ORF72 is a putative transmembrane protein encoded by the ABV genome. Its recombinant form is produced in E. coli and purified with a His-tag for ease of isolation . Key specifications include:
Parameter | Details |
---|---|
Protein Length | Full-length (1–72 amino acids) |
UniProt ID | A4ZUC0 |
Host Species | Acidianus bottle-shaped virus (ABV) |
Purity | >90% (SDS-PAGE validated) |
Storage | Lyophilized powder; store at -20°C/-80°C, avoid repeated freeze-thaw cycles |
Reconstitution | Deionized sterile water (0.1–1.0 mg/mL); add 5–50% glycerol for stability |
The amino acid sequence is:
MANEKTLFYALLGVGAIIVVLSTTGYLNNASNLSIAILFAVFAIAIASVYEFREPEIVVKPAKPTFEEKVIG
.
The ABV genome (23,814 bp, 35% G+C) contains terminal inverted repeats and shares limited homology with other viruses . ORF72 is part of a unique genomic region resembling bacteriophage φ29 and adenovirus, suggesting ancestral gene exchange or convergent evolution .
Recombinant ORF72 proteins differ between viral species:
Source | Length | Host | Expression System |
---|---|---|---|
ABV (A4ZUC0) | 1–72 aa | Acidianus bottle-shaped virus | E. coli (His-tagged) |
Ostreid Herpesvirus 1 | 1–188 aa | Pacific oyster herpesvirus | E. coli (His-tagged) |
The ABV ORF72 is significantly shorter, highlighting species-specific structural adaptations .
ORF72’s transmembrane nature aligns with potential roles in:
Host Membrane Interaction: Facilitating virion attachment or DNA entry into hyperthermophilic archaea.
Viral Assembly: Contributing to the envelope structure or nucleocapsid packaging.
While no direct functional assays are reported, its localization in the virion envelope (based on ABV morphology) supports these hypotheses .
Recombinant ORF72 is used in structural and functional studies, though its utility is limited by:
KEGG: vg:5129809
ORF72 is a 72-amino acid putative transmembrane protein from Acidianus bottle-shaped virus with the sequence: MANEKTLFYALLGVGAIIVVLSTTGYLNNASNLSIAILFAVFAIAIASVYEFREPEIVVKPAKPTFEEKVIG . The protein contains hydrophobic regions consistent with a transmembrane topology, which allows it to be integrated into viral and/or host membranes. Structural analysis suggests ORF72 likely contains at least one membrane-spanning domain, as indicated by its amino acid composition featuring multiple hydrophobic residues arranged in patterns typical of transmembrane segments. Secondary structure predictions suggest a possible alpha-helical conformation spanning the lipid bilayer, which is common among viral transmembrane proteins. This topology would be consistent with the functional requirements for a protein involved in virus-host interactions.
Recombinant ORF72 protein can be produced by cloning the full-length sequence (amino acids 1-72) into an expression vector (such as pET30a) with an N-terminal His-tag . The construct is then transformed into E. coli expression systems (like E. coli transetta DE3) for protein production . Following expression, the protein can be purified using affinity chromatography with Ni²⁺-Sepharose beads, taking advantage of the His-tag . When analyzed by SDS-PAGE, recombinant ORF72 migrates at approximately 25 kDa, which corresponds to its predicted molecular mass . For optimal yield and purity, expression conditions may need optimization regarding temperature, IPTG concentration, and induction time. Purification protocols typically involve lysis under native or denaturing conditions depending on the protein's solubility, followed by multiple washing steps to remove non-specifically bound proteins.
For optimal stability, purified recombinant ORF72 should be stored as a lyophilized powder at -20°C to -80°C upon receipt . Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can compromise protein integrity . For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and for long-term storage, glycerol should be added to a final concentration of 5-50% (with 50% being the default recommended concentration) . Prior to reconstitution, brief centrifugation of the vial is advised to bring contents to the bottom. The recommended storage buffer is Tris/PBS-based with 6% trehalose at pH 8.0, which helps maintain protein stability during storage and freeze-thaw cycles . Proper handling techniques should include using sterile equipment, minimizing exposure to room temperature, and avoiding multiple freeze-thaw cycles by preparing appropriately sized aliquots.
