Recombinant Acidianus filamentous virus 2 Putative transmembrane protein ORF289 (ORF289)

What is Acidianus filamentous virus 2 (AFV2) and how does ORF289 relate to its structure?

Acidianus filamentous virus 2 (AFV2) is a novel filamentous virus isolated from the hyperthermophilic archaeal genus Acidianus from a hot, acidic spring (93°C, pH 2) in a solfataric field in Italy. While structurally similar to lipothrixviruses, AFV2 distinguishes itself through unique terminal and core structures. The virus is classified in a proposed new genus, "Deltalipothrixvirus," within the family Lipothrixviridae. AFV2 virions are filamentous structures measuring 1,100 ± 50 nm in length and 24 ± 1 nm in width .

ORF289 is one of the putative transmembrane proteins encoded by the AFV2 double-stranded DNA genome, which spans 31,787 bp. Based on genome analysis, ORF289 is believed to contribute to the viral envelope structure that encases the viral core. When treated with 0.1% sodium dodecyl sulfate (SDS), AFV2 virion width reduces to 17 ± 1 nm, revealing the envelope where proteins like ORF289 are likely located .

What is known about the protein composition of AFV2?

Analysis of AFV2 virions using SDS-polyacrylamide gel electrophoresis revealed seven proteins with apparent molecular masses of 6, 26, 35, 40, 45, 50, and 65 kDa. Interestingly, no lipids were detected after extraction with chloroform-methanol (volume ratio, 1:1) and thin-layer chromatography analysis, which distinguishes AFV2 from other crenarchaeal viruses. This lack of detectable lipids is part of what makes AFV2 unique within the Lipothrixviridae family .

As a putative transmembrane protein, ORF289 is likely among these seven proteins, potentially playing a role in the virus structure and possibly in host interaction, though specific functional characterization requires further investigation.

How is recombinant ORF289 protein typically produced for research applications?

Recombinant full-length AFV2 ORF289 protein is typically produced using heterologous expression in E. coli systems. The standard protocol involves:

• Cloning the full-length ORF289 gene (encoding amino acids 1-289) into an expression vector

• Adding an N-terminal histidine tag to facilitate purification

• Transforming the construct into an E. coli expression strain

• Inducing protein expression under optimized conditions

• Cell lysis and protein extraction

• Purification using nickel affinity chromatography

• Further purification steps as needed (size exclusion, ion exchange)

• Lyophilization for long-term storage

The resulting product is typically a lyophilized powder containing the purified recombinant ORF289 protein with an N-terminal His-tag. For research applications, the protein can be reconstituted in deionized sterile water to concentrations of 0.1-1.0 mg/mL .

What is the predicted structural and functional role of ORF289 in AFV2 viral assembly and infection?

Based on sequence analysis and the presence of hydrophobic regions consistent with transmembrane domains, ORF289 is predicted to be a viral membrane protein. The amino acid sequence (MAIAKEFLLTVLNYIANGVVNVQSSTTQAVTTLAPYQIIAIMKNNNVTVSRTTITSISVSDVVNASQEETLTIRYSGTDASPFTYTTDEIEIWASTQSALLYKIADIQLQTPLSKTEH DYLNIEYEIIITAGASYTTTSSMSQYTSVVTFRTLVAPILYFFALFLVPAWSTVLKQNPTFPQSQLSNYISPSSYQGINAMYVGSNQVTIVSKLVGFGTTTVSIVVNGEVTSTQVNAPIFIGVTTPS GVLVLAYNYYSGTISKYVSLTVTTTYGSATVINQFETKTTGGTT) contains regions suggesting potential roles in:

• Virion terminal structure formation - possibly contributing to the unusual "bottle brush" terminal structures observed via electron tomography

• Host cell recognition and attachment - though direct evidence of AFV2 virion attachment to host cells has not been documented despite extensive efforts

• Maintaining structural integrity of the viral envelope

The protein lacks detectable sequence homology with characterized proteins outside the Lipothrixviridae family, suggesting a specialized role in these archaeal viruses. Further structural studies using X-ray crystallography or cryo-electron microscopy would be needed to confirm these predictions.

