Recombinant Acidianus two-tailed virus Putative transmembrane protein ORF710

What is Acidianus two-tailed virus and why is it scientifically significant?

Acidianus two-tailed virus (ATV) is a unique archaeal virus discovered in 2003 in thermal springs in Pozzuoli, Italy. It infects crenarchaea of the genus Acidianus living in extremely harsh conditions - temperatures exceeding 85°C and pH levels as low as 1.5. ATV belongs to the Bicaudaviridae family and possesses an extraordinary characteristic that distinguishes it from all other known viruses: it undergoes extracellular morphological transformation, developing two tails at opposite ends of its spindle-shaped body when incubated at temperatures between 75-90°C . This temperature-dependent morphological change occurs without any cellular machinery involvement, making it a uniquely autonomous viral process. ATV can undergo both lysogenic and lytic life cycles, with environmental stress factors like temperature reduction capable of triggering the transition between these states .

What is the ORF710 protein and what is its predicted role in ATV?

ORF710 (also known as Y710_ATV in some databases) is a putative transmembrane protein encoded by the ATV genome . The mature protein spans amino acids 34-710 and contains multiple hydrophobic regions that likely function as membrane-spanning domains . While its exact function remains to be fully characterized, as a transmembrane protein it likely plays crucial roles in viral-host membrane interactions, potentially during entry, assembly, or the unique extracellular tail development process. The protein's significance is highlighted by its absence of homologues in related viruses like Sulfolobus tengchongensis Spindle-shaped Virus 1 (STSV1), which notably does not undergo cell-independent tail development . This correlation suggests ORF710 may be instrumental in ATV's distinctive morphological transformation capability.

What is the genomic context of ORF710 within the ATV genome?

The ATV genome consists of a circular double-stranded DNA of 62,730 base pairs encoding 72 predicted open reading frames (ORFs) . Most of these ORFs, including ORF710, lack identifiable homologues in public sequence databases, highlighting the unique nature of this virus. The genome is likely transcribed by the host RNA polymerase during viral replication . The ORF710 gene encodes a protein that has been identified in virion preparations, suggesting it's a structural component of the mature virus particle. Within the context of the larger viral genomic architecture, ORF710 represents one of several key structural proteins that may collaborate in forming the unique morphology of ATV virions and enabling their temperature-dependent transformations.

What expression systems are optimal for recombinant ORF710 production?

The selection of an appropriate expression system is critical for successful production of recombinant ORF710. While E. coli has been demonstrated to successfully express the recombinant protein with an N-terminal His-tag , researchers should consider several expression systems based on their experimental requirements:

What are the critical parameters for optimizing ORF710 expression in E. coli?

Successful expression of ORF710 in E. coli requires careful optimization of several parameters to maximize yield while ensuring proper folding and membrane insertion. Based on established protocols for membrane protein expression and the specific characteristics of ORF710, researchers should consider the following methodological approach:

First, selection of an appropriate strain is crucial. BL21(DE3) or its derivatives (C41, C43) are often preferred for membrane protein expression due to their reduced protease activity and ability to tolerate toxic membrane proteins. The expression vector should include an N-terminal His-tag for purification, as has been successfully used with ORF710 .

Second, culture conditions require careful tuning. While standard protocols suggest induction at OD600 0.6-0.8, for ORF710, lower temperatures (16-25°C) after induction can slow down protein production and improve proper folding. Additionally, lower inducer concentrations (0.1-0.5 mM IPTG) prevent overwhelming the membrane insertion machinery. Since ORF710 originates from a thermophilic organism, testing expression at various temperatures, including higher temperatures (30-37°C), might yield interesting results regarding protein folding and stability.

Third, media composition can significantly impact expression. Rich media like Terrific Broth supplemented with 1-5% glycerol can enhance membrane protein yields by stabilizing membranes. For ORF710, considering the addition of archaeal lipid extracts might improve proper folding and stability of this extremophilic protein.

What purification strategies yield the highest purity of recombinant ORF710?

Purification of recombinant ORF710 requires specialized strategies to maintain protein stability while achieving high purity. A comprehensive purification workflow should include:

• Membrane preparation: After cell lysis via sonication or French press, separate the membrane fraction by ultracentrifugation (100,000 × g for 1 hour). Solubilize membranes using an appropriate detergent from the table below.

