Recombinant Acanthamoeba polyphaga mimivirus Uncharacterized protein L374 (MIMI_L374), partial

What is Acanthamoeba polyphaga mimivirus and its significance in virology research?

Acanthamoeba polyphaga mimivirus belongs to the Mimiviridae family of nucleocytoplasmic large DNA viruses (NCLDV). It possesses a double-stranded DNA genome that replicates primarily in eukaryotic hosts. The virus has garnered significant attention in virology due to its unusually large genome size and complexity, including genes encoding components of the protein translation machinery typically absent in viruses. Recent metagenomic studies have detected Mimiviridae, including Acanthamoeba polyphaga mimivirus, in clinical samples from upper respiratory tracts of patients, comprising up to 53% of Mimiviridae reads in some samples, suggesting potential clinical relevance beyond its known amoeba host . This virus represents a unique model for studying the evolution of viral genomes and their interaction with host cellular machinery.

How does MIMI_L374 relate to other characterized mimivirus proteins?

MIMI_L374 is situated in proximity to the well-characterized L375 gene in the mimivirus genome. While MIMI_L374 remains uncharacterized, its genomic neighbor L375 encodes a Nudix enzyme that specifically hydrolyzes the 5' m7GpppN mRNA cap, releasing m7GDP as a product . This proximity suggests potential functional relationships, as viral genes in close proximity often participate in related processes. The L375 Nudix enzyme shows sequence similarity to ASFV-DP and eukaryotic Dcp2, which possess mRNA decapping activity, a process thought to accelerate viral and host mRNA turnover and potentially promote shutdown of host protein synthesis . This genomic context provides a starting point for hypothesizing MIMI_L374's potential role in viral RNA metabolism or host-pathogen interactions.

What bioinformatic approaches can help predict the function of uncharacterized mimivirus proteins like MIMI_L374?

For uncharacterized proteins like MIMI_L374, a systematic bioinformatic approach should begin with:

• Sequence-based analysis:

  • Multiple sequence alignment with homologs from other giant viruses

  • Motif identification using databases like PROSITE, PFAM, and InterPro

  • Secondary structure prediction with PSIPRED or JPred

• Structural prediction:

  • Tertiary structure modeling using AlphaFold2 or RoseTTAFold

  • Fold recognition through I-TASSER or Phyre2

  • Active site prediction using CASTp or POCASA

• Functional inference:

  • Gene neighborhood analysis examining nearby genes like L375

  • Co-expression patterns during viral infection

  • Protein-protein interaction network prediction

Given the known function of L375 as an mRNA decapping enzyme , particular attention should be paid to potential roles in RNA metabolism, host translation control, or complementary activities in viral replication cycles.

What expression systems are optimal for recombinant production of mimivirus proteins like MIMI_L374?

The optimal expression system for mimivirus proteins depends on the specific protein's characteristics and research objectives. Based on established protocols for viral proteins:

For MIMI_L374, an initial screening in E. coli with fusion tags (His, MBP, or GST) is recommended, with progression to insect cell systems if solubility issues arise . The proximity to L375, which has been successfully expressed as a recombinant protein for enzymatic studies , suggests that bacterial expression may be feasible for MIMI_L374.

How can expression of recombinant MIMI_L374 be optimized to improve yield and solubility?

Optimization strategies for recombinant MIMI_L374 expression should include:

• Codon optimization: Adjust codon usage based on the expression host to enhance translation efficiency.

• Fusion tag selection: Test multiple fusion partners:

  • MBP (maltose-binding protein) and SUMO tags often enhance solubility

  • GST and His tags facilitate purification

  • Thioredoxin (TrxA) can improve folding

• Expression conditions optimization:

  • Temperature reduction (16-25°C) after induction to slow folding

  • IPTG concentration titration (0.1-1.0 mM)

  • Expression time optimization (4-24 hours)

  • Media supplementation with rare amino acids if limiting

• Co-expression with chaperones:

  • GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor

Based on recombinant protein expression services data, a systematic approach testing multiple expression conditions can achieve success rates exceeding 95% for challenging proteins .

What purification strategy would yield the highest purity MIMI_L374 for structural and functional studies?