How does the genetics of AFV2 ORF289 compare to homologous genes in other archaeal viruses?

Genome analysis of AFV2 revealed that it contains 51 putative open reading frames (ORFs), with approximately 70% arranged in putative operons. While eight ORFs show homology to other lipothrixviral ORFs from the genera Sulfolobus and Acidianus, only three genes have been assigned functions based on sequence matches with public databases .

ORF289 specifically has limited sequence similarity to proteins from related archaeal viruses, reflecting the significant genetic diversity among archaeal viruses even within the same family. When comparing ORF289 to genes in other lipothrixviruses:

This limited homology underscores the unique nature of AFV2 and its proposed classification in a distinct genus ("Deltalipothrixvirus") within the Lipothrixviridae family .

What analytical techniques are most effective for studying protein-protein interactions involving ORF289?

For investigating protein-protein interactions involving the recombinant ORF289 protein, several advanced analytical techniques are particularly suitable:

• Pull-down assays using the His-tag - Leveraging the N-terminal His-tag to isolate ORF289 along with potential binding partners from viral or host protein mixtures

• Cross-linking mass spectrometry (XL-MS) - Especially valuable for membrane proteins like ORF289, this technique can capture transient interactions under near-native conditions by chemically cross-linking interacting proteins before mass spectrometric analysis

• Surface plasmon resonance (SPR) - For quantitative measurement of binding kinetics between ORF289 and candidate interaction partners

• Microscale thermophoresis (MST) - Requires minimal sample amounts and can detect interactions in complex biological matrices

• Biolayer interferometry (BLI) - Provides real-time data on association and dissociation rates

• Yeast two-hybrid screening adapted for membrane proteins - Using split-ubiquitin systems specifically designed for membrane protein interactions

For confirming the transmembrane nature of ORF289 specifically, techniques such as circular dichroism spectroscopy in lipid environments and protease protection assays would provide valuable structural information .

What are the optimal conditions for reconstitution and storage of recombinant ORF289 protein?

The optimal conditions for handling recombinant ORF289 protein are informed by both its archaeal viral origin and transmembrane nature:

Reconstitution Protocol:

• Briefly centrifuge the vial containing lyophilized protein prior to opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• For enhanced stability, add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

Storage Conditions:

• Store working aliquots at 4°C for up to one week

• For long-term storage, keep at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

• Store in Tris/PBS-based buffer with 6% trehalose at pH 8.0 for optimal stability

What are the methodological considerations for studying ORF289 in membrane mimetic systems?

As a putative transmembrane protein, ORF289 requires special consideration when designing experiments to study its structure and function:

• Selection of appropriate membrane mimetics:

  • Detergent micelles (DDM, LDAO, or OG for initial screening)

  • Lipid nanodiscs with archaeal lipid compositions when possible

  • Liposomes with varied lipid compositions to mimic archaeal membranes

  • Amphipols for maintaining native-like folding during structural studies

• Reconstitution protocol optimization:

  • Detergent screening to identify optimal solubilization conditions

  • Gradual detergent removal via dialysis or adsorption to bio-beads

  • Monitoring protein folding throughout reconstitution using circular dichroism

• Functional assays in membrane environments:

  • Proteoliposome-based assays to assess potential ion channel activity

  • Lipid mixing assays to investigate membrane fusion capabilities

  • Protein-lipid interaction analysis using fluorescence techniques

• Structural analysis considerations:

  • Cryo-EM sample preparation modifications for membrane proteins

  • Specialized NMR techniques for membrane-embedded proteins

  • Molecular dynamics simulations using archaeal membrane parameters

Given that AFV2 exists in a stable carrier state with its host and doesn't appear to cause cell lysis, ORF289 might be involved in persistent host interaction rather than lytic functions, which should be considered when designing functional studies .

How can researchers effectively design expression systems for ORF289 to maximize yield and proper folding?