• Immobilized Metal Affinity Chromatography (IMAC): Utilize the N-terminal His-tag for initial purification. Use nickel or cobalt resins with imidazole gradient elution (20-500 mM) for higher purity. All buffers must contain detergent above its critical micelle concentration (CMC).

• Size Exclusion Chromatography (SEC): Essential for removing aggregates and achieving homogeneous protein-detergent complexes. This step also provides valuable information about the oligomeric state of ORF710.

The SMA polymer approach is particularly interesting for ORF710 as it allows extraction with surrounding lipids, potentially preserving functional interactions that might be critical for activity assessment5.

How can the stability of purified ORF710 be maintained for downstream applications?

Maintaining the stability of purified ORF710 is critical for functional and structural studies. Based on the provided information, the following methodological approaches are recommended:

For storage, the recombinant protein should be kept at -20°C/-80°C in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . To prevent freeze-thaw damage, add glycerol to a final concentration of 5-50%. Importantly, multiple freeze-thaw cycles should be strictly avoided as they can severely compromise protein integrity. For working aliquots, store at 4°C for up to one week .

When reconstituting the lyophilized protein, briefly centrifuge vials before opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Given the thermophilic origin of ORF710, exploring thermal stability at elevated temperatures (40-60°C) might yield surprising results, as the protein may exhibit enhanced stability compared to standard storage conditions.

For structural studies, stability enhancements can be achieved by adding specific lipids that mimic the archaeal membrane environment or by exploring detergent mixtures. Alternative membrane mimetics like nanodiscs, amphipols, or styrene maleic acid lipid particles (SMALPs) may provide superior stability by maintaining the protein in a more native-like environment.

What techniques are most appropriate for determining the tertiary structure of ORF710?

Determining the tertiary structure of membrane proteins like ORF710 presents significant challenges. Several complementary methodological approaches should be considered:

For ORF710 specifically, a hybrid approach would be most effective. Initial cryo-EM studies of intact ATV virions could locate ORF710 in context . This could be followed by a domain-based strategy, identifying soluble domains for crystallization. The integrative approach would combine low-resolution data from SAXS with computational modeling to build a comprehensive structural model. The natural thermostability of ORF710 could be advantageous for structural studies, potentially allowing for data collection at elevated temperatures that might reduce conformational heterogeneity.

How might ORF710 contribute to ATV's unique two-tailed morphology?

The contribution of ORF710 to ATV's distinctive two-tailed morphology represents one of the most intriguing questions in the field. While direct experimental evidence is limited, several mechanistic hypotheses can be proposed based on its classification as a putative transmembrane protein:

ORF710 may function as a temperature-dependent molecular switch that undergoes specific conformational changes at temperatures between 75-90°C, which coincides with the temperature range where tail development occurs . These conformational changes could expose domains that initiate or facilitate the extracellular tail development process. Alternatively, ORF710 might serve as a structural scaffold or nucleation site for tail assembly at the viral capsid vertices. Its transmembrane nature could create focal points for the extension of tail proteins from the virion body.

The protein might also possess temperature-sensing capability, functioning as a molecular thermometer that triggers the tail assembly process only within the optimal temperature range of 75-90°C . This would ensure that the morphological transformation occurs only in environments similar to the virus's natural habitat. To investigate these hypotheses, approaches such as temperature-dependent structural studies, interaction mapping with other ATV proteins, and mutagenesis of key ORF710 domains would be valuable.

What comparative insights can be gained from studying ORF710 alongside proteins from related viruses?

Comparative analysis of ORF710 with proteins from related viruses can provide valuable evolutionary and functional insights. The search results indicate that ATV has no homologues in Sulfolobus tengchongensis Spindle-shaped Virus 1 (STSV1), which importantly does not undergo cell-independent tail development . This negative correlation strengthens the hypothesis that ORF710 plays a role in ATV's unique morphological transformation capability.

