Recombinant Acanthamoeba polyphaga mimivirus Putative ankyrin repeat protein L371 (MIMI_L371), partial

What is Acanthamoeba polyphaga mimivirus and its significance in research?

Acanthamoeba polyphaga mimivirus is a giant virus first discovered in 1992 and formally characterized in 2003. It's significant in research due to its exceptionally large genome (approximately 1.2 million base pairs) and its unique position between viruses and cellular organisms. The mimivirus genome encodes numerous proteins that share homology with those found in eukaryotes, bacteria, and archaea, making it a valuable model for studying viral evolution and host-pathogen interactions . The virus infects Acanthamoeba species, which are free-living amoebae found in diverse environments. Mimiviruses challenge traditional definitions of viruses and provide insights into the evolution of cellular life and viruses.

What are ankyrin repeat proteins and why are they important in mimivirus?

Ankyrin repeat proteins contain multiple copies of a conserved structural motif (the ankyrin repeat) that forms helix-turn-helix structures involved in protein-protein interactions. In mimivirus, ankyrin repeats represent the most common motif shared with humans, as evidenced by genomic comparison studies . These proteins are important because they likely mediate interactions between viral and host proteins during infection. The abundance of ankyrin repeat proteins in mimivirus (with at least 81 proteins containing these repeats according to current analyses) suggests they play crucial roles in virus-host interactions, potentially modulating host cellular processes to benefit viral replication and survival .

How does MIMI_L371 compare to other ankyrin repeat proteins in the mimivirus genome?

MIMI_L371 is one of numerous ankyrin repeat proteins encoded by the mimivirus genome. While specific comparative data on MIMI_L371 is limited in the provided search results, genomic analyses have identified a total of at least 81 mimiviral proteins containing ankyrin repeats (48 previously known plus 33 newly identified) . These proteins likely form part of a functional network that mediates virus-host interactions.

The comparison of ankyrin repeat proteins can be visualized in the following table:

What are the optimal expression systems for producing recombinant MIMI_L371 protein?

For expressing recombinant mimivirus proteins, Escherichia coli has proven to be an effective heterologous expression system, particularly for certain mimivirus proteins. Based on research with other mimiviral proteins, E. coli expression systems using vectors such as pET or pGEX offer good protein yields . Unlike some human proteins that express poorly in bacterial systems, certain mimivirus proteins like R699 have shown high expression levels in E. coli, suggesting this might be a suitable system for MIMI_L371 as well .

For optimal expression:

• Select an appropriate expression vector (pET series for high-yield expressions or pGEX for GST-fusion proteins that enhance solubility)

• Transform E. coli strains optimized for protein expression (BL21(DE3), Rosetta, or Arctic Express for proteins that require lower temperature cultivation)

• Optimize induction conditions by testing various IPTG concentrations (0.1-1.0 mM) and induction temperatures (16-37°C)

• Consider co-expression with chaperones if initial expression yields insoluble protein

For proteins that express poorly in E. coli, alternative systems like insect cell lines (Sf9 or High Five cells with baculovirus expression systems) or mammalian cell lines (HEK293 or CHO cells) may be considered, though these typically have lower yields and higher costs.

What purification strategies are most effective for mimivirus ankyrin repeat proteins?

Purification of mimivirus ankyrin repeat proteins requires careful consideration of their structural properties. Based on experiences with similar proteins, the following multi-step purification protocol is recommended:

• Initial capture: Affinity chromatography using tags such as His6, GST, or MBP. His-tagged purification with Ni-NTA resin is often preferred for its efficiency and mild elution conditions.

• Intermediate purification: Ion exchange chromatography based on the protein's theoretical isoelectric point. Ankyrin repeat domains typically have distinct charge distributions that enable effective separation.

• Polishing: Size exclusion chromatography to achieve high purity and remove aggregates, which is particularly important for structural studies.

For MIMI_L371 specifically, maintaining buffer conditions that preserve protein stability throughout purification is critical. Consider including reducing agents (1-5 mM DTT or 1-2 mM β-mercaptoethanol) to prevent disulfide bond formation and protease inhibitors to minimize degradation. Ankyrin repeat proteins can be prone to aggregation, so including stabilizing agents like glycerol (5-10%) in buffers may improve yields of functional protein.

How can researchers validate the structural integrity of purified MIMI_L371?

Validating structural integrity of purified MIMI_L371 requires multiple complementary techniques:

• Circular Dichroism (CD) Spectroscopy: Ankyrin repeat domains have characteristic α-helical content that produces distinctive CD spectra with minima at 208 and 222 nm. This technique provides rapid assessment of secondary structure integrity.

