Recombinant Acidianus filamentous virus 2 Uncharacterized protein ORF413 (ORF413)

How does ORF413 compare structurally to other proteins in Acidianus filamentous viruses?

Although the specific structure of ORF413 has not been fully resolved, research on related proteins from Acidianus filamentous viruses provides valuable comparative insights:

• The major coat proteins (MCPs) of Acidianus filamentous virus 1 (AFV1) display a helical fold with a four-helix-bundle structure that is also found in coat proteins from Sulfolobus islandicus rod-shaped virus (SIRV) .

• The crystal structure of ORF157 from AFV1 reveals an α+β protein with a novel fold remotely resembling nucleotidyltransferase topology, suggesting potential enzymatic activity .

• Highly conserved ORFs among archaeal viruses, such as ORF109 of lipothrixvirus AFV3 and ORFB116 from STIV, are believed to be DNA-binding proteins functioning in transcriptional regulation .

These structural insights from related viral proteins suggest that ORF413 may have a specialized structure adapted to extreme conditions, possibly with DNA-binding capabilities or enzymatic functions.

What expression systems are recommended for producing recombinant AFV2 ORF413?

Based on current research practices with archaeal viral proteins, the following expression systems are recommended:

E. coli Expression System:

• Widely used for recombinant AFV2 ORF413 production with successful expression in strains like Rosetta(DE3)pLysS .

• Typically uses vectors with strong promoters such as T7 or similar to those used for expressing AFV1 proteins .

• Expression protocol typically involves:

  • Transformation into an appropriate E. coli strain

  • Overnight induction with 1 mM IPTG at 25°C

  • Cell harvesting by centrifugation (4,000 × g for 10 min)

Expression Optimization Tips:

• Lower temperature (16-25°C) during induction may improve protein folding

• Addition of rare codon tRNAs (using strains like Rosetta) improves expression of archaeal proteins

• Consider fusion tags that enhance solubility (MBP, SUMO, etc.) in addition to His-tag for purification

What purification protocols yield the highest purity of recombinant ORF413?

A multi-step purification strategy similar to that used for other archaeal viral proteins is recommended:

Step 1: Affinity Chromatography

• Immobilized metal affinity chromatography (IMAC) using a nickel column (e.g., 5 ml His-Trap) is effective for His-tagged ORF413 .

• Purification by FPLC (Fast Protein Liquid Chromatography) systems like Pharmacia Äkta.

Step 2: Tag Removal (Optional)

• If tag removal is desired, incorporate a protease cleavage site (e.g., TEV protease site).

• Incubate with appropriate protease (e.g., TEV at a mass ratio of 1:10 protease:protein at 20°C for 1 hour) .

• Remove cleaved tag by second IMAC run, collecting the flow-through.

Step 3: Size Exclusion Chromatography

• Further purify using gel filtration (e.g., Superdex 200 HR26/60) in appropriate buffer (e.g., 10 mM HEPES, pH 7.5, 500 mM NaCl) .

Step 4: Quality Control

• Analyze purity by SDS-PAGE (>90% purity is typical) .

• Verify identity by MALDI-TOF mass spectrometry and trypsin peptide mass fingerprinting .

• Assess secondary structure by far-UV circular dichroism .

Storage Recommendations:

• Store in Tris/PBS-based buffer with 6% or 50% trehalose/glycerol at pH 8.0 .

• Aliquot and store at -20°C/-80°C, avoiding repeated freeze-thaw cycles .

• For short-term storage, working aliquots can be kept at 4°C for up to one week .

What methodologies can be used to investigate the potential DNA-binding properties of ORF413?

Based on research with similar archaeal viral proteins, several approaches are recommended:

In Vitro DNA Binding Assays:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified ORF413 with labeled DNA fragments

  • Analyze mobility shifts on native PAGE

  • Use competition assays with unlabeled DNA to determine binding specificity

• DNA Filament Formation Assay:

  • Similar to methods used for AFV1 major coat proteins which form filaments when incubated with linear dsDNA

  • Incubate ORF413 with linear dsDNA and observe filament formation using transmission electron microscopy

• Nuclease Protection Assay:

  • If ORF413 binds and protects DNA, incubate protein-DNA complexes with DNase I

  • Identify protected regions by sequencing or footprinting

Site-Directed Mutagenesis Approach:

• Generate mutants of potential DNA-binding residues (typically positively charged residues like lysine and arginine)

• Test mutants for altered DNA binding capabilities

• Follow similar approaches used for AFV1-157 where mutations (E86A and K57A) were used to investigate nuclease activity

How can researchers investigate the potential enzymatic activities of ORF413?

Given that some archaeal viral proteins display enzymatic activities, consider these approaches:

Nuclease Activity Assay:

• Incubate ORF413 with various DNA substrates (linear dsDNA, circular dsDNA, ssDNA)

• Use time-course experiments similar to those performed with AFV1-157

• Include appropriate controls (e.g., BSA)

• Analyze reaction products by agarose gel electrophoresis with ethidium bromide staining

Experimental Protocol Example:

• Incubate protein (250 ng/μl) with DNA substrate (25 ng/μl) at a DNA:protein mass ratio of 1:10

• Perform reaction at archaeal-relevant temperature (e.g., 42°C or higher)

• Include relevant cofactors (e.g., 25 mM MnCl₂ for nuclease activity)

• Sample at different time points (0, 15, 30, 60 min)

• Stop reactions with 50 mM EDTA

• Analyze by gel electrophoresis

Other Potential Enzymatic Activities:

• Test for glycosyltransferase activity (suggested for some AFV proteins)

• Investigate potential phosphatase activity

• Examine possible helicase activity for Holliday junction branch migration

How can the Cre/loxP recombination system be applied to generate marker-free recombinant viruses for ORF413 studies?

