Recombinant Acidianus two-tailed virus Uncharacterized protein ORF134

What is the Acidianus two-tailed virus (ATV) and what makes it unique among archaeal viruses?

The Acidianus two-tailed virus (ATV) is a hyperthermophilic archaeal virus that infects members of the genus Acidianus. What makes ATV particularly distinctive is its extraordinary ability to undergo major morphological development outside of, and independently of, the host cell. After infection, ATV virions are extruded from host cells as lemon-shaped tail-less particles. These particles then develop long tails at each pointed end when exposed to temperatures close to their natural habitat (approximately 85°C), resulting in the characteristic two-tailed morphology .

ATV possesses a circular, double-stranded DNA genome of 62,730 base pairs. The genome is exceptional for a crenarchaeal virus as it carries four putative transposable elements and genes previously associated only with archaeal self-transmissable plasmids. In total, the genome encodes 72 predicted proteins, including 11 structural proteins with molecular masses ranging from 12 to 90 kDa .

Another unique feature of ATV is its ability to establish two distinct infection cycles. Infection with ATV can result either in viral replication followed by cell lysis or in conversion of the infected cell to a lysogen. The lysogenic cycle involves integration of the viral genome into the host chromosome, likely facilitated by a virus-encoded integrase .

What approaches can be used to express recombinant ORF134 protein from ATV?

Expression of recombinant proteins from hyperthermophilic archaeal viruses presents unique challenges due to their extremophilic nature. Based on approaches used for similar viral proteins, the following methodology can be effective:

• Gene synthesis and codon optimization: The ORF134 sequence should be chemically synthesized with codon optimization for the expression host (typically E. coli). This approach was successful for major coat proteins from the related Acidianus filamentous virus 1 (AFV1) .

• Expression vector selection: For thermal-stable proteins like those from hyperthermophilic viruses, vectors with strong inducible promoters (T7 or tac) are recommended. Adding affinity tags (His-tag, as used in the ATSV major coat protein study) facilitates purification .

• Expression host selection: While standard E. coli strains (BL21(DE3)) are commonly used, specialized strains for hyperthermophilic proteins like Rosetta 2 (containing rare codons) may improve expression .

• Expression conditions optimization: Lower induction temperatures (16-25°C) and extended expression times (overnight) can enhance soluble protein production despite the protein's native high-temperature environment.

• Inclusion body recovery and refolding: If inclusion bodies form, solubilization with strong denaturants (8M urea) followed by gradual dialysis at increasing temperatures (approaching 60-75°C) can aid proper folding .

The approach for ORF134 can be informed by successful expression of other archaeal viral proteins, such as the major coat protein of ATSV, which was PCR amplified from total community viral DNA, cloned with a Shine-Dalgarno sequence and N-terminal 6×His tag, and successfully expressed .

What purification methods are most effective for archaeal viral proteins like ORF134?

Purification of recombinant archaeal viral proteins requires specialized approaches to accommodate their unique biochemical properties:

• Heat treatment: Exploiting the thermostability of archaeal proteins, crude extracts can be heated (70-80°C for 15-30 minutes) to precipitate E. coli host proteins while keeping the target protein in solution .

• Affinity chromatography: If expressed with His-tags, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-based resins provides effective initial purification. For the ATSV major coat protein, His-tag purification was successfully implemented .

• Ion exchange chromatography: Given that many archaeal viral proteins interact with DNA (as seen with AFV1 coat proteins), they often have charged regions making ion exchange chromatography (particularly cation exchange for DNA-binding proteins) highly effective .

• Size exclusion chromatography: As a final polishing step, gel filtration helps remove aggregates and provides buffer exchange. This was effective in purifying the helical coat proteins of AFV1 .

• Density gradient ultracentrifugation: For studying virus-like particles or protein-DNA complexes, CsCl gradients can separate based on density, as demonstrated with ATSV purification where gradient fractions were screened for viral DNA by qPCR .

For quality control, SDS-PAGE, Western blotting (using anti-His antibodies or developing specific antisera as done for ATSV), and mass spectrometry should be employed to verify protein identity and purity .

How can researchers assess the DNA-binding properties of ORF134?

Based on studies of related archaeal viral proteins, ORF134 may possess DNA-binding capabilities similar to the coat proteins of AFV1, which were shown to bind DNA and form filaments when incubated with linear dsDNA . The following methodological approaches are recommended for characterizing potential DNA-binding properties:

• Electrophoretic Mobility Shift Assay (EMSA): Incubate purified recombinant ORF134 with labeled DNA fragments (preferably from the ATV genome) at increasing protein concentrations. Analyze the samples by native PAGE to detect mobility shifts indicating protein-DNA complex formation .