A multi-step purification strategy is recommended for high-purity MIMI_L374:

Step 1: Initial capture

• Affinity chromatography based on fusion tag (e.g., IMAC for His-tagged protein)

• Aim for >80% purity at this stage

Step 2: Intermediate purification

• Ion exchange chromatography (choice dependent on theoretical pI of MIMI_L374)

• Hydrophobic interaction chromatography (HIC)

Step 3: Polishing

• Size exclusion chromatography to achieve >95% purity

• Remove aggregates and separate oligomeric states

Step 4: Tag removal (if required)

• TEV, PreScission, or 3C protease cleavage

• Secondary affinity chromatography to remove cleaved tag

Step 5: Final QC

• SDS-PAGE and Western blotting for purity assessment

• Mass spectrometry for identity confirmation

This strategy aligns with approaches used for similar viral proteins, with expected yields of 1-5 mg per liter of culture for bacterial systems and potentially lower yields for eukaryotic systems .

What biochemical assays could determine if MIMI_L374 possesses enzymatic activity?

Given the genomic proximity to L375, which exhibits mRNA decapping activity , and the context of mimivirus translation-related proteins , several targeted biochemical assays should be considered:

• Nuclease/phosphatase activity screening:

  • Test activity against various RNA substrates

  • Monitor nucleotide release using HPLC or coupled enzymatic assays

  • Examine pH and metal ion dependence (particularly Mg²⁺ and Mn²⁺)

• RNA binding assays:

  • Electrophoretic mobility shift assays (EMSA)

  • Surface plasmon resonance (SPR)

  • Fluorescence anisotropy with labeled RNA

• Translation-related activities:

  • In vitro translation inhibition/enhancement assays

  • Interaction with ribosomal components or translation factors

  • Aminoacyl-tRNA interaction assays

• Protein interaction studies:

  • Pull-down assays with L375 and other neighboring proteins

  • Yeast two-hybrid or mammalian two-hybrid screening

  • Co-immunoprecipitation with viral and host factors

The experimental design should include appropriate negative controls and may benefit from comparison with the known L375 activity on capped RNA substrates .

How can I investigate the temporal expression pattern of MIMI_L374 during the mimivirus replication cycle?

Investigating the temporal expression of MIMI_L374 requires a systematic approach:

• Infection time course experiment:

  • Infect Acanthamoeba cells with mimivirus

  • Collect samples at multiple time points (e.g., 0, 2, 4, 8, 12, 16, 24 hours post-infection)

• RNA analysis methods:

  • RT-qPCR targeting MIMI_L374 mRNA

  • Northern blotting for transcript size determination

  • RNA-Seq for transcriptome-wide context

• Protein analysis methods:

  • Western blotting using antibodies against recombinant MIMI_L374

  • Mass spectrometry-based proteomics at different time points

  • Immunofluorescence microscopy for localization

• Comparative analysis:

  • Compare with known early, intermediate, and late viral genes

  • Analyze alongside L375 expression patterns

  • Correlate with viral DNA replication phases

This approach can be adapted based on experimental conditions similar to those used for studying mimivirus translation-related genes under different nutritional conditions , which revealed distinct expression profiles depending on environmental factors.

What cellular localization techniques would be most informative for understanding MIMI_L374 function?

To determine the subcellular localization of MIMI_L374 during infection, employ multiple complementary techniques:

• Immunofluorescence microscopy:

  • Generate specific antibodies against recombinant MIMI_L374

  • Co-stain with markers for viral factories, host organelles

  • Use super-resolution techniques (STED, STORM) for detailed localization

• Biochemical fractionation:

  • Separate nuclear, cytoplasmic, membrane, and viral factory fractions

  • Perform Western blotting on fractions

  • Use gradient centrifugation for refined separation

• Live-cell imaging:

  • Create fluorescent protein fusions (if viable)

  • Monitor dynamics during infection

  • Perform FRAP (Fluorescence Recovery After Photobleaching) for mobility assessment

• Electron microscopy approaches:

  • Immunogold labeling for transmission EM

  • Correlative light and electron microscopy (CLEM)

When designing these experiments, consideration should be given to potential artifacts from overexpression or tag interference. Controls should include known viral proteins with established localization patterns, such as capsid proteins or replication factors.

How might MIMI_L374 interact with host translation machinery based on what we know about other mimivirus proteins?

Analysis of potential interactions between MIMI_L374 and host translation machinery should consider several approaches based on knowledge of mimivirus translation-related proteins:

• Comparative genomic context:
  Mimivirus encodes components of the translation machinery, including tRNAs (leucyl, histidyl, cysteinyl, and tryptophanyl) and aminoacyl-tRNA synthetases (methionyl, arginyl, tyrosyl, and cysteinyl) . This suggests potential roles for proteins like MIMI_L374 in modulating or complementing these functions.

