Recombinant Khujand virus Phosphoprotein (P)

What is Khujand virus and how does its phosphoprotein compare to other lyssavirus P proteins?

Khujand virus (KHUV) is a bat lyssavirus isolated from the whiskered bat (Myotis mystacinus) in Tajikistan in 2001. It belongs to the Lyssavirus genus within the Rhabdoviridae family . Phylogenetically, KHUV is most closely related to European Bat Lyssavirus-2 (EBLV-2), with 79.0% nucleotide identity in the nucleoprotein (N) gene .

The KHUV phosphoprotein (P) consists of 297 amino acids with a molecular weight of 24,373 Da . Like other lyssavirus P proteins, it functions as a multifunctional protein with roles in:

• Acting as a noncatalytic cofactor for viral RNA-dependent RNA polymerase (L protein)

• Antagonizing host interferon (IFN) responses

Sequence analysis of KHUV P protein reveals the characteristic amino acid sequence "MSKIFVNPSAIRAGLADLEMAEETVDLINRNVEDNQAHLQGEPIEVEALPEDRRLHISEQKHSQLDSACGKEEGSDDDFY..." . Comparing this sequence with other lyssavirus P proteins indicates conserved functional domains that are essential for viral replication and immune evasion.

What are the functional domains of Khujand virus phosphoprotein and their significance in research applications?

Based on comparative analysis with other lyssaviruses, particularly rabies virus (RABV), the KHUV phosphoprotein contains several functional domains:

• N-terminal domain: Likely contains regions involved in IFN antagonism. In RABV, amino acids 176-186 are essential for inhibiting IFN induction . Similar regions may exist in KHUV P.

• Central domain: Contains regions for interaction with nucleoprotein (N) to form the viral nucleocapsid complex.

• C-terminal domain: Likely contains the L protein binding site. In RABV, the sequence NPYNE at positions 1929-1933 in the L protein is critical for binding to P protein .

These domains make the P protein valuable for research applications including:

• Studying virus-host interactions

• Developing antiviral strategies targeting P-L interactions

• Understanding mechanisms of immune evasion by lyssaviruses

• Comparative studies of lyssavirus evolution and host adaptation

What are the optimal expression systems for producing functional recombinant Khujand virus P protein?

The recombinant KHUV P protein can be expressed in several systems, each with advantages for different research applications:

E. coli expression system:

• Most commonly used for basic structural studies and antibody production

• The commercially available recombinant KHUV P protein is produced in E. coli with ≥85% purity

• Protocol: The full-length P gene (encoding amino acids 1-297) is cloned into an expression vector (e.g., pET series), transformed into BL21(DE3) cells, induced with IPTG, and purified using affinity chromatography (typically His-tag purification)

• Advantages: High yield, cost-effective, suitable for structural studies

• Limitations: May lack post-translational modifications present in mammalian systems

Mammalian expression systems (HEK293, BHK-21):

• Optimal for functional studies where proper folding and post-translational modifications are critical

• Protocol: The P gene is cloned into mammalian expression vectors (e.g., pcDNA3.1), transfected into mammalian cells, and the protein is purified using affinity tags

• Advantages: Proper protein folding and modifications, suitable for interaction studies

• Similar approaches have been successful for expressing and studying lyssavirus proteins, as demonstrated with ABLV glycoprotein expressed in 293F cells

Baculovirus expression system:

• Provides a balance between yield and proper folding

• Advantages: Higher yield than mammalian systems while maintaining proper folding

• Useful for large-scale production for structural studies

For functional studies investigating P protein interactions with host factors or other viral proteins, mammalian expression systems are recommended despite lower yields.

What are the key considerations for purifying recombinant Khujand virus P protein while maintaining its functional integrity?