• Thermal Shift Assays: These measure protein stability through fluorescence changes as the protein unfolds with increasing temperature. A clear, single melting transition suggests a homogeneous, properly folded protein.

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): This technique verifies the oligomeric state and homogeneity of the purified protein.

• Limited Proteolysis: Properly folded proteins show resistance to proteolytic digestion compared to misfolded variants. Time-course digestion with proteases like trypsin or chymotrypsin followed by SDS-PAGE analysis can reveal structural features.

• Functional Binding Assays: If potential binding partners are known, interaction studies using techniques such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), or pull-down assays can confirm that the protein maintains its binding capacity.

How can MIMI_L371 be investigated for potential interactions with host proteins?

Investigating MIMI_L371's interactions with host proteins requires a systematic approach combining computational predictions with experimental validation:

• In silico prediction:

  • Homology modeling of MIMI_L371 structure based on known ankyrin repeat proteins

  • Molecular docking with potential human protein partners identified through homology with other viral ankyrin repeat proteins

  • Network analysis based on the functional clusters identified in human-mimivirus protein homology studies

• Pull-down assays:

  • Express tagged MIMI_L371 (GST or His-tagged)

  • Incubate with human cell lysates (preferably from amoeba host cells or human cell lines)

  • Identify binding partners through mass spectrometry

  • Validate top candidates with reciprocal pull-downs

• Yeast two-hybrid (Y2H) screening:

  • Use MIMI_L371 as bait against human or amoeba cDNA libraries

  • Validate positive interactions through secondary screening

  • Perform domain mapping to identify specific interaction regions

• Proximity labeling in living cells:

  • Express MIMI_L371 fused to BioID or APEX2 in relevant cell types

  • Identify proximal proteins through streptavidin pull-down and mass spectrometry

  • Compare the interactome of MIMI_L371 with those of other mimivirus ankyrin repeat proteins

These approaches should be complementary, as each has distinct strengths and limitations. The combined data can provide a comprehensive understanding of MIMI_L371's interaction network within host cells.

What functional assays can be used to determine the biological role of MIMI_L371 during mimivirus infection?

Determining the biological role of MIMI_L371 during infection requires multiple functional approaches:

• Temporal expression analysis:

  • Quantify MIMI_L371 mRNA and protein levels at different stages of infection using RT-qPCR and western blotting

  • Determine if MIMI_L371 is an early or late gene, indicating potential roles in host manipulation or virion assembly

• Subcellular localization studies:

  • Express fluorescently tagged MIMI_L371 in host cells

  • Track localization patterns throughout infection using confocal microscopy

  • Co-localize with cellular markers to identify targeted compartments or processes

• Gene knockout or knockdown studies:

  • Generate MIMI_L371-deficient mimivirus using CRISPR-Cas or similar genome editing techniques

  • Compare replication efficiency, virion production, and host response between wild-type and mutant viruses

  • Perform complementation studies to confirm phenotype specificity

• Host signaling pathway analysis:

  • Express MIMI_L371 in host cells independently of infection

  • Examine changes in key signaling pathways using phospho-specific antibodies or reporter assays

  • Compare with changes induced by whole virus infection to determine contribution

• Immunoprecipitation followed by mass spectrometry (IP-MS):

  • Pull down MIMI_L371 from infected cells at different time points

  • Identify co-precipitating proteins and post-translational modifications

  • Map dynamic changes in the MIMI_L371 interactome throughout infection

These complementary approaches can provide insights into both the molecular mechanism and biological significance of MIMI_L371 during the mimivirus infection cycle.

How might the structure of MIMI_L371 inform its potential functions?

The structure of MIMI_L371, as an ankyrin repeat protein, provides significant insights into its potential functions:

• Ankyrin repeat domain architecture:

  • Each ankyrin repeat typically consists of 33 amino acids forming a helix-turn-helix structure

  • Multiple repeats stack together to create a concave binding surface

  • The number of repeats and their arrangement influence binding specificity and affinity

• Structure prediction and molecular modeling:

  • Homology modeling based on known ankyrin repeat protein structures

  • Identification of conserved residues that may be crucial for structural integrity

  • Mapping of variable regions that likely determine binding specificity

• Structural comparison with human ankyrin proteins:

  • Comparing binding surfaces may reveal mimicry of host proteins

  • Identifying unique features that could represent virus-specific adaptations

  • Predicting potential competitive binding with host ankyrin-dependent interactions

• Correlation between structure and evolutionary conservation:

  • Highly conserved residues across mimivirus strains likely indicate structural importance

  • Variable regions may reflect adaptation to different hosts or immune evasion strategies

Understanding these structural features can guide the design of functional experiments, including site-directed mutagenesis of key residues to disrupt specific interactions or functions.

