Recombinant Acanthamoeba polyphaga mimivirus Uncharacterized protein R433 (MIMI_R433)

What is known about the R433 protein in Acanthamoeba polyphaga mimivirus?

R433 is currently classified as an uncharacterized protein in the Acanthamoeba polyphaga mimivirus proteome. Like many Mimivirus proteins, it may be part of the estimated 70% of Mimivirus genes that are either ORFans (open reading frame orphans) or have unknown functions . Preliminary sequence analysis suggests potential structural motifs that may indicate its function, but experimental validation is required. Similar to characterization efforts for other Mimivirus proteins like R458 (translation initiation factor) and gp275 (DNA architectural protein), functional studies would need to be conducted to determine its role in viral replication or structure .

How does R433 compare to other characterized proteins in Mimivirus?

While R433 remains uncharacterized, comparison to other Mimivirus proteins that have been studied can provide research direction. For example, R458 has been experimentally identified as a translation initiation factor with similarities to eukaryotic initiation factor 4a (eIF4a) . The L725 protein was identified as a fiber-associated protein through proteomics and RNA-silencing experiments . Gp275 (R252) has been characterized as an MC1-like architectural protein involved in DNA condensation . Comparative sequence analysis with these characterized proteins may reveal conserved domains or motifs in R433 that could suggest functional similarities or differences.

What bioinformatic approaches should be used for initial characterization of R433?

Initial characterization should include:

• Protein sequence analysis using multiple alignment tools

• Domain prediction using Pfam, InterPro, and SMART databases

• Secondary structure prediction using PSIPRED or similar tools

• Tertiary structure prediction using AlphaFold or similar AI-based tools

• Phylogenetic analysis to identify potential homologs in other viruses or organisms

• Temporal expression pattern analysis based on transcriptomic data, similar to how R458's expression pattern was determined to begin 3 hours post-infection

What are the optimal expression systems for recombinant production of Mimivirus R433?

Based on successful expression of other Mimivirus proteins, Escherichia coli remains the primary expression system for initial characterization. For R433 expression:

• E. coli Expression System:

  • BL21(DE3) or similar strains are recommended for high-level expression

  • Consider using pET vectors with an N-terminal His-tag for purification

  • Temperature optimization is critical, with reduced temperatures (16-18°C) often yielding better soluble protein

Similar approaches were successful for expressing Mimivirus L725 (an ORFan gene product) and putative glycosyltransferases like L193, R139, R363, R654, and R707 .

How should solubility issues with recombinant R433 be addressed?

Solubility challenges are common with viral proteins. A methodological approach includes:

• Optimization Strategies:

  • Testing multiple fusion tags (His, GST, MBP, SUMO)

  • Screening expression conditions (temperature, induction time, IPTG concentration)

  • Co-expression with chaperones (GroEL/ES, DnaK/J)

  • Testing detergents for membrane-associated proteins

• Alternative Approaches:

  • Expressing protein fragments based on domain predictions

  • Using cell-free expression systems

  • Expression in insect cells using baculovirus systems for complex proteins

The success with expressing L725 as a His-fusion protein suggests this approach may work for other Mimivirus proteins including R433 .

What purification strategy yields the highest purity and activity for Mimivirus proteins?

A multi-step purification approach is recommended:

• Initial Capture:

  • Affinity chromatography using His-tag (IMAC) similar to approaches for other Mimivirus proteins

• Intermediate Purification:

  • Ion exchange chromatography (IEX) based on theoretical pI of R433

• Polishing:

  • Size exclusion chromatography (SEC) to remove aggregates and achieve high purity

• Quality Assessment:

  • SDS-PAGE to verify purity

  • Western blotting with anti-His antibodies

  • Dynamic light scattering (DLS) to assess homogeneity

For Mimivirus L725, recognition by specific antibodies was used to confirm successful expression, suggesting immunological methods should be part of validation .

What techniques are most effective for determining the oligomeric state of R433?

