Recombinant Lumpy skin disease virus E3 ubiquitin-protein ligase LAP (LW010), partial

What is the structural composition of recombinant LSDV E3 ubiquitin-protein ligase LAP (LW010)?

Recombinant LSDV E3 ubiquitin-protein ligase LAP (LW010) is a full-length viral protein consisting of 78 amino acids (1-78aa), typically expressed with an N-terminal His tag in E. coli expression systems. The amino acid sequence is MEGSDNTNTHCWICKDEYNVSTNFCNCKNEFKIVHKNCLEEWINFSHNTKCKICNGKYNIKKNKKSCLRWKCSFMYCN, which contains cysteine-rich regions characteristic of RING-finger domains commonly found in E3 ubiquitin ligases . The protein functions as a RING-type E3 ubiquitin transferase and is also known as Leukemia associated protein (LAP). The molecular structure features zinc-binding motifs typical of RING domains that facilitate protein-protein interactions essential for its ligase activity .

How does LW010 relate to other poxvirus proteins genetically?

LW010 (designated as LSDV010 in genomic analyses) shows significant homology to proteins in other poxviruses, particularly within the Chordopoxvirinae subfamily. Comparative genomic analyses reveal that LSDV010 shares 38% amino acid identity with the M153R protein from Swinepox virus (SPV) and approximately 25-30% identity with similar proteins from other poxviruses . The gene encoding LW010 is located in the terminal genomic regions of LSDV, where colinearity with other poxviruses is often disrupted and sequence identity is lower (averaging 43%) compared to genes in the central region (averaging 65%) . This positioning in the terminal region is consistent with genes involved in viral virulence and host range determination, suggesting LW010's potential role in these functions.

What are the optimal storage and reconstitution conditions for recombinant LW010 protein?

Recombinant LW010 protein requires specific handling conditions to maintain stability and functionality. The protein is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use scenarios . For reconstitution, the protein should be briefly centrifuged prior to opening and then reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Addition of 5-50% glycerol (with 50% being standard) to the reconstituted protein is recommended for long-term storage at -20°C/-80°C . Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided to preserve protein integrity and activity .

What role does LW010 play in LSDV virulence and host immune evasion?

LSDV010 (LW010) likely plays a significant role in viral virulence and host range determination based on its genomic location and functional classification. As an E3 ubiquitin ligase, LW010 is involved in protein ubiquitination, a process that can target host proteins for degradation through the proteasome pathway . This mechanism allows the virus to modulate host cell processes and evade immune responses. Comparative genomic analyses show that LW010 is positioned in the terminal regions of the LSDV genome, which typically contain genes responsible for viral virulence and host-specific interactions .

The protein contains a LAP/PHD-finger domain and transmembrane regions, suggesting membrane association and potential interaction with host factors . While direct experimental evidence of LW010's specific role in LSDV pathogenesis is limited, related E3 ubiquitin ligases in other poxviruses have been shown to target key components of host antiviral responses, including immune signaling molecules and transcription factors. Advanced research should focus on identifying the specific host targets of LW010 and elucidating how these interactions contribute to LSDV virulence and immune evasion strategies.

What are the challenges and considerations in expressing full-length LW010 in prokaryotic systems?

Expression of recombinant LW010 in prokaryotic systems presents several challenges that researchers should address. The protein contains cysteine-rich regions typical of RING finger domains, which can form disulfide bonds critical for proper folding and function . E. coli expression systems, which are commonly used for recombinant protein production, have a reducing cytoplasmic environment that may impact the formation of these disulfide bonds.

To optimize expression and purification of functional LW010 protein, researchers should consider the following:

• Expression strain selection: Use specialized E. coli strains designed for expression of proteins with disulfide bonds, such as Origami or SHuffle strains.

• Fusion tag optimization: While His-tags are commonly used (as seen in the commercial product), alternative fusion partners like GST or MBP may improve solubility.

• Inclusion body recovery: If LW010 forms inclusion bodies, protocols for solubilization and refolding should be optimized to recover functional protein.

• Purification strategy: Multi-step purification protocols may be necessary to achieve >90% purity, typically including initial capture via affinity chromatography followed by size exclusion or ion exchange chromatography.

• Functional verification: Activity assays specific to E3 ligase function should be established to confirm that the recombinant protein retains its enzymatic activity.

What purification protocols are recommended for isolating high-purity LW010 protein for structural studies?

For structural studies of LW010 protein, high-purity preparations are essential. Based on established methods for similar viral proteins, the following purification protocol is recommended:

• Initial extraction: After expression in E. coli, harvest cells by centrifugation (4,000 g, 15 minutes, 4°C) and resuspend in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1 mM DTT, and protease inhibitor cocktail .