• Filter binding assays: This quantitative approach can determine binding affinity (Kd) by measuring retention of labeled DNA on nitrocellulose filters in the presence of varying ORF134 concentrations.

• Fluorescence anisotropy: Using fluorescently labeled DNA oligonucleotides, measure changes in rotational diffusion upon protein binding to determine binding kinetics and thermodynamics.

• Atomic Force Microscopy (AFM) or Transmission Electron Microscopy (TEM): These techniques can visualize protein-DNA complexes and potential filament formation, as observed with AFV1 coat proteins . Sample preparation would involve incubating ORF134 with linear DNA at conditions mimicking the extreme environment (pH 2-3, 75-85°C).

• DNA protection assays: If ORF134 binds to specific DNA sequences, DNase I footprinting can identify protected regions, providing insights into sequence specificity.

When designing these experiments, consider testing various DNA templates (single-stranded, double-stranded, curved, or specific ATV genomic regions) and environmental conditions (pH, temperature, salt concentration) relevant to the natural hyperthermophilic acidic habitat of ATV .

How might the structure of ORF134 relate to its function in ATV's unique extracellular morphological development?

The extraordinary extracellular morphological development of ATV, where tailless particles develop tails at high temperatures, suggests specialized structural proteins are involved in this process. While specific information about ORF134 structure is not available in the provided resources, we can propose an analytical approach based on related archaeal viral proteins:

• Computational structure prediction: Begin with homology modeling using structures of characterized archaeal viral proteins. The AFV1 coat proteins, which display a four-helix-bundle fold despite low sequence identity with other viral proteins, provide a potentially relevant structural template . Programs like AlphaFold2 or I-TASSER can generate preliminary models of ORF134.

• Structural characterization: For experimental structure determination, X-ray crystallography has been successful for archaeal viral proteins, as demonstrated with AFV1-132 (determined at 1.95Å resolution) . Alternatively, Cryo-EM could be particularly valuable if ORF134 forms oligomeric assemblies.

• Temperature-dependent structural changes: Given ATV's temperature-dependent morphological changes, circular dichroism (CD) spectroscopy should be employed to monitor secondary structure changes across a temperature gradient (25-90°C), potentially revealing structural transitions corresponding to tail formation temperatures.

• Identification of structural motifs: Based on the finding that several larger proteins in ATV are rich in coiled-coil and/or low complexity sequence domains (unusual for archaea), particular attention should be paid to identifying such features in ORF134 . The P800 protein of ATV, which resembles intermediate filament proteins and undergoes modification specifically in two-tailed virions, might provide insights into structural proteins involved in tail development .

• Structural comparison with characterized viral proteins: The structural similarity between AFV1-132 and the coat protein of Sulfolobus islandicus rod-shaped virus (SIRV) despite only 13% sequence identity suggests convergent evolution of functionally important folds . Similar structural comparisons with ORF134 could reveal functional relationships.

By combining computational predictions with experimental structural data across different temperatures, researchers can generate hypotheses about ORF134's potential role in ATV's unique morphological development process.

What methods can be used to investigate protein-protein interactions between ORF134 and other ATV structural proteins?

Understanding the interaction network of ORF134 within the ATV virion is crucial for elucidating its function in viral assembly and the unique extracellular morphological development. Based on approaches used for similar systems, the following methods are recommended:

• Co-immunoprecipitation (Co-IP): Generate specific antibodies against purified recombinant ORF134 (similar to the approach used for ATSV major coat protein) . Use these antibodies to immunoprecipitate ORF134 from ATV-infected Acidianus lysates, followed by mass spectrometry to identify co-precipitating proteins.

• Pull-down assays: Immobilize His-tagged ORF134 on Ni-NTA resin and incubate with ATV virion lysate or individual recombinant ATV proteins to identify interaction partners.

• Yeast two-hybrid (Y2H) screening: While traditional Y2H may be challenging for hyperthermophilic proteins, modified systems like the bacterial two-hybrid system might be more suitable for screening ORF134 against a library of other ATV proteins.

• Proximity labeling: Fusion of ORF134 with enzymes like BioID or APEX2 could enable identification of proximal proteins in reconstituted systems or in vitro assemblies.

• Crosslinking mass spectrometry (XL-MS): Chemical crosslinking combined with mass spectrometry can identify proteins in close proximity to ORF134 and provide spatial constraints for modeling interactions, particularly valuable for transient interactions during morphological development.

• Fluorescence microscopy approaches: Techniques similar to the catalyzed reporter deposition - fluorescence in situ hybridization (CARD-FISH) method adapted for viral proteins and hosts could visualize co-localization of ORF134 with other viral components .