Successful purification of functional recombinant KHUV P protein requires careful attention to several factors:

Affinity tag selection:

• His-tag (6×His): Most commonly used, enables purification using Ni-NTA agarose

• GST-tag: Alternative approach useful for solubility enhancement and pull-down assays

• The tag position (N or C-terminal) should be selected based on known functional domains to minimize interference

Buffer optimization:

• Phosphate buffers (pH 7.4-8.0) with 150-300 mM NaCl are typically suitable

• Addition of 5-10% glycerol and 1-5 mM DTT or 2-ME helps maintain stability

• For proteins prone to aggregation, low concentrations (0.05-0.1%) of non-ionic detergents (Triton X-100, NP-40) may be included

Purification protocol:

• Cell lysis: Sonication or French press in buffer containing protease inhibitors

• Clarification: Centrifugation at 15,000-20,000×g for 30 minutes

• Affinity chromatography: Binding to Ni-NTA resin for His-tagged proteins

• Washing: Multiple washes with increasing imidazole concentrations (10-40 mM)

• Elution: With 250-300 mM imidazole

• Dialysis: Against storage buffer to remove imidazole

• Quality control: SDS-PAGE and Western blotting to confirm purity

Functional validation:

• Circular dichroism to assess secondary structure

• Thermal shift assays to evaluate stability

• Binding assays with known interaction partners (e.g., L protein fragments)

For studying interactions with host proteins, additional purification steps such as size exclusion chromatography may be necessary to ensure high purity (>95%).

How can researchers effectively use recombinant Khujand virus P protein to study its interaction with host immune factors?

Studying KHUV P protein interactions with host immune factors requires specialized experimental approaches:

Co-immunoprecipitation (Co-IP) assays:

• Express tagged recombinant KHUV P protein in mammalian cells (e.g., HEK293T)

• Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and protease inhibitors

• Incubate lysates with antibodies against the tag or suspected interaction partners

• Precipitate using protein A/G beads and analyze by Western blotting

• This approach has successfully identified interactions between RABV P and pattern recognition receptors like RIG-I, MDA5, and Lgp2

Yeast two-hybrid screening:

• Useful for discovering novel interaction partners

• Clone KHUV P into bait vector and screen against human cDNA libraries

• Validate hits using co-IP or GST pull-down assays

Luciferase reporter assays for IFN antagonism:

• Transfect cells with IFN-β promoter-luciferase reporter construct

• Co-transfect with KHUV P expression plasmid

• Stimulate cells with poly(I:C) or other IFN inducers

• Measure luciferase activity to quantify IFN inhibition

• Similar approaches have demonstrated that RABV P protein inhibits poly(I:C)-induced IFN-β promoter activity

Confocal microscopy for subcellular localization:

• Express fluorescently tagged KHUV P in mammalian cells

• Co-stain for cellular compartments or co-express fluorescently tagged host factors

• Analyze colocalization using confocal microscopy

• This approach can reveal interactions with IRF3, STAT1, or other immune signaling proteins

These methods can be used to compare KHUV P protein with P proteins from other lyssaviruses to understand species-specific differences in immune evasion strategies.

What are the most effective approaches for developing monoclonal antibodies against Khujand virus P protein?

Developing high-quality monoclonal antibodies against KHUV P protein requires systematic approaches:

Immunization strategy:

• Prepare 100 μg of purified recombinant KHUV P protein emulsified with Freund's complete adjuvant for initial immunization

• Boost with the same amount of protein mixed with Freund's incomplete adjuvant every 2 weeks (twice)

• Final boost with purified protein before hybridoma fusion

• This approach has been successful for generating mAbs against lyssavirus L protein

Hybridoma generation protocol:

• Isolate splenocytes from immunized mice (BALB/c preferred)

• Fuse with SP2/0 myeloma cells using polyethylene glycol 2000

• Culture in RPMI 1640 medium containing HAT selection medium

• Screen hybridomas by indirect ELISA using purified KHUV P protein (2 μg/mL in carbonate buffer, pH 9.6)

• Subclone positive hybridomas three times by limiting dilution

• This methodology has been validated for lyssavirus proteins

Antibody characterization:

• Determine isotypes using isotyping kits

• Test reactivity by Western blot and immunofluorescence assay

• Assess cross-reactivity with P proteins from other lyssaviruses

• Map epitopes using truncated protein fragments

Epitope mapping strategy:

• Generate overlapping fragments of KHUV P protein

• Express fragments as fusion proteins with His or GST tags

• Test reactivity of mAbs with each fragment by Western blot

• Progressively narrow down the epitope by expressing smaller fragments

• Confirm the minimal epitope using alanine scanning mutagenesis

• This approach can identify conserved epitopes across lyssaviruses

For optimal results, consider developing antibodies against both linear and conformational epitopes by using both denatured and native protein for screening.

How can researchers investigate the role of Khujand virus P protein in viral RNA synthesis and viral replication?

Investigating KHUV P protein's role in viral RNA synthesis requires specialized techniques:

Minigenome assays:

• Construct a KHUV-specific minigenome containing a reporter gene (luciferase or GFP) flanked by KHUV leader and trailer sequences

• Co-transfect cells with the minigenome and plasmids expressing KHUV N, P, and L proteins

• Measure reporter gene expression as an indicator of viral RNA synthesis

• Create P protein mutants to identify functional domains

• This approach has been used successfully with rabies virus to evaluate P protein function as a cofactor of viral RNA polymerase

Protein-protein interaction analysis:

• Express and purify recombinant KHUV P and L proteins

• Perform in vitro binding assays to map interaction domains

• Generate truncated or mutated versions of P to identify essential regions

• In RABV, the sequence NPYNE at positions 1929-1933 in the L protein is critical for binding P protein , suggesting similar critical regions may exist in KHUV

Trans-complementation assays:

• Generate P-deficient KHUV or use P-deficient RABV as a surrogate system

• Complement with wild-type or mutant KHUV P protein

• Measure viral replication efficiency

• This approach can identify functionally important regions of P protein

Structural analysis approaches:

• Use X-ray crystallography or cryo-EM to determine the structure of KHUV P protein alone or in complex with N or L protein fragments

• Perform molecular dynamics simulations to understand structural flexibility

• Use hydrogen-deuterium exchange mass spectrometry to identify regions involved in protein-protein interactions

These approaches together provide comprehensive insights into the roles of KHUV P protein in viral RNA synthesis and replication.

What methodologies are most effective for comparative analysis of interferon antagonism between Khujand virus P protein and other lyssavirus phosphoproteins?

Comparing interferon antagonism activities of KHUV P with other lyssavirus P proteins requires systematic approaches:

Luciferase reporter assays:

• Transfect cells with reporters containing IFN-β promoter, ISRE (Interferon-Stimulated Response Element), or GAS (Gamma-Activated Sequence) controlling luciferase expression

• Co-transfect with expression plasmids for P proteins from different lyssaviruses (KHUV, RABV, EBLV-1, EBLV-2, etc.)

• Stimulate with appropriate inducers (poly(I:C) for IFN-β, IFN-α/β for ISRE, IFN-γ for GAS)

• Measure luciferase activity to quantify and compare inhibition levels

• Similar approaches have shown that RABV P protein isoforms inhibit poly(I:C)-induced IFN-β promoter activity

IRF3 phosphorylation and nuclear translocation assays:

• Express KHUV P and other lyssavirus P proteins in cells

• Stimulate with poly(I:C) or RIG-I ligands

• Analyze IRF3 phosphorylation by Western blot using phospho-specific antibodies

• Examine IRF3 nuclear translocation by immunofluorescence or nuclear/cytoplasmic fractionation

• RABV P has been shown to bind to an activation-intermediate form of IRF3, preventing its full activation

Quantitative RT-PCR for IFN and ISG expression:

• Express P proteins in appropriate cell lines (e.g., human or bat cells)

