Recombinant Acidianus filamentous virus 2 Uncharacterized protein ORF83 (ORF83)

What is Acidianus filamentous virus 2 (AFV2) and how is it classified taxonomically?

Acidianus filamentous virus 2 (AFV2) is a novel filamentous virus that infects the hyperthermophilic archaeal genus Acidianus. While it shows structural similarity to lipothrixviruses, it differs from them in its unusual terminal and core structures. Based on these distinctive features, AFV2 has been classified in a new genus, "Deltalipothrixvirus," within the family Lipothrixviridae .

The Lipothrixviridae family consists of enveloped, rod-shaped viruses with the following characteristics:

• Capsid diameter: 24-38 nm

• Length: 410-1950 nm

• Linear dsDNA genome: 15.9 to 56 kb

• Genome organized in operons

The classification of AFV2 as a separate genus was based on its distinct properties compared to other genera within Lipothrixviridae:

• Alphalipothrixvirus (e.g., TTV1)

• Betalipothrixvirus (e.g., SIFV)

• Gammalipothrixvirus (e.g., AFV1)

• Deltalipothrixvirus (e.g., AFV2)

What are the genomic features of AFV2 and where is ORF83 located?

AFV2 possesses a double-stranded DNA genome containing 31,787 bp. Notable genomic features include:

• Eight open reading frames homologous to other lipothrixviruses

• A single tRNA Lys gene containing a 12-bp archaeal intron

• A 1,008-bp repeat-rich region near the center of the genome

The genome contains approximately 51 putative ORFs (>40 amino acids), with about 70% arranged in putative operons. ORF83 is located downstream of the unusual 1,008-bp region, flanked by ORF67 upstream and ORF66 downstream .

The unusual 1,008-bp region (positions 8608 to 9615) contains:

• Two large 46-bp direct repeats

• Multiple imperfect short repeats

• Strongly biased base composition (one DNA strand containing only 7% guanosines)

• Bordered by the inverted repeat GTCACTGACATAATA

What is known about the structure and function of the ORF83 protein?

ORF83 is currently classified as an uncharacterized protein with a length of 83 amino acids . Unlike some other AFV2 proteins, ORF83 has not been assigned a specific function based on sequence matches with public databases.

While direct experimental data on ORF83 function is limited, its genomic location near a region with unusual sequence features (the 1,008-bp region) suggests it may play a role in viral replication or genome organization. The proximity to this region, which has been hypothesized to constitute a replication initiation site, may indicate ORF83's involvement in DNA replication processes .

What methods are used to express and purify recombinant ORF83 protein?

The recombinant ORF83 protein can be expressed using the following methodology:

Expression System:

• Host organism: Escherichia coli

• Expression vector: His-tagged expression vectors (such as pQE80L) are commonly used

• The full-length ORF83 protein (amino acids 1-83) is typically expressed with a His-tag for purification purposes

Purification Protocol:

• Cell Lysis: After induction of protein expression, bacterial cells are harvested and lysed using methods such as sonication or chemical lysis.

• Affinity Chromatography: The His-tagged ORF83 protein can be purified using nickel-nitrilotriacetic acid (Ni-NTA) resin.

• Protein Analysis: The purified protein is typically analyzed by:

  • SDS-PAGE to confirm size and purity

  • Western blotting using anti-His antibodies or specific antisera against viral proteins

  • Mass spectrometry to verify the protein identity

This approach is similar to methods used for other viral proteins from archaeal viruses, such as the AFV1 coat proteins (ORF132 and ORF140), which have been successfully expressed in E. coli and characterized structurally .

How can researchers detect and characterize interactions between ORF83 and other viral or host proteins?

To investigate potential protein-protein interactions involving ORF83, researchers can employ the following methodologies:

In Vitro Interaction Studies:

• Pull-down Assays: Using recombinant His-tagged ORF83 as bait to identify interacting partners from viral or host cell lysates.

• Co-immunoprecipitation (Co-IP): Using antibodies against ORF83 to precipitate protein complexes that can be further analyzed by mass spectrometry.

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics between ORF83 and potential binding partners.

In Vivo Interaction Studies:

• Yeast Two-Hybrid (Y2H) Screening: To identify potential interacting partners from genomic libraries.

• Bimolecular Fluorescence Complementation (BiFC): To visualize protein interactions in living cells.

• Proximity-based Labeling Methods: Such as BioID or APEX2, which can detect transient or weak interactions in the native cellular environment.

Structural Analysis:

• X-ray Crystallography: To determine the three-dimensional structure of ORF83 alone or in complex with interaction partners.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: Particularly useful for smaller proteins like ORF83 (83 amino acids).