• BiFC (Bimolecular Fluorescence Complementation): Though challenging in extremophilic systems, modified approaches could visualize protein interactions in reconstituted systems.

When designing these experiments, consider the potential temperature-dependent nature of the interactions, since ATV undergoes morphological changes at temperatures around 85°C . Experiments should ideally incorporate temperature shifts to capture interaction dynamics during the tail development process.

How can researchers assess the role of ORF134 in the biphasic life cycle of ATV?

The Acidianus two-tailed virus exhibits a unique biphasic life cycle, capable of both lytic replication and lysogeny . Investigating ORF134's potential role in this process requires specialized approaches for extremophilic archaeal systems:

• Generation of recombinant ATV lacking functional ORF134:

  • Develop a genetic system for ATV similar to those established for other archaeal viruses

  • Create ORF134 deletion or point mutation variants

  • Assess the impact on viral replication, lysogeny establishment, and morphological development

• Temporal expression analysis:

  • Perform time-course experiments of ATV infection in Acidianus cultures

  • Use quantitative PCR and Western blotting to track ORF134 expression levels throughout the infection cycle

  • Correlate expression patterns with viral morphological changes and transition between lytic and lysogenic phases

  • Follow approaches similar to those used for Acidianus sp. virus infection studies where samples were collected every 4-8 hours post-infection

• Localization studies:

  • Develop specific antibodies against ORF134 for immunolocalization

  • Track protein localization during different stages of infection and virion development

  • Correlate localization with viral morphological changes, particularly during tail development

• Interaction with host factors:

  • Identify potential host interaction partners using pull-down assays with recombinant ORF134

  • Focus on interactions with host ESCRT system components, as other archaeal viruses show interplay with ESCRT machinery

  • Examine whether ORF134 interacts with the viral integrase or host chromosomal components during lysogeny

• Temperature-shift experiments:

  • Since ATV morphological development occurs at high temperatures, perform temperature-shift experiments to identify when ORF134 is active

  • Monitor ORF134 modifications (posttranslational modifications like phosphorylation or cleavage) during temperature shifts

  • Compare to the P800 protein of ATV, which is modified specifically in two-tailed but not in tail-less virion particles

These approaches should be integrated with electron microscopy to correlate molecular findings with structural observations, similar to the TEM imaging performed in Acidianus virus infection studies where samples were dialyzed into 5 mM glycine (pH 2.5) for visualization .

What expression systems and conditions are optimal for producing functional recombinant ORF134?

Producing functional recombinant proteins from hyperthermophilic archaeal viruses presents unique challenges requiring specialized approaches:

Expression Systems Comparison:

Optimization Protocol:

• Vector design considerations:

  • Include a strong inducible promoter (T7 or tac)

  • Add an N-terminal 6×His tag following a Shine-Dalgarno sequence (similar to ATSV major coat protein expression)

  • Consider thermal stability elements in the construct design

  • For archaeal protein expression, include a short linker between tag and protein

• Induction optimization matrix:

  Temperature	IPTG Concentration	Duration	Expected Outcome
  37°C	0.1-1.0 mM	3-4 hours	High yield, possible inclusion bodies
  25-30°C	0.1-0.5 mM	5-8 hours	Balanced yield and solubility
  16-18°C	0.05-0.1 mM	12-18 hours	Maximized solubility, lower yield

• Buffer optimization for purification:

  • Test pH range 5.0-8.0 (archaeal proteins often have acidic optima)

  • Include stabilizing agents (glycerol 10-20%, specific ions)

  • For heat-stable proteins like ORF134, incorporate a heat treatment step (65-75°C for 15-30 minutes) to remove E. coli proteins

• Refolding strategy if inclusion bodies form:

  • Solubilize in 8M urea or 6M guanidine-HCl

  • Gradual dialysis with decreasing denaturant concentration

  • Incorporate a temperature gradient during refolding (25°C → 50°C → 75°C)

  • Add stabilizing agents (arginine, low concentrations of detergents)

• Functional validation:

  • Thermal stability assessment via differential scanning fluorimetry

  • DNA binding assays at different temperatures (mimicking the 85°C natural environment of ATV)

  • Oligomerization state analysis by size exclusion chromatography

This comprehensive approach draws on successful strategies for expressing archaeal viral proteins while addressing the unique challenges of ORF134 from the extremophilic ATV.

How can researchers design experiments to investigate ORF134's potential role in ATV's unique morphological changes?