• Stimulate with IFN inducers

• Measure mRNA levels of IFN-β, Mx1, OAS1, and other ISGs by qRT-PCR

• This approach has revealed that RABV P protein mutants lacking amino acids 176-186 cannot inhibit IFN induction

Co-immunoprecipitation to identify interactions with host factors:

• Express tagged P proteins from different lyssaviruses

• Immunoprecipitate and identify bound host factors by Western blot or mass spectrometry

• Compare binding profiles to identify conserved and species-specific interactions

• RABV P has been shown to interact with RIG-I-like receptors (RLRs) and IRF3

A comprehensive table comparing the interferon antagonism mechanisms of different lyssavirus P proteins would help identify conserved strategies and virus-specific adaptations.

How can truncated forms of Khujand virus P protein be generated and utilized to study domain-specific functions?

Generating and utilizing truncated forms of KHUV P protein involves several methodological considerations:

Design and generation of truncation mutants:

• Analyze sequence alignments of lyssavirus P proteins to identify conserved domains

• Design truncation mutants targeting specific functional regions:

  • N-terminal truncations (similar to natural P2, P3, P4, P5 isoforms in RABV)

  • C-terminal truncations

  • Internal deletions of specific domains

• Clone into expression vectors with appropriate tags (His, FLAG, HA) for detection and purification

• Express in E. coli or mammalian cells depending on the experimental goals

• Similar approaches with RABV have identified that truncated P proteins (P2, P3) function as IFN antagonists

Functional characterization of truncation mutants:

Expression of naturally occurring isoforms:

• Identify potential alternative start codons in KHUV P gene (similar to positions 20, 53, 69, and 83 in RABV P gene )

• Generate constructs expressing each potential isoform (P1, P2, P3, etc.)

• Compare their functions in:

  • IFN antagonism (reporter assays)

  • Subcellular localization (immunofluorescence)

  • Host protein interactions (co-IP)

• Research with RABV has shown that these truncated isoforms contribute to pathogenesis through IFN antagonism

Creation of chimeric proteins:

• Generate chimeric proteins swapping domains between KHUV P and other lyssavirus P proteins

• Compare functions to identify species-specific adaptations

• Use in minigenome systems to assess polymerase cofactor functions

• Test in IFN antagonism assays to map domains responsible for host immune evasion

These approaches provide comprehensive insights into domain-specific functions of KHUV P protein and their conservation across the lyssavirus genus.

What are common challenges in working with recombinant Khujand virus P protein and strategies to overcome them?

Researchers working with recombinant KHUV P protein often encounter several technical challenges:

Low solubility and protein aggregation:

• Challenge: P proteins can form aggregates during expression and purification

• Solutions:

  • Reduce induction temperature to 16-18°C

  • Use solubility-enhancing tags (MBP, SUMO, or GST)

  • Add 5-10% glycerol to all buffers

  • Include low concentrations (0.05-0.1%) of non-ionic detergents

  • Consider refolding approaches if inclusion bodies form

Protein instability:

• Challenge: P protein may degrade during purification or storage

• Solutions:

  • Include protease inhibitors in all buffers

  • Add reducing agents (1-5 mM DTT or 2-ME)

  • Store at -80°C in small aliquots with 10-15% glycerol

  • Avoid repeated freeze-thaw cycles

Non-specific binding in interaction studies:

• Challenge: P proteins can exhibit non-specific interactions with cellular proteins

• Solutions:

  • Increase salt concentration (300-500 mM NaCl) in binding buffers

  • Include 0.1-0.5% BSA as a blocking agent

  • Use more stringent washing conditions

  • Include appropriate negative controls (unrelated proteins with similar tags)

Inconsistent functional activity:

• Challenge: Variable results in functional assays

• Solutions:

  • Verify protein folding by circular dichroism

  • Confirm activity in well-established assays before proceeding to more complex experiments

  • Use freshly purified protein for critical experiments

  • Include positive controls (e.g., RABV P protein) in parallel experiments

Cross-reactivity issues with antibodies:

• Challenge: Antibodies may cross-react with P proteins from other lyssaviruses

• Solutions:

  • Carefully validate antibody specificity using multiple lyssavirus P proteins

  • Consider epitope mapping to identify unique regions for antibody development

  • Use tagged proteins and tag-specific antibodies for detection when possible

Implementing these strategies can significantly improve the success rate of experiments involving recombinant KHUV P protein.

