 mM imidazole gradient

• Intermediate purification using ion-exchange chromatography:

  • Dialyze IMAC eluate to reduce salt concentration

  • Apply to appropriate ion-exchange column based on theoretical pI

  • Elute with salt gradient (0-1 M NaCl)

• Polishing step using size-exclusion chromatography:

  • Apply concentrated sample to Superdex 75 column

  • Use buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl

  • Collect fractions and analyze by SDS-PAGE

For quality control, each purification step should be monitored by SDS-PAGE, and the final protein should be verified by western blot and mass spectrometry.

What methods are most effective for functional characterization of MIMI_R213?

Given the uncharacterized nature of MIMI_R213, a comprehensive approach to functional characterization should include:

• Bioinformatic analysis:

  • Sequence homology searches against protein databases

  • Structure prediction using tools like Phyre2 (similar to approach used for L442)

  • Identification of conserved domains and motifs

• Protein-protein interaction studies:

  • Pull-down assays with other mimivirus proteins

  • Yeast two-hybrid screening

  • Cross-linking mass spectrometry to identify interaction partners

• Localization studies:

  • Generation of specific antibodies against MIMI_R213

  • Immunofluorescence microscopy at different stages of infection

  • Fractionation of viral particles to determine if R213 is structural

• Functional assays:

  • DNA/RNA binding assays if bioinformatic analysis suggests nucleic acid interaction

  • Enzymatic activity assays based on predicted functions

  • Microinjection experiments with and without R213 to assess its necessity for viral replication

Studies with other mimivirus proteins have shown that proteinase K treatment of viral DNA prevents successful viral production after transfection , suggesting protein-DNA interactions are critical. Similar approaches could determine if R213 plays such a role.

How might MIMI_R213 be involved in mimivirus genome packaging and virion assembly?

Mimivirus employs a sophisticated genome packaging mechanism that differs from other viral systems and shares similarities with prokaryotic chromosome segregation machinery . While the specific role of MIMI_R213 in this process is not established, several possibilities exist:

Mimivirus genome packaging involves several components:

• A packaging ATPase related to prokaryotic FtsK/SPOIIIE/HerA motors

• Three putative recombinases

• A putative type II topoisomerase

MIMI_R213 could potentially:

• Interact with the packaging ATPase as a regulatory factor

• Function as a structural component of the two-portal system unique to mimivirus

• Assist in genome segregation prior to packaging

• Participate in DNA condensation to facilitate packaging of the large genome

Research has shown that certain mimivirus proteins (L442, L724, L829, R387, and R135) remain associated with viral DNA and are essential for successful transfection and virus production . MIMI_R213 might have a similar DNA-associated function.

What techniques are most appropriate for studying MIMI_R213 localization during infection?

To determine the localization of MIMI_R213 during mimivirus infection, several complementary approaches are recommended:

• Immunofluorescence microscopy:

  • Generate specific antibodies against MIMI_R213

  • Perform time-course experiments during infection

  • Co-stain with markers for viral factories, capsids, and DNA

• Electron microscopy techniques:

  • Immunogold labeling for transmission electron microscopy

  • Correlative light and electron microscopy to combine fluorescence and ultrastructural data

• Biochemical fractionation:

  • Separate infected cells into subcellular components

  • Isolate viral particles at different maturation stages

  • Analyze fractions by western blotting for MIMI_R213

• Live-cell imaging (if feasible):

  • Create fluorescently tagged MIMI_R213 constructs

  • Microinject into amoeba as described in published protocols

  • Track localization throughout infection cycle

The microinjection technique described in the literature for mimivirus DNA could potentially be adapted for fluorescently labeled MIMI_R213 studies, though the technical challenges (low success rate of approximately 4%) should be considered .

How can researchers optimize transfection protocols when studying MIMI_R213 in amoeba hosts?

Transfecting Acanthamoeba species presents unique challenges. Based on published methods for mimivirus DNA transfection, the following protocol can be adapted for studies involving MIMI_R213:

• Microinjection approach:

  • Prepare amoebae at low concentration (10^3 cells/ml) in starvation medium

  • Use femtotips and micromanipulator for precise injection

  • Include fluorescent markers (e.g., rhodamine-dextran) to confirm successful injection

  • Monitor cell viability (maintenance of trophozoite state)

• Optimization considerations:

  • Cell preparation is critical - use gently tapped cells after 48h incubation

  • Perform washes in starvation medium before injection

  • Allow recovery in appropriate medium post-injection

  • Be prepared for low success rates (~24% successful experiments, with only a fraction producing viable virus)

• Controls and validation:

  • Include positive controls (known functional constructs)

  • Use appropriate fluorescent markers to track transfection efficiency

  • Confirm expression through microscopy and/or western blotting

  • Verify effects on viral replication using flow cytometry and electron microscopy

What mass spectrometry methods are most suitable for characterizing MIMI_R213 interactions?