Determining the membrane topology of ORF72 requires multiple complementary approaches. Computational predictions using algorithms like TMDET can provide initial orientation hypotheses, though experimental validation is crucial . One effective approach combines protease protection assays with site-directed epitope tagging, where tags are inserted at different positions in the protein sequence, and their accessibility to antibodies or proteases on either side of the membrane is assessed. Fluorescence-based methods using environment-sensitive probes attached to specific residues can also reveal membrane-embedded regions. Additionally, techniques like substituted cysteine accessibility method (SCAM) can identify membrane-spanning segments by testing the accessibility of engineered cysteine residues to membrane-impermeable sulfhydryl reagents. The data from these experiments should be integrated with the TOPDB database, which collects topology information for transmembrane proteins . For definitive structural characterization, cryo-electron microscopy studies of the virus particles may help visualize ORF72 in its native context, though this approach is complicated by the heterogeneity of spindle-shaped virions .
Pull-down assays using recombinant ORF72 as bait have identified several potential interacting partners in host cells . Notably, ORF72 shows strong interactions with tubulin subunits (both alpha and beta chains), suggesting involvement with the host cytoskeleton . Additional interacting proteins include 1,4-alpha-glucan branching enzymes, tyrosine-protein phosphatases, and ABC transmembrane domain-containing proteins . To comprehensively study these interactions, researchers should employ multiple complementary approaches: (1) Co-immunoprecipitation using antibodies against ORF72 followed by mass spectrometry; (2) Yeast two-hybrid screening against host cDNA libraries; (3) Proximity labeling approaches such as BioID or APEX2; and (4) Surface plasmon resonance or microscale thermophoresis to quantify binding affinities. Validation of identified interactions should include reciprocal pull-downs, co-localization studies using confocal microscopy, and functional assays to assess the biological significance of these interactions. Protein interaction networks can be constructed using STRING database analysis followed by K-means clustering to identify functional protein groups .
Based on protein interaction studies, ORF72 appears to function primarily with tubulin components of the host cytoskeleton, suggesting a role in intracellular viral transport mechanisms . The interaction pattern differs from other viral membrane proteins like ORF25, which tends to function synergistically with actins . This specialized interaction profile indicates ORF72 may participate in microtubule-dependent trafficking of viral components during infection. The protein's transmembrane nature suggests it could be incorporated into the viral envelope, potentially mediating membrane fusion events during viral entry or egress. Additionally, antibodies targeting ORF72 have been shown to inhibit viral replication, further supporting its essential role in the viral life cycle . Mechanistically, ORF72 might function in conjunction with other viral proteins to modify host cell membranes or create specialized replication compartments. Acidianus bottle-shaped virus, as a member of the spindle-shaped virus family, may utilize ORF72 in the remarkable structural transitions these viruses undergo during maturation, potentially contributing to the variable dimensions observed in the spindle head (170 ± 50 nm long by 100 ± 30 nm wide) or tail (20 ± 9 nm wide) .
Studying ORF72 in archaeal systems presents unique challenges due to the extreme environments inhabited by Acidianus hosts and the specialized nature of archaeal molecular biology. An effective experimental strategy should begin with gene deletion or silencing approaches to assess the protein's essentiality for viral replication. CRISPR-Cas systems adapted for archaeal hosts can provide precise genetic manipulation capabilities. For functional complementation studies, researchers should create point mutations in conserved regions of ORF72 to identify critical functional residues. Time-course experiments during viral infection are crucial, monitoring ORF72 expression, localization, and interaction dynamics at different infection stages. For host-pathogen interaction studies, fluorescently tagged ORF72 can be expressed in archaeal cells to track its subcellular localization using high-temperature-adapted microscopy setups. When designing fusion proteins, researchers must carefully position tags to avoid disrupting membrane topology or protein function, preferably validating multiple tag positions. Additionally, heterologous expression systems can be developed using related thermophilic organisms if direct manipulation of Acidianus species proves challenging. All experiments should include appropriate controls for the extreme temperature and pH conditions under which these archaeal viruses naturally operate.