How does codon usage in the Khujand virus P gene compare to other lyssaviruses, and what are the implications for recombinant expression?

Analysis of codon usage in the KHUV P gene reveals important patterns with implications for recombinant expression:

Codon usage patterns:

• The KHUV P gene, like other lyssavirus P genes, shows distinct codon usage bias

• The GC content of KHUV P gene is approximately 50.50%, with GC1>GC3>GC2 pattern typical for lyssaviruses

• At the third codon position, T3% is highest, followed by A3%, C3%, and G3%

• Effective Number of Codons (ENC) value for KHUV P gene is around 50.50-55.80, indicating moderate codon bias

Comparative analysis with other lyssaviruses:

Data adapted from codon usage analysis of Rhabdoviridae genomes

Implications for recombinant expression:

• Expression system selection: Codon usage differences between KHUV and expression hosts (E. coli, yeast, insect cells) may affect expression efficiency

• Codon optimization strategies:

  • For E. coli expression: Optimize rare codons (particularly those encoding Arg, Leu, Ile, and Pro)

  • For mammalian expression: Less optimization may be needed as mammalian cells generally accommodate lyssavirus codon usage

• Expression construct design:

  • Consider incorporating a strong Kozak sequence for mammalian expression

  • For bacterial expression, reduce secondary structure in the 5' region of the mRNA

Recommended approach:

• Generate both native and codon-optimized constructs

• Compare expression levels in different systems

• Assess protein functionality to ensure optimization doesn't affect folding or activity

• For structural or functional studies, verify that codon optimization doesn't introduce artifacts

Understanding these patterns helps researchers optimize expression strategies for obtaining high yields of functional recombinant KHUV P protein.

What are emerging techniques for studying the structural biology of Khujand virus P protein and its interactions?

Recent advancements offer new opportunities for studying KHUV P protein structure and interactions:

Cryo-electron microscopy (Cryo-EM):

• Allows visualization of P protein complexes with N or L proteins without crystallization

• Can resolve dynamic regions that are challenging for X-ray crystallography

• Single-particle analysis enables determination of structures at near-atomic resolution

• Sample preparation: Purify protein complexes to high homogeneity and optimize buffer conditions to prevent aggregation

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Maps protein-protein interaction surfaces by detecting changes in hydrogen-deuterium exchange rates

• Identifies flexible regions and conformational changes upon binding

• Particularly valuable for studying P protein interactions with host factors

• Protocol optimization: Test various deuterium labeling times (10 seconds to 24 hours) to capture fast and slow exchanging regions

AlphaFold2 and other AI-based structure prediction:

• Provides accurate structural models without experimental structure determination

• Can predict structures of P protein domains and their complexes

• Useful for generating hypotheses about functional domains and guiding mutagenesis

• Validation approach: Compare predictions with experimental data from limited proteolysis or HDX-MS

Integrative structural biology approaches:

• Combining multiple techniques (SAXS, NMR, cross-linking/mass spectrometry)

• Creates comprehensive structural models of P protein complexes

• Particularly useful for intrinsically disordered regions in P protein

• Data integration: Use specialized software (e.g., IMP, HADDOCK) to combine various structural constraints

Proximity labeling techniques:

• BioID or TurboID fused to P protein identifies proximal proteins in living cells

• APEX2 fusion allows electron microscopy visualization of P protein localization

• Maps the dynamic interactome of P protein during infection

• Optimization: Compare different tagging positions to minimize functional disruption