Investigating ORF134's role in ATV's remarkable extracellular morphological development requires specialized experimental designs that account for the extreme conditions under which these changes occur:

• Temperature-triggered morphological transformation assays:

  • Purify ATV virions as lemon-shaped tail-less particles

  • Establish a temperature-shift protocol mimicking natural conditions (raising temperature to 85°C in acidic media, pH 2-3)

  • Compare wild-type virions with particles treated with anti-ORF134 antibodies

  • Quantify tail development using electron microscopy and measure key parameters (tail length, formation time)

  • Use gradient ultracentrifugation similar to methods for ATSV, where CsCl gradients were used and fractions screened by qPCR

• In vitro reconstitution experiments:

  • Express and purify recombinant ORF134 along with other key structural proteins

  • Test combinations of proteins for filament formation with DNA substrates

  • Observe structures using transmission electron microscopy with negative staining (1.5% uranyl acetate as used for ATSV)

  • Compare to the filament formation capacity of known structural proteins like AFV1 coat proteins

  • Analyze the effect of temperature shifts on reconstituted complexes

• Time-resolved structural analysis:

  • Isolate ATV particles at various stages of tail development

  • Perform proteomics analysis to track modifications of ORF134

  • Look for patterns similar to P800, which is modified specifically in two-tailed but not tail-less particles

  • Use Cryo-EM to capture structural transitions during morphogenesis

• Mutagenesis studies:

  • Identify conserved domains in ORF134 through bioinformatic analysis

  • Create targeted mutations in potential functional domains

  • Express mutant proteins and assess their ability to:

    • Bind to viral DNA

    • Interact with other structural proteins

    • Form proper oligomeric structures

    • Support tail development in reconstitution experiments

• Domain swap experiments:

  • Create chimeric proteins by swapping domains between ORF134 and related proteins from viruses lacking extracellular development

  • Test these chimeras in reconstitution assays to identify domains responsible for tail formation

• Environmental parameter optimization:

  • Systematically vary pH, temperature, and ionic strength to define optimal conditions for ORF134 function

  • Create a phase diagram of conditions supporting tail development

  • Compare with natural hot spring conditions where ATV is found

This experimental design framework enables researchers to systematically investigate ORF134's potential contribution to ATV's unique extracellular morphological development while addressing the technical challenges of working with extremophilic archaeal systems.

What computational approaches can predict functional domains in ORF134?

Given that ORF134 is uncharacterized, computational approaches are essential first steps toward functional annotation. A systematic computational workflow should include:

• Sequence-based analysis:

  • PSI-BLAST and HHpred searches against protein databases to identify distant homologs

  • Multiple sequence alignment with other archaeal viral proteins, particularly focusing on ATV proteins and those from related archaeal viruses

  • Search for conserved domains using CDD, PFAM, and InterPro databases

  • Look specifically for coiled-coil regions and low complexity domains that are present in several larger ATV proteins

  • Analyze for structural features similar to P800, which resembles intermediate filament proteins and is implicated in viral tail development

• Structural prediction approaches:

  • Generate 3D models using AlphaFold2, RoseTTAFold, or I-TASSER

  • Compare predicted structures with the four-helix bundle fold observed in AFV1 coat proteins and SIRV coat protein

  • Despite potential low sequence identity (AFV1-132 and SIRV-134 share only 13% sequence identity but identical fold), structural similarity could suggest functional relationships

  • Analyze surface electrostatics to identify potential DNA-binding regions

  • Look for structural motifs that could be involved in protein-protein interactions

• Functional site prediction:

  • Identify potential DNA-binding regions using DNA-Binding Domain predictor (DBD-Hunter) and BindUP

  • Predict protein-protein interaction interfaces using SPPIDER or WHISCY

  • Analyze for potential membrane-interacting regions that might be involved in the virion's lipid outer shell interactions, similar to AFV1-140's lipophilic C-terminal domain

• Integrated analysis approaches:

  • Combine sequence conservation, structural features, and predicted functional sites to generate hypotheses about ORF134 function

  • Map conserved residues onto the predicted 3D structure to identify potential functional hotspots

  • Generate a consensus prediction based on multiple computational methods

• Comparative genomics:

  • Compare ORF134 positioning in the genome relative to other functional elements

  • Analyze gene neighborhood conservation across related viruses

  • Look for co-evolution patterns with other viral proteins that might suggest functional associations

By systematically applying these computational approaches, researchers can generate testable hypotheses about ORF134 function, particularly its potential role in ATV's unique morphological development, and guide the design of targeted experiments.

How can researchers interpret structural data for ORF134 in the context of archaeal virus evolution?

