Recombinant Acidianus two-tailed virus Putative transmembrane protein ORF80

What is Acidianus Two-Tailed Virus Putative Transmembrane Protein ORF80?

Acidianus Two-Tailed Virus (ATV) Putative Transmembrane Protein ORF80 is a small protein encoded by the ATV genome. It consists of 80 amino acids with the sequence: MNPAVVLFLALITSIILIILFTATIAIYHDFVTIEQNYNMTAYAQQQNTQANNVLVTGFRFLFIGLIGVTIALIIYQRRT . The protein is characterized as putative transmembrane protein due to its hydrophobic domains consistent with membrane insertion capability. ATV itself belongs to the large tailed spindle virus superfamily that infects hyperthermophilic archaea of the genus Acidianus .

How does ATV ORF80 differ from other viral structural proteins in the Acidianus virus family?

The ATV ORF80 protein differs from other viral structural proteins in the Acidianus virus family through both its structural organization and functional properties. Unlike the major coat proteins of Acidianus filamentous virus 1 (AFV1), which contain a four-helix-bundle fold in their C-terminal domains and demonstrate DNA-binding capabilities , the ATV ORF80 is primarily characterized by its transmembrane domains.

While AFV1 coat proteins (132 and 140 amino acids) bind DNA and form filaments when incubated with linear dsDNA , the ATV ORF80 likely functions within the viral envelope as a membrane-embedded element. The distinction is further emphasized by the architectural differences between the two viruses - AFV1 has a filamentous structure with a lipidic outer shell and linear dsDNA genome, whereas ATV possesses a spindle-shaped head with a circular dsDNA genome packaged in a capsid with a single extending tail .

What are the predicted structural domains of ATV ORF80?

Based on the amino acid sequence analysis of ATV ORF80, several structural domains can be predicted:

What expression systems are most effective for producing recombinant ATV ORF80?

For producing recombinant ATV ORF80, E. coli expression systems have been demonstrated to be effective, as evidenced by the commercial availability of His-tagged recombinant full-length ATV ORF80 protein expressed in E. coli . The methodological approach typically involves:

• Gene synthesis or PCR amplification of the ORF80 coding sequence

• Cloning into an appropriate expression vector with a histidine tag for purification

• Transformation into an E. coli expression strain (commonly BL21(DE3) or similar)

• Induction of protein expression using IPTG or auto-induction media

• Cell lysis and protein purification using affinity chromatography

For researchers working with archaeal viral proteins, it's important to note that codon optimization may be necessary due to the different codon usage preferences between archaea and E. coli. Temperature optimization during expression may also be critical, considering the thermophilic origin of the Acidianus host.

What purification strategies yield the highest purity and activity of recombinant ATV ORF80?

To obtain high-purity and functionally active recombinant ATV ORF80, a multi-step purification strategy is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins for His-tagged protein variants

• Intermediate Purification: Ion exchange chromatography to separate based on charge properties

• Polishing Step: Size exclusion chromatography to remove aggregates and achieve high purity

Specific buffer considerations for ATV ORF80:

• Inclusion of non-ionic detergents (0.1-0.5% n-dodecyl β-D-maltoside or digitonin) to maintain solubility of this putative transmembrane protein

• Addition of glycerol (10-50%) for stability during storage

• Tris-based buffer systems with pH optimized for protein stability

While specific activity assays for ATV ORF80 are not detailed in the search results, quality control typically includes SDS-PAGE, Western blotting, and potentially circular dichroism to confirm proper secondary structure formation.

How can researchers assess the membrane integration properties of ATV ORF80?

To assess the membrane integration properties of ATV ORF80, researchers can employ several complementary techniques:

• Membrane Fraction Analysis:

  • Separate membrane and soluble fractions after expression in a suitable host

  • Analyze protein distribution by Western blotting with antibodies against ATV ORF80

  • Compare with known membrane and soluble protein controls

• Liposome Reconstitution:

  • Purify ATV ORF80 in the presence of detergents

  • Reconstitute into artificial liposomes of varying lipid compositions

  • Assess integration using flotation assays or protease protection assays

• Fluorescence-Based Approaches:

  • Generate fluorescent protein fusions (similar to EGFP fusion strategies used for other viral proteins )

  • Observe subcellular localization in eukaryotic or prokaryotic cells

  • Perform FRET analysis with known membrane markers

• Biophysical Techniques:

  • Circular dichroism to assess secondary structure in membrane-mimicking environments

  • NMR studies in detergent micelles to determine topology

  • Hydrogen-deuterium exchange mass spectrometry to map membrane-embedded regions

These methodologies would help determine not only if ATV ORF80 integrates into membranes but also its orientation and structural changes upon membrane insertion.