• Cell disruption: Use sonication (20W, 40-second pulses with cooling intervals) or French press to lyse cells, followed by centrifugation at 40,000 g for 30 minutes to remove cellular debris .

• Affinity chromatography: Apply the clarified lysate to a Ni-NTA column equilibrated with binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole). Wash extensively and elute with a gradient of imidazole (10-250 mM) .

• Secondary purification: Further purify using size exclusion chromatography with a Superdex 75 column in a buffer containing 20 mM Tris-HCl pH a and 150 mM NaCl to achieve >95% purity for structural studies.

• Buffer exchange: For crystallography studies, exchange the protein into a low-salt buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl) using dialysis or ultrafiltration.

How can researchers verify the functionality of recombinant LW010 protein?

Verifying the functionality of recombinant LW010 protein is critical for ensuring experimental validity. As an E3 ubiquitin ligase, several assays can assess its activity:

• In vitro ubiquitination assay: This gold standard assay requires purified E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), ubiquitin, ATP, and potential substrate proteins. The reaction products are analyzed by SDS-PAGE and western blotting with anti-ubiquitin antibodies to detect ubiquitinated products.

• E3 ligase auto-ubiquitination test: Many E3 ligases can self-ubiquitinate in the absence of substrate. Incubate purified LW010 with E1, E2, ubiquitin, and ATP, then analyze by western blotting to detect higher molecular weight forms of LW010 indicating auto-ubiquitination.

• Substrate binding assays: Use pull-down assays, surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC) to assess binding between LW010 and potential substrate proteins identified through proteomic approaches.

• Structural integrity assessment: Circular dichroism (CD) spectroscopy can verify proper protein folding by analyzing secondary structure elements characteristic of RING-finger domains.

• Zinc binding verification: As a RING finger protein, LW010 should bind zinc ions. Atomic absorption spectroscopy or colorimetric assays using 4-(2-pyridylazo)resorcinol (PAR) can confirm zinc incorporation, which is essential for structural integrity and function.

What are the most effective techniques for studying LW010's interactions with host proteins?

Understanding LW010's interactions with host proteins is crucial for elucidating its role in viral pathogenesis. Several complementary techniques are recommended:

• Yeast two-hybrid screening: This approach can identify potential host protein interaction partners from a bovine cDNA library. Confirmation of interactions should be performed using additional methods.

• Co-immunoprecipitation (Co-IP): Transfect mammalian cells (preferably of bovine origin) with tagged LW010 constructs, then immunoprecipitate the protein complex and identify interacting partners by mass spectrometry.

• Proximity-based labeling: BioID or APEX2 fusion constructs of LW010 can be used to biotinylate proximal proteins in living cells, followed by streptavidin pulldown and mass spectrometry identification.

• Surface plasmon resonance (SPR): This technique can provide quantitative binding data, including association and dissociation constants for LW010 and its interaction partners.

• Cellular ubiquitination assays: Transfect cells with LW010 and candidate substrate proteins, then analyze ubiquitination status of the substrates through immunoprecipitation and western blotting.

• Fluorescence microscopy: Co-localization studies using fluorescently tagged LW010 and cellular proteins can provide insights into subcellular localization and potential interaction sites.

What methodology should be employed for detecting LW010 expression in LSDV-infected cells?

Detection of native LW010 expression in LSDV-infected cells requires specific approaches due to potential low expression levels:

• Quantitative PCR: Design primers specific to the LW010 gene sequence (similar to the H3L primers mentioned in the research: H3L forward: AAAACGGTATATGGAATAGAGTTGGAA, H3L reverse: AAATGAAACCAATGGATGGGATA) . For LW010-specific detection, primers should be designed based on the gene sequence provided in the genome analysis.

• Immunofluorescence microscopy: Generate specific antibodies against recombinant LW010 protein for immunostaining of infected cells. This approach allows visualization of protein expression and subcellular localization.

• Western blotting: Prepare cellular extracts from infected cells at various time points post-infection, separate by SDS-PAGE, and detect using anti-LW010 antibodies. This method provides information about expression kinetics and protein integrity.

• Mass spectrometry: Employ a proteomic approach similar to that described for virion analysis . Infected cells can be lysed, fractionated, and analyzed by LC-MS/MS to detect LW010 and potential modified forms.

• Ribosome profiling: This technique can detect active translation of the LW010 gene during infection, providing insights into expression timing.

How should researchers design experiments to study the role of LW010 in LSDV pathogenesis?