The structural analysis of archaeal viral proteins has revealed surprising evolutionary connections despite minimal sequence conservation. For ORF134, interpreting structural data requires specialized analytical approaches:

• Structural homology detection beyond sequence similarity:

  • The surprising structural similarity between AFV1-132 and SIRV-134 (identical four-helix bundle fold despite only 13% sequence identity) demonstrates that structure can reveal evolutionary connections when sequence fails

  • Use structure comparison tools (DALI, FATCAT) to compare ORF134 models or structures with known archaeal viral proteins

  • Look beyond simple fold similarities to arrangement of functional residues and surface properties

• Identification of viral lineages through structural conservation:

  • The structural similarity between coat proteins from different archaeal virus families (Lipothrixviridae and Rudiviridae) suggests they could have derived from a common ancestor

  • Position ORF134 within this emerging structural classification system

  • Analyze if ORF134 belongs to previously identified or novel structural lineages

• Structure-guided functional analysis:

  • For helical bundle proteins like those in AFV1, specific functions can be assigned to different domains:

    • AFV1-140's basic N-terminus binds genomic DNA

    • Its lipophilic C-terminal domain embeds in the lipidic outer shell

  • Similar domain-specific functional assignment should be attempted for ORF134

• Structural adaptation to extreme environments:

  • Analyze features that confer thermostability (increased salt bridges, disulfide bonds, compactness)

  • Compare with mesophilic viral proteins to identify archaeal-specific adaptations

  • Look for structural features enabling function at the extreme pH (2-3) and temperature (85°C) conditions where ATV thrives

• Integration with virion architecture models:

  • In the topological model of AFV1 filament core, the genomic dsDNA superhelix wraps around the AFV1-132 basic protein

  • Develop similar topological models for ORF134 within the ATV virion

  • Analyze potential structural transitions during the unique extracellular tail development

• Evolutionary implications data table:

  Structural Feature	Evolutionary Interpretation	Implication for ORF134
  Four-helix bundle fold	Ancient viral protein fold preserved across archaeal virus families	If present in ORF134, suggests membership in this viral lineage
  DNA-binding domains	Conserved function despite sequence divergence	Potential role in genome packaging
  Lipid-interacting domains	Adaptation to membrane-containing virions	Possible involvement in ATV's lipidic outer shell
  Thermostable structural elements	Adaptation to extreme environments	Essential for function during extracellular development at 85°C
  Coiled-coil domains	Unusual in archaea but present in ATV structural proteins	Potential role in tail assembly if present in ORF134

By placing ORF134 structural data in this evolutionary context, researchers can gain insights into archaeal virus diversification while illuminating the protein's specific role in ATV's unique biology.

What are the future research directions for understanding ORF134 and similar uncharacterized archaeal viral proteins?

The study of ORF134 and similar uncharacterized archaeal viral proteins represents an exciting frontier in understanding extremophilic viruses and their unique biology. Future research should focus on several key directions:

• Integrated structural and functional characterization:

  • Combining high-resolution structural studies with functional assays will be essential to decode the precise role of ORF134 in ATV biology

  • The surprising structural conservation despite sequence divergence observed between AFV1 and SIRV coat proteins suggests that structural approaches may be particularly revealing for archaeal viral proteins

• Development of genetic systems for archaeal viruses:

  • Creating tools for genetic manipulation of ATV would enable definitive functional studies through targeted mutations and complementation assays

  • This would allow researchers to directly test hypotheses about ORF134's role in the remarkable extracellular morphological development of ATV

• Environmental viromics approaches:

  • Expanding metagenomic studies of high-temperature acidic hot springs could reveal natural variants of ORF134 and related proteins

  • This approach successfully identified ATSV from Yellowstone National Park hot springs and could reveal the diversity and evolution of ATV-like viruses

• Host-virus interaction studies:

  • Investigating potential interactions between ORF134 and host Acidianus proteins could reveal how these viruses manipulate their hosts

  • Particular attention should be paid to potential interactions with the host ESCRT system, as seen with other archaeal viruses

• Comparative analyses across archaeal virus families:

  • The unexpected structural similarity between proteins from different archaeal virus families suggests previously unrecognized evolutionary connections

  • Systematic comparison of proteins like ORF134 across archaeal virus families could help define new viral lineages

• Biotechnological applications:

  • The extreme stability and unique properties of archaeal viral proteins make them promising candidates for biotechnological applications

  • ORF134, once characterized, could provide novel DNA-binding domains or thermostable structural components for synthetic biology applications

• Development of in vitro reconstitution systems:

  • Creating systems to reconstitute ATV morphological development in vitro would allow detailed mechanistic studies of the role of individual proteins like ORF134