Additionally, comparison with Acidianus tailed spindle virus (ATSV), another member of the large tailed spindle virus superfamily, may be particularly informative. ATSV contains a 70,812-bp circular dsDNA genome with 96 ORFs and forms a spindle-shaped capsid with a single tail extending from one end . Unlike ATV where spindle heads contract as tails extend, ATSV does not show correlation between spindle volume and tail length . Comparing ORF710 with analogous proteins in ATSV might reveal conserved structural elements important for tail formation and highlight differences that explain the distinct morphological transformations of these viruses.

How can temperature-dependent structural changes in ORF710 be experimentally monitored?

Monitoring temperature-dependent structural changes in ORF710 is crucial for understanding its potential role in ATV's unique temperature-triggered morphological transformation. Several complementary methodological approaches can be employed:

Circular dichroism (CD) spectroscopy provides a straightforward approach to monitor secondary structure changes as a function of temperature. By measuring CD spectra at temperatures ranging from ambient to 90°C, researchers can detect transitions in alpha-helical and beta-sheet content that might correlate with ATV's morphological transformation temperature range (75-90°C) .

Differential scanning calorimetry (DSC) can identify thermal transitions and stability thresholds, potentially revealing temperature points where ORF710 undergoes conformational changes. This technique could establish whether ORF710 exhibits unusual thermal behavior in the critical 75-90°C range where tail formation occurs.

For more detailed structural information, hydrogen-deuterium exchange mass spectrometry (HDX-MS) at different temperatures can map regions of the protein that become more exposed or protected during temperature increases. This technique requires minimal protein amounts and can identify specific domains involved in temperature-dependent conformational changes.

Fluorescence-based approaches offer additional insights. Intrinsic tryptophan fluorescence and thermal shift assays using environment-sensitive dyes can detect subtle conformational changes and provide information about hydrophobic exposure during thermal transitions. Additionally, site-directed spin labeling coupled with electron paramagnetic resonance (EPR) spectroscopy can monitor distance changes between specific residues during temperature shifts, providing detailed information about domain movements.

What methods can establish the membrane topology of ORF710?

Determining the membrane topology of ORF710 is fundamental to understanding its function. Several complementary experimental approaches can establish which regions of the protein are exposed to the cytoplasm, which span the membrane, and which face the extracellular/luminal space:

Protease protection assays represent a classical approach to topology determination. By treating intact virions or membrane vesicles containing ORF710 with proteases, only exposed regions are digested. Mass spectrometry analysis of the protected fragments can identify transmembrane and luminal domains. This approach can be complemented with detergent solubilization to progressively expose protected regions.

Cysteine scanning mutagenesis combined with accessibility studies offers a more refined analysis. By systematically introducing cysteine residues throughout ORF710 and then assessing their accessibility to membrane-permeable and membrane-impermeable sulfhydryl reagents, researchers can map which regions are exposed to which side of the membrane. This approach is particularly powerful when combined with site-directed mutagenesis to replace native cysteines.

Fluorescence spectroscopy with environment-sensitive probes attached to specific regions can provide dynamic information about membrane insertion. Changes in fluorescence properties can indicate whether a region is in an aqueous or lipid environment, providing evidence for transmembrane domains.

Computational prediction should complement experimental approaches. Transmembrane prediction algorithms (TMHMM, Phobius, MEMSAT) can generate initial topology models based on hydrophobicity analysis and the positive-inside rule. These predictions can guide experimental design and be refined based on experimental results.

How can site-directed mutagenesis be used to investigate ORF710 function?

Site-directed mutagenesis represents a powerful approach to investigate the function of ORF710. A systematic mutagenesis strategy should target several key features of the protein:

First, predicted transmembrane regions should be investigated through substitution of key hydrophobic residues with charged amino acids. Such mutations would disrupt membrane insertion and could reveal which transmembrane domains are essential for function. The final 20 amino acids of ORF710 (YGTKIWIGVFIFAIS) appear particularly hydrophobic and would be prime candidates for this approach.

Second, the numerous tyrosine-rich regions in the ORF710 sequence (e.g., YMINQYGTLLY, YYYYYNSLKY) warrant special attention. Tyrosine residues often participate in specific interactions or function in signal transduction. Systematic alanine substitution of these tyrosines could identify those critical for function or protein-protein interactions.