Multiple complementary techniques should be employed:

• Size Exclusion Chromatography (SEC):

  • Compare elution volumes against protein standards

• Dynamic Light Scattering (DLS):

  • Measure hydrodynamic radius in solution

• Native PAGE:

  • Assess oligomeric state under non-denaturing conditions

• Analytical Ultracentrifugation (AUC):

  • Provide definitive data on molecular weight and shape

• Chemical Crosslinking coupled with Mass Spectrometry:

  • Identify interaction interfaces

This multi-technique approach is important as demonstrated with gp275, which was shown to be oligomeric through biochemical characterization .

What are the challenges in obtaining structural data for Mimivirus R433?

Several challenges must be addressed:

• Crystallization Challenges:

  • Viral proteins often have flexible regions inhibiting crystallization

  • Solution: Construct optimization by removing disordered regions predicted by bioinformatics

• Structural Heterogeneity:

  • Potential for multiple conformational states

  • Solution: Biochemical and biophysical screening to identify stabilizing conditions

• Limited Structural Homologs:

  • Many Mimivirus proteins lack structural homologs

  • Solution: Integrate cryo-EM approaches with computational prediction methods

• Sample Requirements:

  • High concentration requirements for structural studies can lead to aggregation

  • Solution: Optimize buffer conditions and use additives to improve stability

How can Cryo-EM be utilized for structural determination of R433?

Cryo-EM approaches offer advantages for viral proteins:

• Sample Preparation Optimization:

  • Protein concentration typically 0.5-5 mg/ml

  • Grid type selection (Quantifoil, C-flat)

  • Freezing conditions optimization

• Data Collection Strategy:

  • Motion correction and CTF estimation parameters

  • Collection of tilted datasets for preferred orientation issues

• Processing Workflow:

  • 2D classification to identify homogeneous particles

  • Ab initio model generation followed by 3D refinement

  • Local resolution estimation and map sharpening

• Model Building and Validation:

  • Integrating AlphaFold predictions with experimental density

  • Validation using MolProbity and EMRinger

What methods are effective for determining the function of uncharacterized Mimivirus proteins like R433?

A systematic multi-pronged approach is recommended:

• Gene Silencing Studies:

  • siRNA-based silencing similar to R458 studies to observe phenotypic effects

  • Examining viral fitness and protein expression profiles in silenced vs. wild-type viruses

• Protein-Protein Interaction Analysis:

  • Pull-down assays with viral and host proteins

  • Proximity labeling (BioID, APEX) in infected cells

  • Yeast two-hybrid screening

• Subcellular Localization:

  • Immunofluorescence microscopy with specific antibodies

  • GFP-fusion protein expression and tracking during infection

• Biochemical Activity Assays:

  • Nucleic acid binding assays (EMSA, filter binding)

  • Enzymatic activity screening (kinase, protease, glycosyltransferase)

  • Structural modification activities (like DNA bending seen with gp275)

How can RNA silencing approaches be optimized for studying R433 function?

Based on successful R458 silencing experiments, the following methodological approach is recommended:

• siRNA Design:

  • Multiple siRNA sequences targeting different regions of R433 mRNA

  • Control siRNAs with scrambled sequences

  • Validation of specificity using sequence alignment tools

• Transfection Protocol:

  • Lipofectamine-based transfection of Acanthamoeba polyphaga

  • Timing: transfection 3 hours post-infection as established for other Mimivirus genes

  • Concentration optimization through dose-response experiments

• Validation of Silencing:

  • RT-PCR to confirm reduction in target mRNA levels

  • Western blotting to confirm protein reduction if antibodies are available

• Phenotypic Analysis:

  • Growth curve analysis and viral particle production assessment

  • Microscopic examination of viral factory formation

  • Comparative proteomic analysis using 2D-DIGE to identify deregulated proteins

What advanced methods can reveal interactions between R433 and host cell components?