A multi-technique approach is essential for comprehensive characterization of ORF72. For structural analysis, X-ray crystallography of the purified protein can provide atomic-level details, though crystallization of membrane proteins presents challenges . Circular dichroism spectroscopy offers insights into secondary structure content, particularly useful for confirming alpha-helical content typical of transmembrane domains. Nuclear magnetic resonance (NMR) spectroscopy of isotopically labeled ORF72 can provide dynamic structural information in membrane-mimetic environments. For interaction studies, isothermal titration calorimetry (ITC) can quantify thermodynamic parameters of binding to host proteins. Protein crosslinking coupled with mass spectrometry can capture transient interactions and identify interaction interfaces. AlphaFold2 or similar prediction tools can generate structural models to guide experimental design, particularly for identifying potential coiled-coil structures or leucine zipper motifs that might mediate protein-protein or protein-nucleic acid interactions . For visualization in the viral context, cryo-electron microscopy and tomography approaches similar to those used for ATSV (Acidianus tailed spindle virus) would be valuable, potentially allowing subtomographic averaging to enhance resolution of structurally heterogeneous particles .
Researchers working with ORF72 frequently encounter several challenges that require systematic troubleshooting. First, membrane protein expression often results in inclusion body formation in E. coli systems. This can be addressed by: (1) using specialized E. coli strains designed for membrane protein expression; (2) lowering induction temperature to 16-20°C; (3) reducing IPTG concentration; or (4) adding membrane-mimetic compounds to the growth medium. Second, protein aggregation during purification can be mitigated by including appropriate detergents in purification buffers, with screening of multiple detergent types (non-ionic, zwitterionic) recommended to identify optimal conditions. Third, difficulties in reconstitution may occur, requiring testing of different buffer compositions, pH conditions, and reconstitution methods (dialysis vs. direct dilution). For functional studies, the extreme thermophilic nature of the native host environment presents challenges for in vitro assays, necessitating careful temperature control and the use of thermostable reagents. Additionally, the small size of ORF72 (72 amino acids) may complicate detection in some analytical techniques, potentially requiring specialized antibodies or high-sensitivity detection methods . When troubleshooting protein-protein interaction studies, varying salt concentrations, pH, and detergent conditions is recommended to eliminate non-specific interactions.
Comparisons between ORF72 from Acidianus bottle-shaped virus and analogous proteins from other archaeal viruses reveal interesting evolutionary patterns. The ORF72 protein appears to belong to a broader class of small transmembrane proteins found in several archaeal viruses with spindle-shaped morphology . While sequence conservation may be limited, functional similarities suggest convergent evolution driven by the constraints of virus-host interactions in extreme environments. Notably, members of the large tailed spindle virus superfamily, including ATSV (Acidianus tailed spindle virus), encode similar putative transmembrane proteins that likely contribute to their unique morphology and assembly . Comparative genomic analysis indicates that while these proteins share functional characteristics, they may have evolved independently to fulfill similar roles in different viral lineages. Unlike some other viral membrane proteins that have recognizable homologs across viral families, ORF72-like proteins often show virus-specific adaptations, reflecting the specialized host range and extreme environmental conditions of archaeal viruses. This evolutionary pattern contrasts with more conserved viral proteins involved in nucleic acid metabolism, suggesting that membrane proteins evolve more rapidly to adapt to specific host membrane compositions found in extreme environments.
Functional comparison between ORF72 and other viral membrane proteins reveals specialized roles in host-virus interactions. Pull-down experiments demonstrate that while both ORF72 and ORF25 interact with components of the host cytoskeleton, they exhibit distinct preferences: ORF72 primarily associates with tubulins, whereas ORF25 shows stronger interactions with actins . This functional differentiation suggests these proteins may cooperate to manipulate different aspects of the host cytoskeleton during infection. Antibody inhibition studies indicate both proteins contribute to viral replication, but potentially through different mechanisms . In terms of expression and regulation, archaeal viruses often organize their genes into functional modules with coordinated expression. Similar to genes encoding anti-defense functions in other archaeal viruses, such as those found in Sulfolobales archaeal virus and Sulfolobus spindle-shaped virus (SSV) genomes, ORF72 may be part of a regulatory network controlling viral gene expression during infection . The specialized interaction profile of ORF72 with host tubulins suggests it might function in microtubule-dependent trafficking of viral components, while other viral membrane proteins might be more involved in entry, assembly, or immune evasion. This functional specialization represents an efficient evolutionary strategy allowing even viruses with limited genome capacity to manipulate multiple host cellular systems.