Third, temperature-sensing regions should be investigated through thermoswitch analysis. If ORF710 functions as a molecular thermometer that triggers tail formation at specific temperatures, certain regions might undergo critical conformational changes in the 75-90°C range. Creating chimeric proteins with thermostable domains or introducing rigidifying mutations could identify regions involved in temperature sensing.

Finally, potential interaction interfaces should be probed through charge-reversal mutations. If ORF710 interacts with other viral proteins during tail formation, disrupting these interactions through strategic mutations of charged residues could prevent proper assembly and tail formation.

What assays can determine if ORF710 interacts with other viral proteins?

Investigating protein-protein interactions involving ORF710 is essential for understanding its role in ATV biology. Several complementary methods can be employed:

Co-immunoprecipitation (Co-IP) experiments using antibodies against ORF710 can pull down interacting partners from virion lysates. This approach requires developing specific antibodies against ORF710, but offers the advantage of capturing interactions in near-native conditions. Mass spectrometry analysis of co-precipitated proteins can identify interaction partners.

Yeast two-hybrid (Y2H) screening represents a powerful approach to identify binary interactions. By creating a library of ATV proteins fused to activation domains and testing against ORF710 fused to a DNA-binding domain, researchers can systematically screen for interactors. Split-ubiquitin Y2H variants are particularly suited for membrane proteins like ORF710.

Surface plasmon resonance (SPR) provides quantitative binding data. By immobilizing purified ORF710 on a sensor chip and flowing other purified ATV proteins over the surface, researchers can measure binding kinetics and affinities. This requires purification of multiple viral proteins but provides detailed interaction parameters.

For high-throughput analysis, protein arrays containing the complement of ATV proteins can be probed with labeled ORF710. This approach allows simultaneous testing of multiple interactions and requires relatively small amounts of protein.

Crosslinking mass spectrometry (XL-MS) can capture interactions in intact virions. By treating purified ATV particles with crosslinking reagents and analyzing the resulting crosslinked peptides by mass spectrometry, researchers can identify proteins in proximity to ORF710 and map interaction interfaces at amino acid resolution.

How can researchers assess the role of ORF710 in ATV's temperature-dependent morphological transformation?

Investigating ORF710's role in ATV's unique temperature-dependent tail formation requires a multi-faceted experimental approach:

Cryo-electron microscopy (cryo-EM) of wild-type and ORF710-mutant virions at different stages of tail development could provide direct structural evidence of ORF710's involvement. By preparing samples at different time points during incubation at 75-90°C, researchers could track the progressive changes in virion morphology . Comparison between wild-type and mutant virions could reveal structural differences attributed to ORF710 function.

Immunolocalization studies using gold-labeled antibodies against ORF710 could determine its spatial distribution during tail formation. If ORF710 is directly involved in tail assembly, it might relocalize or concentrate at the tail formation sites during the temperature-induced transformation.

In vitro reconstitution assays could test whether purified ORF710, potentially in combination with other viral proteins, can induce membrane remodeling or tubulation at elevated temperatures. Using artificial liposomes or membrane vesicles, researchers could observe whether ORF710 alone or in combination with other factors is sufficient to generate tail-like structures when heated to 75-90°C.

Mutagenesis studies targeting predicted temperature-sensing regions of ORF710 could identify domains crucial for the thermal trigger. Creating temperature-insensitive mutants (that fail to induce tail formation despite high temperatures) or temperature-hypersensitive mutants (that form tails at lower temperatures) would provide compelling evidence for ORF710's role as a thermal switch in ATV morphogenesis.

How can computational approaches predict ORF710 structure-function relationships?

Computational approaches offer powerful tools to predict structure-function relationships for ORF710, especially given the challenges of experimental structural determination for membrane proteins:

Molecular dynamics (MD) simulations can provide insights into temperature-dependent behavior of ORF710. By simulating the protein at different temperatures, researchers can identify regions that undergo significant conformational changes in the critical 75-90°C range where ATV tail formation occurs . Coarse-grained simulations can extend timescales to capture large conformational transitions that might be relevant to ORF710's function.

Protein-protein docking simulations can predict potential interactions between ORF710 and other ATV proteins. Tools like HADDOCK or ClusPro can generate models of protein complexes that might form during virion assembly or tail formation. These predictions can guide experimental validation through targeted mutagenesis of predicted interface residues.