Optimizing expression systems for recombinant ORF289 requires addressing several challenges associated with archaeal viral membrane proteins:

• Expression vector selection:

  • Vectors with tunable promoter strength to prevent toxicity

  • Incorporation of fusion partners that enhance membrane protein folding (e.g., MBP, SUMO)

  • Inclusion of C-terminal GFP fusion as a folding indicator

  • Codon optimization for E. coli expression while preserving critical folding elements

• Host strain optimization:

  • C41(DE3) or C43(DE3) strains specifically developed for membrane protein expression

  • Strains with enhanced membrane capacity like Lemo21(DE3)

  • Consideration of archaeal expression hosts for difficult-to-express constructs

• Expression condition parameters:

  Parameter	Recommended Range	Rationale
  Temperature	16-30°C	Lower temperatures slow folding and prevent aggregation
  Induction OD	0.6-0.8	Optimal cell density for membrane protein expression
  Inducer concentration	0.1-0.5 mM IPTG	Lower concentrations reduce aggregation
  Media supplements	1% glucose	Provides energy for membrane protein insertion
  Expression time	16-20 hours	Extended time allows proper membrane insertion

• Extraction and purification considerations:

  • Gentle cell lysis methods to preserve membrane integrity

  • Screening multiple detergents for optimal extraction

  • Two-step purification using affinity chromatography followed by size exclusion

  • Quality control using size exclusion chromatography profiles and thermal stability assays

What functional domains have been identified in ORF289 and how do they contribute to its putative transmembrane role?

Analysis of the ORF289 amino acid sequence (289 amino acids) reveals several potential functional domains that support its classification as a transmembrane protein:

• N-terminal hydrophilic domain (aa 1-60):

  • Contains several charged residues

  • Likely oriented toward either the virion interior or exterior

  • May participate in protein-protein interactions with other viral components

• Central hydrophobic regions (approximately aa 61-200):

  • Multiple hydrophobic segments consistent with transmembrane helices

  • The segment "LYFFALFLVPAWSTVL" is particularly characteristic of a transmembrane domain

  • These regions likely anchor the protein within the viral envelope

• C-terminal domain (aa 201-289):

  • More hydrophilic character

  • Contains the sequence "VNAPIFIGVTTPSGVLVLAYNYYSGTISKYVSLTVTTTYGSATVINQFETKTTGGTT"

  • Potentially involved in specific recognition functions or structural roles in virion assembly

No enzymatic domains have been definitively identified in ORF289, suggesting its primary role is structural rather than catalytic. This is consistent with the observation that many viral structural proteins in archaeal viruses have highly specialized functions without clear homologs in other viral families .

How does the extreme environmental origin of AFV2 affect the biochemical properties of ORF289?

The origin of AFV2 from extreme environments (93°C, pH 2) has significant implications for the biochemical properties of ORF289:

• Thermostability considerations:

  • Higher than average content of hydrophobic amino acids that contribute to protein core stability

  • Potential disulfide bonds that remain stable at high temperatures

  • Compact structural domains with reduced loop regions susceptible to thermal denaturation

• Acid stability features:

  • Reduced number of acid-labile peptide bonds

  • Strategic distribution of charged residues to maintain folding at low pH

  • Potential acid-resistant post-translational modifications in the native protein

• Experimental implications:

  Property	Native AFV2 ORF289	Recombinant E. coli-produced ORF289
  Thermostability	Likely stable at temperatures >80°C	Potentially less thermostable due to lack of archaeal-specific folding machinery
  pH stability	Functional at pH 2-3	May retain some acid tolerance but likely optimized for pH 7-8
  Structural integrity	Maintained in extreme conditions	May require stabilizing buffers and additives
  Post-translational modifications	Possible archaeal-specific modifications	Lacks archaeal-specific modifications

What evidence supports the transmembrane nature of ORF289, and what methods would confirm this prediction?

Several lines of evidence support the classification of ORF289 as a transmembrane protein:

• Sequence-based predictions:

  • Hydropathy plot analysis showing multiple hydrophobic regions of sufficient length to span a membrane

  • The presence of charged residue distributions typical of transmembrane proteins (positive inside rule)

  • Secondary structure predictions indicating alpha-helical segments of appropriate length for membrane spanning

• Structural context:

  • AFV2 virions have an envelope structure visible after SDS treatment

  • ORF289 is likely one of the seven proteins detected in purified virions

  • The protein's predicted properties align with requirements for maintaining viral envelope structure

To definitively confirm the transmembrane nature of ORF289, researchers would need to employ multiple complementary approaches:

These approaches would not only confirm the transmembrane nature of ORF289 but could also provide detailed information about its topology and orientation within membranes .