Understanding virus-host interactions requires sophisticated approaches:

• Proximity-dependent Biotinylation (BioID/TurboID):

  • Fusion of R433 with biotin ligase

  • Identification of proximity partners by mass spectrometry

• Co-immunoprecipitation coupled with Mass Spectrometry:

  • Pull-down of R433 complexes from infected cells

  • Identification of interaction partners

• Fluorescence Microscopy Techniques:

  • FRET to detect direct protein-protein interactions

  • FRAP to assess dynamics of R433 in infected cells

• Cross-linking Mass Spectrometry (XL-MS):

  • Capture transient interactions during infection

  • Identify interaction interfaces at amino acid resolution

• Cryo-Electron Tomography:

  • Visualize R433 in the context of infected cells or viral particles

  • Correlative light and electron microscopy for targeted analysis

How should phylogenetic analysis of R433 be approached?

A comprehensive phylogenetic analysis requires:

• Sequence Collection:

  • BLAST searches against viral, bacterial, archaeal, and eukaryotic databases

  • Inclusion of proteins with similar domain architecture

• Multiple Sequence Alignment:

  • Use of MUSCLE, MAFFT, or T-Coffee algorithms

  • Manual curation of alignments to improve quality

• Phylogenetic Tree Construction:

  • Maximum likelihood methods (RAxML, IQ-TREE)

  • Bayesian inference (MrBayes)

  • Selection of appropriate evolutionary models

• Tree Visualization and Interpretation:

  • Mapping of functional domains onto phylogenetic trees

  • Identification of potential horizontal gene transfer events

  • Assessment of selection pressures using dN/dS analysis

This approach can help determine if R433 is an ORFan unique to Mimivirus or has homologs in other organisms, similar to analyses done for R458 and gp275 .

What can comparative analysis with other giant viruses reveal about R433?

Comparative genomics approaches provide evolutionary context:

• Ortholog Identification:

  • Systematic search for R433 orthologs in other Mimiviridae and NCLDV families

  • Analysis of synteny to identify genomic context conservation

• Conservation Analysis:

  • Identification of conserved motifs and domains

  • Assessment of selection pressures on different protein regions

• Expression Timing Comparison:

  • Analysis of transcription timing in different viral species

  • Correlation with virus replication cycle phases

• Functional Inference:

  • Transfer of functional annotations from characterized homologs

  • Prediction of biological roles based on evolutionary patterns

How can structural information about R433 contribute to understanding Mimivirus assembly?

Structural insights can illuminate viral assembly mechanisms:

• Interface Analysis:

  • Identification of protein-protein interaction surfaces

  • Docking simulations with potential binding partners

• Structural Comparison:

  • Comparison with architectural proteins like gp275

  • Assessment of potential roles in genome packaging or capsid assembly

• In situ Structural Studies:

  • Cryo-electron tomography of infected cells

  • Subtomogram averaging to visualize R433 in cellular context

• Structure-guided Mutagenesis:

  • Design of mutations to disrupt specific functions

  • Assessment of effects on viral assembly and maturation

What experimental approaches can determine if R433 is involved in host-range determination?

Host-range determination studies require:

• Comparative Infection Assays:

  • Testing infection efficiency in different Acanthamoeba species

  • Assessment of entry, replication, and release in permissive vs. non-permissive hosts

• Chimeric Virus Construction:

  • Generation of recombinant viruses with R433 variants

  • Assessment of host-range alterations

• Host Factor Identification:

  • Pull-down assays with host proteins from different amoeba species

  • Yeast two-hybrid screening against host protein libraries

• CRISPR-based Host Modification:

  • Modification of potential host receptors

  • Assessment of R433 interaction with modified host factors

How can biophysical techniques determine if R433 interacts with nucleic acids?

Multiple complementary approaches should be employed:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Testing binding to different nucleic acid substrates (dsDNA, ssDNA, RNA)

  • Determining binding specificity through competition assays

• Microscale Thermophoresis (MST):

  • Quantitative measurement of binding affinities

  • Assessment of binding under various buffer conditions

• Surface Plasmon Resonance (SPR):

  • Real-time binding kinetics determination

  • Association and dissociation rate constants measurement

• DNA/RNA Protection Assays:

  • Nuclease footprinting to identify binding sites

  • Analysis of structural changes upon binding (similar to DNA bending by gp275)

• Atomic Force Microscopy (AFM):

  • Direct visualization of R433-nucleic acid complexes

  • Assessment of structural changes upon binding