• Potential interaction mechanisms:

  • Direct binding to host ribosomes or translation factors

  • Modification of host mRNAs (similar to L375's decapping activity)

  • Competition with host factors for binding sites

  • Alteration of tRNA modification or aminoacylation

• Experimental approach:

  • Ribosome binding assays with purified components

  • Polysome profiling during infection with/without L374 knockdown

  • In vitro translation assays with addition of recombinant MIMI_L374

  • SILAC-based proteomics to identify binding partners

• Translational impact analysis:
  Analysis of amino acid usage patterns between Acanthamoeba and mimivirus isolates has revealed distinct profiles , suggesting specialized adaptation of viral translation machinery that proteins like MIMI_L374 might influence.

What CRISPR-based approaches could be employed to study the function of MIMI_L374 in the context of viral infection?

CRISPR-based approaches offer powerful tools for studying MIMI_L374 function:

• Genome editing of the mimivirus genome:

  • Design sgRNAs targeting MIMI_L374

  • Create knockout, knockdown, or tagged versions

  • Introduce point mutations in predicted functional domains

• Technical considerations:

  • Delivery methods for CRISPR-Cas9 components into viral factories

  • Selection of edited viral variants

  • Verification of edits through sequencing

• Phenotypic analysis of edited viruses:

  • One-step growth curves to assess replication efficiency

  • Electron microscopy for virion morphology

  • Transcriptome analysis for effects on gene expression

  • Proteomics for altered protein composition

• Host factor identification:

  • CRISPR screens in host cells to identify factors interacting with MIMI_L374

  • CRISPRi/CRISPRa to modulate host genes potentially affected by MIMI_L374

This strategy would need to account for challenges in manipulating the large mimivirus genome and the potential for compensatory mechanisms through functionally redundant proteins.

How can structural biology approaches contribute to understanding MIMI_L374 function?

Structural biology approaches offer critical insights into protein function, particularly for uncharacterized proteins like MIMI_L374:

• X-ray crystallography workflow:

  • High-purity protein preparation (>95%)

  • Crystallization screening (vapor diffusion, batch methods)

  • Data collection at synchrotron facilities

  • Structure determination and refinement

• Cryo-electron microscopy:

  • Single-particle analysis for protein complexes

  • Sub-3Å resolution achievable for well-behaved samples

  • Visualization of MIMI_L374 in complex with potential binding partners

• NMR spectroscopy:

  • Solution structure determination for smaller domains

  • Dynamics studies to identify flexible regions

  • Binding site mapping with isotopically labeled ligands

• Integrated structural approach:

  • Combining experimental data with computational modeling

  • Molecular dynamics simulations to study conformational changes

  • Virtual screening for potential inhibitors

The structural information could be particularly valuable in comparison with L375, which possesses Nudix enzyme activity for mRNA decapping . Structural similarities or differences could provide insights into whether MIMI_L374 has related functions in RNA metabolism or entirely different roles.

What strategies can overcome expression challenges when MIMI_L374 forms inclusion bodies?

When MIMI_L374 forms inclusion bodies, consider this systematic approach to recover properly folded protein:

• Prevention strategies:

  • Lower expression temperature (16°C)

  • Reduce inducer concentration

  • Use slower promoters (trc vs. T7)

  • Co-express with molecular chaperones

  • Fusion to solubility enhancers (MBP, SUMO, TrxA)

• Solubilization methods:

  • Mild detergents (0.1% Triton X-100)

  • High salt conditions (300-500 mM NaCl)

  • Solubility screening with different buffers and additives

• Refolding protocols:

  • Gradual dilution method

  • Dialysis with decreasing denaturant concentration

  • On-column refolding during affinity purification

  • Pulsatile refolding with redox conditions for disulfide formation

Successful protein renaturation from inclusion bodies can achieve functional protein with purity exceeding 85%, as demonstrated in custom recombinant protein services . For MIMI_L374, the refolding strategy should consider the protein's predicted secondary structure and any known functional domains.

How can RNA contamination be managed when purifying RNA-binding proteins like potentially MIMI_L374?

Managing RNA contamination for potential RNA-binding proteins like MIMI_L374 requires specific strategies:

• Prevention during extraction:

  • High salt extraction buffers (500-750 mM NaCl)

  • Addition of nuclease inhibitors (EDTA, DTT, β-mercaptoethanol)

  • RNase-free reagents and equipment

• Removal during purification:

  • Strategic RNase A treatment (0.1-0.5 mg/ml) at specific steps

  • Polyethyleneimine precipitation (0.15-0.5%) to selectively remove nucleic acids

  • Heparin affinity chromatography as a dual purification/RNA removal step

• Quality control methods:

  • A260/A280 ratio monitoring (<1.0 for pure protein)

  • Agarose gel analysis for RNA contamination

  • TBE-Urea PAGE for small RNA detection

If contaminating RNA cannot be removed without affecting protein function, consider characterizing the bound RNA through sequencing, which may provide functional insights. This approach proved valuable in studying the L375 Nudix enzyme, which showed specific interaction with RNA substrates .