What experimental approaches can determine if ATV ORF80 interacts with host or viral proteins?

To investigate protein-protein interactions involving ATV ORF80, researchers should consider these methodological approaches:

• Affinity Purification Coupled with Mass Spectrometry (AP-MS):

  • Express tagged ATV ORF80 in host cells or reconstituted systems

  • Perform pull-down experiments to capture protein complexes

  • Identify interacting partners by mass spectrometry analysis

• Yeast Two-Hybrid Screening:

  • Create bait constructs using ATV ORF80

  • Screen against prey libraries derived from host Acidianus species or viral proteins

  • Validate positive interactions with secondary binding assays

• Proximity Labeling Techniques:

  • Fuse ATV ORF80 to BioID or APEX2 enzymes

  • Express in relevant cellular contexts

  • Identify proximal proteins through biotinylation and subsequent purification

• Cross-linking Mass Spectrometry:

  • Apply chemical cross-linkers to preserve transient interactions

  • Digest and analyze by mass spectrometry

  • Map interaction interfaces through identification of cross-linked peptides

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize purified ATV ORF80

  • Test binding to candidate interacting proteins

  • Determine binding kinetics and affinity constants

These approaches would help construct an interaction network for ATV ORF80, potentially revealing its role in viral assembly, host manipulation, or other functions.

How is ATV ORF80 evolutionarily related to similar proteins in other archaeal viruses?

The evolutionary relationships of ATV ORF80 can be analyzed through comparative genomics and phylogenetic approaches. While the search results don't directly address the evolutionary history of ATV ORF80, we can draw insights from related archaeal viral proteins:

• Sequence-Based Comparisons:

  • Perform multiple sequence alignments with putative homologs

  • Construct phylogenetic trees to visualize evolutionary relationships

  • Identify conserved domains that may suggest functional conservation

• Structural Homology:

  • Compare predicted structural features of ATV ORF80 with experimentally determined structures

  • Analyze fold conservation across archaeal viral proteins

  • Identify structural motifs that persist despite sequence divergence

An interesting comparative point emerges from search result , which describes ORF80 from Sulfolobus islandicus. Despite sharing the same name designation, this protein represents a novel type of basic leucine zipper DNA-binding protein with no sequence homology to characterized proteins. This illustrates the challenge in tracking evolutionary relationships solely based on ORF numbering conventions, as ORF80 appears to be a designation used across different archaeal viral genomes for potentially unrelated proteins.

What structural similarities exist between ATV ORF80 and other archaeal viral transmembrane proteins?

When analyzing structural similarities between ATV ORF80 and other archaeal viral transmembrane proteins, researchers should consider:

While the major coat proteins of Acidianus filamentous virus 1 (AFV1) show a four-helix-bundle fold that appears identical to the coat protein of Sulfolobus islandicus rod-shaped virus (SIRV) despite low sequence identity , similar detailed structural analysis of ATV ORF80 would require experimental determination of its structure or advanced computational modeling approaches.

How can ATV ORF80 be utilized in engineered viral delivery systems for extreme environments?