How can MIMI_L371 be utilized in studying human-virus protein interactions?

MIMI_L371 offers several valuable applications for studying human-virus protein interactions:

• Model system for ankyrin repeat-mediated interactions:

  • The abundance of ankyrin repeat proteins in both humans and mimiviruses makes MIMI_L371 an excellent model for studying how these domains mediate cross-species interactions

  • Comparative studies with human ankyrin proteins can reveal convergent evolutionary strategies

• Investigation of mimivirus-human protein homology:

  • Building on the established homology between mimivirus and human proteins , MIMI_L371 can serve as a probe for identifying novel human protein functions

  • The interactive genome-wide comparison website (https://guolab.shinyapps.io/app-mimivirus-publication/) can facilitate identification of human homologs

• Development of protein interaction inhibitors:

  • Characterizing MIMI_L371's interaction interfaces could guide the design of peptides or small molecules that disrupt viral-host protein interactions

  • These inhibitors could serve as tools for studying infection mechanisms and potentially as templates for antiviral therapeutics

• Understanding host range determination:

  • Comparing MIMI_L371's interaction with proteins from permissive and non-permissive hosts may reveal determinants of host range

  • This could enhance our understanding of virus-host co-evolution and species barriers

These applications highlight how MIMI_L371 research extends beyond understanding mimivirus biology to broader questions in protein-protein interactions and host-pathogen relationships.

What are the challenges in studying the evolutionary relationship between mimivirus ankyrin repeat proteins and their human homologs?

Studying the evolutionary relationship between mimivirus ankyrin repeat proteins and human homologs presents several significant challenges:

• Sequence divergence and convergence issues:

  • Ankyrin repeats have relatively short consensus sequences, making it difficult to distinguish homology from convergent evolution

  • Standard sequence alignment tools may fail to detect distant relationships between viral and human proteins

  • Specialized approaches like Domain Enhanced Lookup Time Accelerated BLAST (DELTA-BLAST) are required for accurate identification

• Complex evolutionary history:

  • The origin of mimivirus genes remains controversial (horizontal gene transfer vs. ancient common ancestry)

  • Determining whether mimivirus acquired ankyrin repeat domains from hosts or vice versa

  • Accounting for different evolutionary rates between viral and host proteins

• Functional validation challenges:

  • Sequence similarity does not guarantee functional similarity

  • Experimental confirmation of homologous functions is resource-intensive

  • Differences in cellular context between mimivirus and humans may affect protein function

• Structural analysis limitations:

  • Few mimivirus protein structures have been experimentally determined

  • Reliance on computational modeling introduces uncertainties

  • Subtle structural differences may have significant functional consequences

These challenges necessitate an integrated approach combining advanced computational methods, experimental validation, and careful interpretation of results.

How might genomic and proteomic approaches be combined to better understand the role of MIMI_L371?

An integrated genomic and proteomic approach offers powerful insights into MIMI_L371's role:

• Comparative genomics strategy:

  • Analyze MIMI_L371 conservation across different mimivirus strains and related giant viruses

  • Identify selective pressures through dN/dS ratio analysis

  • Map genetic variations to predicted functional domains

  • Apply methods similar to those used in the human-mimivirus homology studies

• Transcriptomics integration:

  • RNA-seq of host cells during mimivirus infection to track expression patterns

  • Correlate MIMI_L371 expression with changes in host gene expression

  • Identify co-expressed viral genes for potential functional relationships

• Proteomics approaches:

  • Global proteome analysis of mimivirus-infected cells with and without functional MIMI_L371

  • Quantitative proteomics to identify proteins affected by MIMI_L371 expression

  • Post-translational modification mapping to understand regulation

  • Protein-protein interaction network construction using affinity purification-mass spectrometry

• Structural proteomics:

  • Cryo-electron microscopy of MIMI_L371 complexes with binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Cross-linking mass spectrometry to capture transient interactions

• Integrated data analysis:

  • Network modeling combining genomic, transcriptomic, and proteomic data

  • Machine learning approaches to predict function from integrated datasets

  • Visualization tools similar to the interactive genome-wide comparison website

This multi-omics approach can overcome limitations of individual techniques and provide a comprehensive understanding of MIMI_L371's biological context and function.