What control experiments are essential when characterizing the biochemical activity of MIMI_L374?

Essential control experiments for biochemical characterization of MIMI_L374 include:

• Negative controls:

  • Heat-inactivated MIMI_L374

  • Catalytic site mutant (based on bioinformatic prediction)

  • Unrelated protein of similar size/properties

  • Buffer-only reactions

• Positive controls:

  • Known enzymes with similar predicted functions

  • L375 protein (if RNA interaction is suspected)

  • Commercial enzymes that perform expected reactions

• Specificity controls:

  • Substrate range testing (various RNA structures, lengths)

  • Competition assays with unlabeled substrates

  • Testing at different pH, temperature, and ion conditions

• Validation experiments:

  • Multiple detection methods for the same activity

  • Dose-dependency analysis

  • Kinetic parameter determination (Km, Vmax)

  • Inhibitor studies

These controls are particularly important given the uncharacterized nature of MIMI_L374 and should be designed based on any predicted functions from bioinformatic analysis and its genomic context near L375 .

How might systems biology approaches advance our understanding of MIMI_L374's role in mimivirus infection?

Systems biology offers integrative approaches to understanding MIMI_L374's function:

• Multi-omics integration:

  • Transcriptomics, proteomics, metabolomics during infection

  • Network analysis to place MIMI_L374 in functional pathways

  • Temporal correlation analysis with other viral and host factors

• Modeling approaches:

  • Constraint-based models of virus-host metabolism

  • Dynamic models of viral replication incorporating MIMI_L374

  • Protein-protein interaction network simulations

• High-throughput phenotypic analyses:

  • CRISPR screens of host factors

  • Chemical genomics to identify modulators of MIMI_L374 activity

  • Synthetic genetic array analysis if yeast models are applicable

• Data integration framework:

  • Machine learning approaches to predict functional interactions

  • Literature mining for related viral proteins

  • Evolutionary analysis across the Mimiviridae family

This systems-level understanding would contextualize MIMI_L374 within the broader virus-host interaction network, potentially revealing its role in mimivirus infection dynamics and host response modulation.

What are the implications of Mimiviridae detection in human respiratory samples for MIMI_L374 research?

The detection of Mimiviridae, including Acanthamoeba polyphaga mimivirus, in human respiratory samples opens new research directions for MIMI_L374:

• Clinical relevance investigations:

  • Expression analysis of MIMI_L374 in clinical samples

  • Correlation with disease severity and outcomes

  • Potential biomarker development

• Host range considerations:

  • MIMI_L374 interaction with human cell components

  • Functionality in different cellular environments

  • Comparison with amoeba-specific interactions

• Immunological aspects:

  • Antigenic properties of MIMI_L374

  • Potential immune recognition and response

  • Cross-reactivity with human proteins

• Methodological approaches:

  • Development of specific detection methods for MIMI_L374 in clinical samples

  • Cell culture models using respiratory epithelial cells

  • Animal models for mimivirus respiratory infection

Recent metagenomic studies showing Mimiviridae comprising up to 24% of virome reads in respiratory samples highlight the importance of understanding proteins like MIMI_L374 in potential human health contexts.

How can evolutionary analysis of MIMI_L374 homologs inform functional predictions?

Evolutionary analysis provides a powerful framework for functional prediction:

• Phylogenetic profiling:

  • Identification of homologs across Mimiviridae and other NCLDV families

  • Conservation analysis of key residues

  • Correlation with viral adaptation to different hosts

• Evolutionary rate analysis:

  • Calculation of dN/dS ratios to identify selection pressures

  • Identification of rapidly evolving domains

  • Coevolution analysis with interacting proteins

• Domain architecture comparison:

  • Analysis of domain gain, loss, or shuffling events

  • Identification of lineage-specific insertions or deletions

  • Correlation with functional adaptation

• Ancestral sequence reconstruction:

  • Inference of ancestral MIMI_L374-like proteins

  • Experimental characterization of reconstructed proteins

  • Tracing functional evolution through time

This approach could reveal whether MIMI_L374 represents a conserved function across giant viruses or a specialized adaptation in Mimivirus, potentially informing both basic virology research and applications in synthetic biology or biotechnology.