The potential applications of ATV ORF80 in engineered viral delivery systems targeting extreme environments stem from its origin in a virus that infects hyperthermophilic, acidophilic archaea. Methodological approaches for such applications could include:

• Chimeric Viral Particle Construction:

  • Incorporate ATV ORF80 into designed viral nanoparticles

  • Evaluate stability and assembly under extreme temperature and pH conditions

  • Test delivery efficiency of cargo molecules (DNA, RNA, or proteins)

• Membrane Fusion Studies:

  • Assess whether ATV ORF80 contributes to membrane fusion events

  • Engineer fusion constructs combining ATV ORF80 with cargo-carrying components

  • Measure fusion efficiency under varying environmental conditions

• Stabilization of Delivery Vehicles:

  • Incorporate ATV ORF80 into liposomes or other lipid-based delivery systems

  • Test thermal and pH stability improvements

  • Evaluate protection of cargo from degradation in extreme environments

• Cross-Domain Delivery Systems:

  • Explore functionality of ATV ORF80 in eukaryotic or bacterial systems

  • Develop hybrid viral systems combining elements from archaeal and non-archaeal viruses

  • Test species barrier crossing potential

Drawing from the methodology used in the EGFP reporter system described in search result , researchers could develop tracking systems to monitor the performance of ATV ORF80-enhanced delivery vehicles in real-time using fluorescence microscopy.

What methodologies are most effective for studying ATV ORF80 function in the context of the complete viral replication cycle?

To study ATV ORF80 function within the complete viral replication cycle, researchers would need to implement these methodological approaches:

• Gene Knockout and Complementation:

  • Develop a recombination system similar to that described for Orf virus in search result

  • Generate ATV ORF80 deletion mutants

  • Perform complementation studies with wild-type and mutated versions

• Fluorescent Tagging and Live Imaging:

  • Create fluorescently tagged versions of ATV ORF80 (using EGFP or similar reporters )

  • Track protein localization during infection using confocal microscopy

  • Correlate localization with specific stages of the viral cycle

• Cryo-Electron Microscopy Studies:

  • Visualize ATV ORF80 within the virion structure

  • Compare wild-type and mutant viral particles

  • Integrate structural data with functional analyses

• Time-Course Transcriptomics and Proteomics:

  • Monitor expression of ATV ORF80 throughout the infection cycle

  • Correlate with other viral and host gene expression patterns

  • Identify temporal regulation mechanisms

• Host-Virus Interaction Studies:

  • Identify host factors that interact with ATV ORF80

  • Characterize effects of ATV ORF80 on host cell physiology

  • Determine whether ATV ORF80 modulates host defense mechanisms

The approach would be similar to that described for studying ORFV replication , where fluorescent reporter systems significantly reduced the time needed for recombinant virus isolation and purification while enabling visualization of virus-host interactions at the cellular level.

What are the common pitfalls in recombinant expression of ATV ORF80 and how can they be addressed?

Researchers working with recombinant ATV ORF80 often encounter these challenges:

Based on available commercial preparations , successful expression strategies typically involve E. coli systems with His-tag purification approaches, storage in glycerol-containing buffers, and careful attention to buffer composition to maintain protein stability.

How can researchers overcome the challenges of studying protein-protein interactions involving membrane proteins like ATV ORF80?

Studying protein-protein interactions for membrane proteins like ATV ORF80 presents unique methodological challenges that can be addressed through:

• Detergent Selection and Optimization:

  • Screen multiple detergent types and concentrations

  • Test native membrane-mimicking systems (nanodiscs, amphipols, SMALPs)

  • Validate protein folding and function in each detergent system

• Modified Yeast Two-Hybrid Approaches:

  • Implement split-ubiquitin membrane yeast two-hybrid systems

  • Use MYTH (Membrane Yeast Two-Hybrid) for topology-specific interaction studies

  • Screen against cDNA libraries from host organisms

• In-Membrane Protein Crosslinking:

  • Apply membrane-permeable crosslinkers to intact systems

  • Identify interaction partners through immunoprecipitation followed by mass spectrometry

  • Map interaction sites through MS/MS analysis of crosslinked peptides

• Bimolecular Fluorescence Complementation:

  • Split fluorescent proteins (e.g., split GFP) fused to potential interaction partners

  • Monitor reconstitution of fluorescence upon protein-protein interaction

  • Visualize interactions in cellular contexts

• Thermal Shift Assays Adaptations:

  • Develop thermal shift assays compatible with detergent-solubilized proteins

  • Screen potential interaction partners by monitoring thermal stability changes

  • Identify stabilizing conditions for complex formation

These approaches build upon the recombinant protein methodology described in the search results while addressing the specific challenges posed by transmembrane proteins.