Designing experiments to study LW010's role in LSDV pathogenesis requires strategic approaches:

• Generation of recombinant viruses: Create LW010 deletion mutants and point mutants that disrupt E3 ligase activity using bacterial artificial chromosome (BAC) technology. Compare the phenotype of these mutants to wild-type virus in vitro and in vivo.

• Cell culture infection models: Establish bovine cell line infection models (such as MDBK cells) to compare wild-type and mutant virus replication kinetics, cytopathic effects, and host cell responses .

• Identification of cellular targets: Perform comparative proteomics between cells infected with wild-type and LW010-mutant viruses to identify host proteins whose stability is affected by LW010 expression.

• Immune response analysis: Assess the impact of LW010 on innate immune signaling pathways by measuring activation of key transcription factors (e.g., NF-κB, IRF3) and expression of type I interferons and proinflammatory cytokines in response to wild-type versus mutant virus infection.

• In vivo studies: Where ethically appropriate and legally permitted, animal studies comparing pathogenesis of wild-type and LW010-mutant viruses can provide definitive evidence of this protein's role in disease.

What are the critical considerations for analyzing protein-protein interactions involving LW010?

When analyzing protein-protein interactions involving LW010, researchers should consider:

• Confirmation across multiple methods: No single method for detecting protein-protein interactions is definitive; therefore, potential interactions should be validated using at least two independent techniques (e.g., yeast two-hybrid followed by co-immunoprecipitation).

• Appropriate controls: Include negative controls (unrelated proteins) and positive controls (known interacting protein pairs) in all interaction experiments.

• Domain mapping: After identifying interacting partners, determine which specific domains or residues within LW010 mediate these interactions through deletion and point mutation analysis.

• Physiological relevance: Confirm that identified interactions occur during actual LSDV infection, not just in overexpression systems.

• Functional consequences: Determine how each interaction affects the function of the host protein partner (e.g., degradation, altered localization, inhibition of activity).

• Host species considerations: Since LSDV primarily infects cattle, validation of interactions in bovine cells or with bovine proteins is essential for establishing physiological relevance.

What are common challenges in working with recombinant LW010 and how can they be addressed?

Researchers working with recombinant LW010 may encounter several challenges:

• Protein solubility issues: If LW010 forms inclusion bodies during expression, optimize conditions by:

  • Lowering expression temperature (16-20°C)

  • Using weaker promoters to slow expression

  • Expressing as a fusion with solubility-enhancing tags (MBP, SUMO)

  • Adding solubilizing agents (0.1% Triton X-100, 1M urea) to extraction buffers

• Protein stability problems: If purified LW010 aggregates or precipitates:

  • Optimize buffer conditions (test different pH values and salt concentrations)

  • Add stabilizing agents like glycerol (5-20%) or reducing agents (1-5 mM DTT)

  • Store at higher concentrations to prevent surface adsorption

  • Add carrier proteins (0.1% BSA) for dilute solutions

• Enzymatic activity issues: If purified LW010 shows poor E3 ligase activity:

  • Verify proper zinc incorporation using atomic absorption spectroscopy

  • Test different E2 enzymes, as E3 ligases often show specificity for particular E2 partners

  • Ensure buffers contain reducing agents to maintain cysteine residues in reduced state

  • Add zinc or other metal ions that might be required for activity

• Antibody cross-reactivity: When generating antibodies against LW010:

  • Design peptide antigens from unique regions of LW010 not conserved in host proteins

  • Validate antibody specificity against recombinant protein and in uninfected vs. infected cells

  • Pre-adsorb antibodies with host cell lysates to remove cross-reactive antibodies

How can researchers interpret conflicting data regarding LW010 function?

When faced with conflicting data regarding LW010 function, consider these analytical approaches:

• Experimental system differences: Results may vary between different expression systems (bacterial vs. mammalian) or cell types (bovine vs. human). Prioritize data from systems that most closely match LSDV's natural environment.

• Protein modification status: Post-translational modifications absent in recombinant systems may affect function. Compare proteins expressed in prokaryotic vs. eukaryotic systems.

• Concentration effects: LW010 may have different effects at different concentrations. Ensure experiments use physiologically relevant protein levels similar to those in infection.

• Temporal considerations: Some functions may be time-dependent. Analyze results across different time points post-infection or post-treatment.

• Genetic variation: Consider whether conflicting results stem from sequence variations between LSDV strains. Compare the exact sequence used in each study.

• Technical validation: Evaluate the technical robustness of conflicting results by assessing:

  • Statistical analysis and sample sizes

  • Reproducibility across independent experiments

  • Controls used in each experimental setup

  • Sensitivity and specificity of detection methods

• Integration of multiple data types: When possible, integrate data from diverse approaches (genetic, biochemical, structural) to develop a more comprehensive understanding of LW010 function.