How might ORF289 be used as a model for studying archaeal virus-host interactions?

ORF289 presents several valuable opportunities for advancing our understanding of archaeal virus-host interactions:

• Investigation of extremophilic viral attachment mechanisms:

  • As a putative membrane protein from a virus that exists in a stable carrier state with its host, ORF289 may participate in the non-lytic, persistent infection strategy observed in AFV2

  • Using fluorescently labeled recombinant ORF289, researchers could assess binding to Acidianus cell surface components

  • Developing ORF289 mutants could help identify specific regions responsible for host recognition

• Evolution of viral membrane proteins in extreme environments:

  • Comparative analysis of ORF289 with membrane proteins from mesophilic viruses could reveal adaptations specific to hyperthermophilic conditions

  • Ancestral sequence reconstruction approaches could trace the evolutionary pathway of ORF289 and related proteins

• Model system for studying viral persistence:

  • AFV2 establishes a stable carrier state without causing cell lysis, making ORF289 a potential component in mechanisms of viral persistence

  • Expression of ORF289 in heterologous hosts could help determine if it plays a role in establishing or maintaining persistent infection

  • Development of ORF289-based tools could enable manipulation of virus-host dynamics in archaeal systems

What research gaps need to be addressed to better understand the structure-function relationship of ORF289?

Several critical knowledge gaps currently limit our understanding of ORF289:

• High-resolution structural information:

  • No crystal or cryo-EM structure currently exists for ORF289

  • Structural studies are needed to confirm transmembrane topology and identify potential functional sites

  • Comparative modeling is limited by the lack of close homologs with known structures

• Functional characterization:

  • The precise role of ORF289 in the viral life cycle remains speculative

  • Experimental evidence is needed to confirm interactions with host cell components

  • The contribution of ORF289 to the unusual terminal structures of AFV2 requires investigation

• Methodological challenges:

  Challenge	Potential Solution	Expected Outcome
  Lack of genetic systems for AFV2	Development of CRISPR-based tools for archaeal viruses	Ability to create ORF289 mutants in viral context
  Difficulty culturing Acidianus hosts	Optimization of growth conditions and development of reporter systems	Improved ability to study virus-host interactions
  Limited structural data	Application of advanced membrane protein structural techniques	High-resolution structure of ORF289
  Unknown binding partners	Proximity labeling approaches in native context	Identification of host and viral interaction partners

Addressing these gaps would significantly advance our understanding of archaeal virus biology and potentially reveal novel mechanisms of virus-host interaction in extreme environments .

How does research on ORF289 contribute to the broader understanding of viral evolution and extremophilic adaptations?

Research on ORF289 offers unique insights into several broader scientific questions:

• Viral adaptation to extreme environments:

  • AFV2 thrives in conditions (93°C, pH 2) that would rapidly denature most proteins

  • Understanding how ORF289 maintains structural integrity under these conditions could reveal fundamental principles of protein thermostability

  • Comparative analysis with mesophilic viral proteins could identify specific adaptations to extreme conditions

• Evolutionary insights:

  • The limited sequence similarity between ORF289 and proteins from other lipothrixviruses highlights the rapid evolution of archaeal viruses

  • Studying the genomic context of ORF289 within the unusual 31,787 bp genome of AFV2 provides insights into viral genome evolution

  • The unusual terminal structures of AFV2 virions, which may involve ORF289, represent potentially novel solutions to the challenges of viral assembly and host entry

• Applications in synthetic biology:

  • Extremophilic viral proteins like ORF289 represent untapped resources for designing proteins stable under harsh conditions

  • Understanding the structure-function relationship of ORF289 could inform the design of novel membrane proteins for biotechnological applications

  • The non-lytic, persistent relationship between AFV2 and its host could inspire new approaches to engineering stable host-vector systems