• Cryo-electron Microscopy: For larger protein complexes or to visualize ORF83 in the context of the viral structure.

These approaches have been successfully applied to other archaeal viral proteins, such as the coat proteins of AFV1 which were found to interact with DNA and form filaments when incubated with linear dsDNA .

Advanced Research Questions

While direct experimental evidence for ORF83's role is limited, analysis of its genomic context and comparison with other archaeal viruses allows for hypothesis generation regarding its potential functions:

Virion Assembly Hypothesis:

Host Interaction Hypothesis:

• Regulatory Function: ORF83 could function similarly to ORF3 proteins in coronaviruses, which are known to play roles in virulence and modulation of host responses .

• Anti-defense Function: Recent research has identified viral proteins that counter host defense mechanisms. ORF83 could potentially function as an anti-defense gene (ADG) similar to those identified in rudiviruses and other archaeal viruses .

To test these hypotheses, researchers could employ the following approaches:

• Generation of ORF83 deletion mutants to assess impacts on viral replication and virion morphology

• Immunolocalization studies to determine ORF83's subcellular location during infection

• Protein-protein interaction studies to identify binding partners

• Host transcriptome/proteome analysis upon expression of ORF83

How might the unique genomic context of ORF83 influence its expression and function?

The genomic organization surrounding ORF83 has several unusual features that may influence its expression and function:

Regulatory Context:

• Proximity to the 1,008-bp Region: ORF83 is located downstream of an unusual 1,008-bp region that contains multiple repeat structures and has a strongly biased base composition. This region has been hypothesized to function as a replication initiation site .

• Potential Operonic Structure: About 70% of AFV2 ORFs are arranged in putative operons, suggesting coordinated expression. Understanding which operon contains ORF83 and which genes are co-expressed with it would provide insights into its functional context .

Expression Regulation Hypotheses:

• Transcriptional Regulation: The unusual sequence features near ORF83 might influence transcription through:

  • Formation of complex secondary structures in DNA

  • Binding sites for viral or host transcription factors

  • Effects on local DNA topology that impact transcription initiation or elongation

• Translational Regulation: Similar to the upstream ORFs in Rous sarcoma virus that modulate viral gene expression , ORF83's expression might be regulated at the translational level through mechanisms involving ribosome scanning or shunting.

Experimental Approaches to Test These Hypotheses:

• Transcriptome Analysis: RNA-Seq during different stages of AFV2 infection to determine when ORF83 is expressed and which genes show coordinated expression patterns.

• Reporter Gene Assays: Fusion of the ORF83 promoter/regulatory region to reporter genes to study the influence of the surrounding sequence context on expression.

• SHAPE Analysis (Selective 2'-hydroxyl acylation analyzed by primer extension): To determine RNA secondary structures in the ORF83 mRNA region that might influence translation.

• Ribosome Profiling: To assess translation efficiency and identify potential regulatory mechanisms at the translational level.

What challenges exist in working with recombinant archaeal viral proteins and how can they be overcome?

Researchers face several challenges when working with archaeal viral proteins like ORF83:

Expression Challenges:

• Codon Usage Bias: Archaea and their viruses often have distinct codon preferences compared to standard expression hosts like E. coli.

  • Solution: Use codon-optimized synthetic genes or specialized expression strains with rare codon tRNAs.

• Protein Folding in Mesophilic Hosts: Proteins from hyperthermophilic organisms (growing at 75°C and pH 3) may not fold properly at lower temperatures.

  • Solution: Express proteins with solubility tags (MBP, SUMO, GST) or attempt refolding from inclusion bodies. Consider expression at higher temperatures (30-37°C).

• Post-translational Modifications: Archaeal-specific modifications may be absent in bacterial expression systems.

  • Solution: Consider archaeal expression systems or cell-free protein synthesis systems supplemented with archaeal components.

Purification and Stability Challenges:

• Protein Stability: Proteins from hyperthermophiles may have unusual stability properties.

  • Solution: Add stabilizing agents (glycerol, specific ions) to buffers and consider purification at higher temperatures.

• Solubility Issues: Hydrophobic proteins or domains may aggregate during purification.

  • Solution: Use detergents or amphipathic agents for membrane-associated proteins; screen various buffer conditions.

Functional Analysis Challenges:

• In vitro Activity Assays: Archaeal proteins often function optimally at high temperatures and low pH.

  • Solution: Develop assays that can function under these extreme conditions or identify compromise conditions where function is retained.

• Lack of Genetic Tools: Limited genetic manipulation tools for archaeal virus hosts.

  • Solution: Develop heterologous systems or consider CRISPR-based approaches being developed for archaea.

How can researchers study the interaction between ORF83 and nucleic acids?