Based on successful approaches with other mimivirus proteins, several mass spectrometry techniques are recommended for MIMI_R213 interaction studies:

• MALDI-TOF-MS and LC-MS for protein identification:

  • These techniques successfully identified mimivirus proteins L442, R135, R387, L724, and L829 from gel bands

  • For MIMI_R213, in-gel digestion followed by MALDI-TOF-MS can confirm identity

  • LC-MS provides higher sensitivity for low-abundance samples

• Affinity purification coupled with LC-MS/MS:

  • Express tagged MIMI_R213 and use it as bait to capture interaction partners

  • Digest captured complexes with trypsin

  • Analyze by LC-MS/MS to identify co-purifying proteins

  • Quantify interactions using label-free or SILAC approaches

• Cross-linking mass spectrometry:

  • Treat MIMI_R213-containing complexes with cross-linking reagents

  • Identify cross-linked peptides to map interaction interfaces

  • This approach can provide structural constraints for modeling interactions

The published studies show that LC-MS identified mimivirus proteins with varying peptide coverage (11% for L442, 16% for R135) , suggesting similar approaches would be effective for MIMI_R213.

What computational approaches can predict the function of MIMI_R213?

In the absence of direct experimental data, computational approaches can provide valuable insights into potential MIMI_R213 functions:

• Sequence-based predictions:

  • PSI-BLAST for detecting remote homologs

  • PFAM and InterPro for domain identification

  • FoldIndex for predicting intrinsically disordered regions

• Structure prediction and analysis:

  • Phyre2 for tertiary structure prediction (similar to approach used for L442)

  • AlphaFold2 for more accurate structural models

  • CASTp for binding pocket identification

  • ProFunc for function prediction from structure

• Protein-protein interaction prediction:

  • STRING database to identify potential interaction partners

  • PRISM for structural interface prediction

  • Evolutionary coupling analysis to identify co-evolving residues

• Integrative approaches:

  • Combine multiple prediction methods using meta-servers

  • Incorporate experimental data as it becomes available

  • Refine predictions iteratively as more information emerges

For another mimivirus protein (L442), Phyre2 analysis suggested similarity to human ATP-dependent DNA helicase, indicating possible involvement in DNA metabolism . A similar approach might reveal functional insights for MIMI_R213.

How might post-translational modifications affect MIMI_R213 function?

Post-translational modifications (PTMs) could significantly influence MIMI_R213 function:

• Potential PTMs in mimivirus proteins:

  • Glycosylation: Some mimivirus proteins (L829 and R135) are glycosylated and function in viral fibrils

  • Phosphorylation: Could regulate activity or interactions

  • Proteolytic processing: May activate or modulate function

• Detection methods:

  • Mass spectrometry with enrichment for specific modifications

  • Western blotting with modification-specific antibodies

  • Site-directed mutagenesis of predicted modification sites

• Functional implications:

  • Localization: PTMs might direct MIMI_R213 to specific subcellular locations

  • Interactions: Modifications could modulate binding to other proteins or DNA

  • Activity: Enzymatic activity might be regulated by reversible modifications

Research has shown that glycosylated mimivirus proteins mediate adhesion to host cells through interactions with mannose and N-acetylglucosamine . If MIMI_R213 undergoes similar modifications, it might play a role in virus-host interactions.

What are the most promising research avenues for understanding MIMI_R213 function?

Several research directions hold particular promise for elucidating MIMI_R213 function:

• Comprehensive interactome analysis:

  • Identify all proteins that interact with MIMI_R213 during different stages of infection

  • Map the interaction network to place R213 in functional context

  • Determine if R213 is part of known mimivirus complexes

• Structural biology approaches:

  • X-ray crystallography or cryo-EM structures of MIMI_R213

  • Structural comparisons with proteins of known function

  • Structure-guided functional predictions

• Genetic manipulation:

  • Development of systems for targeted gene disruption in mimivirus

  • Creation of R213 mutants to assess phenotypic effects

  • Complementation studies to verify function

• Integration with systems biology:

  • Transcriptomic and proteomic profiling during infection

  • Temporal analysis of R213 expression and localization

  • Metabolic impact of R213 presence/absence

• Investigation of potential host interactions:

  • Identification of host factors that interact with R213

  • Assessment of impact on host cellular processes

  • Potential immune evasion or modulation functions

How might understanding MIMI_R213 contribute to broader knowledge of giant virus biology?

Research on MIMI_R213 has potential to advance several areas of giant virus biology:

• Evolution of viral complexity:

  • Understanding how uncharacterized proteins like R213 contribute to the sophisticated life cycle of giant viruses

  • Investigating the origins of these proteins through comparative genomics

  • Assessing whether R213 represents a repurposed cellular function or a novel viral innovation

• Virus-host interaction mechanisms:

  • Determining if R213 plays a role in host manipulation

  • Understanding how mimivirus proteins cooperate to establish successful infection

  • Identifying potential targets for intervention

• Viral factory dynamics:

  • Elucidating the organization and function of viral factories

  • Understanding protein recruitment and localization during infection

  • Clarifying the roles of viral proteins in compartmentalization

• Genome packaging innovations:

  • Contributing to knowledge of the unique genome packaging mechanisms in giant viruses

  • Understanding how proteins like R213 might interact with the packaging machinery

  • Comparative analysis with other viral and cellular systems

The study of mimivirus has already revealed remarkable parallels between its genome packaging system and prokaryotic chromosome segregation mechanisms . Further research on proteins like MIMI_R213 may uncover additional unexpected connections between these seemingly disparate biological systems.