The Cre/loxP system, as demonstrated with other viruses, offers a powerful approach for removing selection markers:

Protocol Outline:

• Generate initial recombinant virus with EGFP marker flanked by loxP sites

• Transfect host cells with a plasmid expressing Cre recombinase (e.g., pBS185 CMV-Cre)

• 24 hours post-transfection, infect the cells with the recombinant virus (~1 MOI)

• Harvest recombinant viruses ~48 hours post-infection

• Perform additional passage in Cre-expressing cells

• Select foci lacking GFP expression through plaque assays

• Verify marker removal by whole genome sequencing

Application to ORF413 Studies:

• This system can be used to create ORF413 deletion mutants

• It can also generate recombinants where ORF413 is replaced with modified versions (point mutations, domain deletions)

• The marker-free approach minimizes potential artifacts caused by reporter genes

What bioinformatic approaches can predict potential functions of ORF413?

Given the uncharacterized nature of ORF413, computational approaches are valuable for function prediction:

Sequence-Based Analysis:

• Profile-Sequence Comparisons:

  • PSI-BLAST against multiple databases (NCBI nr, UniProt)

  • HHpred for detection of remote homology

  • HMMER searches against Pfam/InterPro

• Motif Identification:

  • Search for known functional motifs using PROSITE, PRINTS, or BLOCKS

  • Identify conserved residues that might be involved in catalysis or DNA binding

Structure-Based Prediction:

• Protein Structure Prediction:

  • Use AlphaFold or RoseTTAFold to predict 3D structure

  • Compare predicted structure to known folds using DALI or FATCAT

• Function Prediction from Structure:

  • Identify potential binding pockets or catalytic sites

  • Compare with structural databases of proteins with known function

Comparative Genomics:

• Analyze gene neighborhood conservation across related viruses

• Identify syntenic regions and co-occurrence patterns with genes of known function

What approaches are effective for studying ORF413 protein-protein interactions?

Understanding protein interactions is crucial for elucidating function:

In Vitro Methods:

• Pull-Down Assays:

  • Use His-tagged ORF413 as bait protein

  • Incubate with host cell lysates or viral protein extracts

  • Identify binding partners by mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize purified ORF413 on a sensor chip

  • Flow potential interacting proteins over the surface

  • Measure binding kinetics and affinity

In Vivo Methods:

• Yeast Two-Hybrid (Y2H):

  • Create fusion constructs of ORF413 with DNA-binding domain

  • Screen against a library of host proteins fused to activation domain

  • Note: May require adaptation for archaeal proteins

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse ORF413 to a biotin ligase

  • Express in host cells and identify biotinylated proximity partners

Crosslinking Mass Spectrometry:

• Treat viral particles or infected cells with crosslinking reagents

• Digest and identify crosslinked peptides by mass spectrometry

• Map interaction interfaces between ORF413 and partner proteins

What challenges exist in crystallizing viral proteins from extremophile viruses like AFV2?

Crystallizing proteins from extremophilic viruses presents unique challenges:

Extremophile-Specific Challenges:

• Protein Stability:

  • Proteins may be unstable under non-extreme conditions used for crystallization

  • May require high salt, low pH, or high temperature during purification and crystallization

• Unconventional Folding:

  • Extremophile proteins often have unusual structural features

  • May require specialized crystallization conditions

Technical Approaches:

• Buffer Optimization:

  • Test various buffers that mimic native conditions (acidic pH, high salt)

  • Consider additives that stabilize extremophilic proteins

• Truncation Constructs:

  • Generate multiple constructs targeting structured domains

  • Remove flexible regions that may hinder crystallization

• Surface Entropy Reduction:

  • Identify and mutate surface residue clusters with high conformational entropy

  • Replace flexible residues (Lys, Glu) with residues promoting crystal contacts (Ala)

• Co-crystallization:

  • Attempt crystallization with potential binding partners

  • Consider co-crystallization with DNA for potential DNA-binding proteins

Case Study Insights:

• Success with AFV1 coat proteins crystallization provides valuable precedent

• Consider similar approaches used for crystallizing ORF157 from AFV1

What are the most promising research directions for understanding ORF413 function?

Based on current knowledge of Acidianus filamentous viruses, several research avenues show particular promise:

Integrative Structural Biology:

• Combine X-ray crystallography, cryo-EM, and computational modeling

• Determine structure in context of viral particles

• Compare with structures of other viral proteins with known functions

Functional Genomics:

• Generate ORF413 deletion mutants and characterize phenotypes

• Perform RNA-seq to identify genes affected by ORF413 deletion

• Conduct ChIP-seq if evidence suggests DNA-binding activity

Proteomic Approaches:

• Identify interaction partners in host and viral proteomes

• Map temporal changes in interactions during infection cycle

• Correlate with functional data from genetic studies

Evolutionary Analysis:

• Compare ORF413 with homologs across archaeal viruses

• Identify conserved features that may indicate functional importance

• Analyze patterns of selection pressure on different protein regions