Based on the functions of other archaeal viral proteins, ORF83 may interact with nucleic acids. Several methods can be employed to investigate such interactions:

Biochemical Approaches:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified ORF83 with labeled DNA/RNA of various sequences

  • Analyze complex formation by native gel electrophoresis

  • Include competition assays with unlabeled nucleic acids to determine specificity

• Filter Binding Assays:

  • Quantify binding of labeled nucleic acids to ORF83 immobilized on filters

  • Determine binding kinetics and affinity constants

• Crosslinking Studies:

  • Use UV or chemical crosslinking to capture transient interactions

  • Analyze crosslinked complexes by mass spectrometry to identify binding sites

Biophysical Approaches:

• Isothermal Titration Calorimetry (ITC):

  • Measure thermodynamic parameters of ORF83-nucleic acid interactions

  • Determine binding stoichiometry, affinity, and thermodynamic parameters

• Surface Plasmon Resonance (SPR):

  • Real-time measurement of association and dissociation kinetics

  • Test various nucleic acid sequences to determine binding specificity

• Microscale Thermophoresis (MST):

  • Measure interactions under near-native conditions

  • Requires small amounts of sample

Structural Approaches:

• X-ray Crystallography of Complexes:

  • Determine three-dimensional structure of ORF83-nucleic acid complexes

  • Identify specific contacts and binding motifs

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Map interaction surfaces on both protein and nucleic acid

  • Study dynamics of interaction

These approaches have successfully been applied to other archaeal viral proteins, such as the AFV1 coat proteins that were shown to bind DNA and form filaments when incubated with linear dsDNA .

What are the promising avenues for future research on ORF83 and other uncharacterized archaeal viral proteins?

Several promising research directions could advance our understanding of ORF83 and similar archaeal viral proteins:

Structural and Functional Characterization:

• High-resolution Structure Determination: Solving the three-dimensional structure of ORF83 would provide insights into its function and evolutionary relationships.

• Functional Genomics Approaches:

  • CRISPR interference in archaeal hosts to assess the impact of ORF83 knockdown

  • Transposon mutagenesis to identify genetic interactions

  • Systematic protein-protein interaction mapping to place ORF83 in functional networks

• Development of In Vitro Reconstitution Systems: Establishing systems to reconstitute archaeal virus assembly or replication processes in vitro to identify the role of ORF83.

Evolutionary and Comparative Studies:

• Expanded Metagenomic Analyses: Identifying ORF83 homologs in uncharacterized archaeal viruses from extreme environments.

• Phylogenetic Profiling: Correlating the presence/absence of ORF83-like proteins with specific viral phenotypes or host ranges.

• Ancestral Sequence Reconstruction: Reconstructing ancestral sequences of ORF83-like proteins to understand their evolutionary trajectory and functional shifts.

Application-Oriented Research:

• Biotechnological Applications: Exploring the potential use of thermostable archaeal viral proteins in biotechnology, particularly for processes requiring extreme conditions.

• Development as Research Tools: Investigating whether ORF83 or related proteins could be developed as tools for molecular biology, similar to the development of CRISPR-Cas systems from microbial defense mechanisms.

How might systems biology approaches enhance our understanding of ORF83's role in viral replication and host interaction?

Systems biology approaches offer powerful frameworks for understanding the complex roles of proteins like ORF83 in viral replication cycles:

Integrative Omics Approaches:

• Multi-omics Integration: Combining transcriptomics, proteomics, and metabolomics data from AFV2-infected cells to identify networks and pathways affected by viral proteins including ORF83.

• Temporal Resolution Studies: Analyzing the dynamics of viral and host processes throughout the infection cycle to place ORF83 function in temporal context.

• Spatial Proteomics: Determining the subcellular localization of ORF83 and other viral proteins throughout infection to understand spatial organization of viral processes.

Computational Modeling:

• Protein-Protein Interaction Networks: Constructing virus-host protein interaction networks to identify the functional module containing ORF83.

• Genome-Scale Metabolic Models: Incorporating viral components into host metabolic models to understand how viruses like AFV2 redirect host resources.

• Machine Learning Approaches: Using machine learning to predict protein function based on sequence features, genomic context, and evolutionary patterns.

Host-Pathogen Interface Studies:

• CRISPR Screens in Host Cells: Identifying host factors required for ORF83 function or AFV2 replication.

• Comparative Analysis Across Host Species: Studying how ORF83 function varies when AFV2 infects different Acidianus species or strains.

• Anti-Defense Mechanisms: Investigating whether ORF83 functions in countering host defense systems, similar to recently discovered anti-defense genes in other archaeal viruses .

Such approaches could place ORF83 in the broader context of archaeal virus-host interactions and potentially reveal unexpected functions and connections.