Recombinant High plains virus Capsid protein

What is the High Plains wheat mosaic virus (HPWMoV) and its capsid protein?

High Plains wheat mosaic virus (HPWMoV) is one of 21 definitive species in the genus Emaravirus in the family Fimoviridae. Originally called High Plains virus, maize red stripe virus, or wheat mosaic virus, it causes High Plains disease characterized by severe mosaic and necrosis symptoms in corn and wheat . The virus contains a 32 kDa nucleoprotein that encapsidates multiple RNA species and forms the nucleocapsid (NC) of the virus particles. This nucleoprotein is encoded by RNA 3 of the virus's octapartite negative-sense RNA genome and has been used as a diagnostic feature in virus detection .

How does the structure of the HPWMoV capsid protein compare to other viral capsid proteins?

The HPWMoV nucleocapsid protein (32 kDa) forms thread-like ribonucleoproteins that encapsidate multiple RNA species within double-membrane virus particles ranging from 80-200 nm in size . Unlike typical icosahedral viral capsids, HPWMoV particles possess a unique double-membrane structure, as confirmed by electron microscopy examination of leaf-dip preparations. The nucleocapsid protein protects viral genomic RNAs by forming nucleocapsids, which is a common function among viral capsid proteins, but the presence of two variants of RNA 3 (encoding the nucleocapsid protein) in HPWMoV is distinctive, though the significance of these variants in viral biology remains unknown .

What is the genomic organization of HPWMoV and how does it relate to capsid protein expression?

HPWMoV possesses an octapartite single-stranded negative-sense RNA genome, with each RNA segment encoding a single open reading frame (ORF). The capsid protein (nucleoprotein) is specifically encoded by RNA 3 . The virus produces both virus-sense and virus-complementary (vc)-sense genomic RNA copies in infected tissue at a ratio of 10-20:1. Additionally, shorter-than-genome length RNAs of vc sense were detected for genomic RNAs 3, 4, 7, and 8, suggesting these represent subgenomic mRNAs for the expression of ORFs, including the nucleocapsid protein gene. Each genomic RNA appears to produce genomic-length virus- and vc-sense RNAs and subgenomic mRNAs of vc sense for gene expression .

What expression systems are most effective for producing recombinant HPWMoV capsid protein?

Based on similar viral capsid protein studies, baculovirus expression systems using SF21 insect cells provide effective production platforms for viral capsid proteins . While the search results don't specifically address HPWMoV capsid protein expression systems, the successful expression of the similar EHDV VP7 capsid protein suggests that baculovirus systems would be appropriate for HPWMoV capsid protein expression. The baculovirus system, utilizing the polyhedrin promoter, enables high-level expression of recombinant proteins in insect cells, as demonstrated by the EHDV VP7 protein, which showed visible bands of approximately 39 kDa in SDS-PAGE analysis of infected cell lysates .

How does the position of affinity tags affect the expression and functionality of recombinant capsid proteins?

The position of affinity tags significantly impacts both expression levels and functionality of recombinant viral capsid proteins. From research on related viral proteins, N-terminal histidine tags generally result in higher expression levels comparable to untagged constructs, while C-terminal histidine tags can adversely affect protein expression . This effect was observed with the EHDV VP7 protein, where N-terminal six-histidine tagged protein showed expression levels similar to untagged protein, but C-terminal tagged protein exhibited significantly reduced expression. The reduced expression with C-terminal tags may be attributed to conformational changes induced by the tag placement, as has been observed with other proteins .

What are the optimal conditions for maximizing expression of recombinant HPWMoV capsid protein?

For optimal expression of recombinant viral capsid proteins like HPWMoV nucleoprotein, several factors should be considered based on similar protein production systems. These include:

• Expression vector design: Utilizing baculovirus vectors with strong promoters like polyhedrin promoter

• Tag placement: Preferring N-terminal tagging over C-terminal tagging for histidine affinity tags

• Infection parameters: Harvesting infected cells approximately 3 days post-infection for optimal protein accumulation

• Cell line selection: Using SF21 insect cells which support high-level expression

• Codon optimization: Adapting the coding sequence to the codon usage bias of the expression host

The optimal conditions should be empirically determined through expression trials, but harvesting cells 3 days post-infection has been shown to yield good results for similar viral capsid proteins .

What are the most efficient purification strategies for recombinant HPWMoV capsid protein?

For efficient purification of histidine-tagged recombinant viral capsid proteins, nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography provides a rapid, one-step purification method . Based on related viral capsid protein purification strategies, the following approach would be effective for HPWMoV capsid protein:

• Cell lysis: Harvesting infected cells and disrupting them under native or denaturing conditions

• Affinity chromatography: Applying the clarified lysate to Ni-NTA resin

• Washing: Removing non-specifically bound proteins with wash buffers containing low imidazole concentrations

• Elution: Recovering purified protein with buffer containing higher imidazole concentrations

• Quality assessment: Confirming purity by SDS-PAGE and functionality by immunological methods

For untagged capsid proteins, alternative methods including rate zonal sucrose density gradient centrifugation followed by caesium sulphate isopycnic gradient centrifugation have been successfully employed for HPWMoV virion purification, which could be adapted for recombinant protein purification .

How can researchers confirm the identity and structural integrity of purified recombinant HPWMoV capsid protein?

Researchers can confirm the identity and structural integrity of purified recombinant HPWMoV capsid protein using multiple complementary techniques:

• SDS-PAGE analysis: Verifying molecular weight (expected around 32 kDa for HPWMoV nucleoprotein)

• Western blot analysis: Using specific antibodies against the nucleoprotein or against the histidine tag for tagged constructs

• ELISA: Testing antigenicity with HPWMoV-specific antibodies

• Mass spectrometry: Confirming protein identity through techniques like matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry

• Functional assays: Assessing the ability to bind viral RNA or to be recognized by virus-specific antibodies

Western blotting with both protein-specific antibodies and tag-specific antibodies (for tagged constructs) provides dual confirmation of protein identity and integrity, as demonstrated with similar viral capsid proteins .

What analytical techniques are most suitable for studying HPWMoV capsid protein interactions with viral RNA?

To study HPWMoV capsid protein interactions with viral RNA, several analytical techniques can be employed:

• Electrophoretic mobility shift assay (EMSA): To detect direct binding between the nucleoprotein and viral RNA sequences

• RNA immunoprecipitation: To isolate protein-RNA complexes using antibodies against the capsid protein

• Surface plasmon resonance (SPR): To quantitatively measure binding kinetics and affinity

• Isothermal titration calorimetry (ITC): To determine thermodynamic parameters of binding

• Fluorescence anisotropy: To investigate binding in solution

• Cryo-electron microscopy: To visualize the structural organization of nucleoprotein-RNA complexes

While the search results don't directly address these methods for HPWMoV, the presence of thread-like ribonucleoproteins within HPWMoV particles suggests strong interactions between the nucleoprotein and viral RNA that could be studied using these approaches .

How can recombinant HPWMoV capsid protein be utilized in development of diagnostic assays?

Recombinant HPWMoV capsid protein can be utilized in developing various diagnostic assays for the detection of HPWMoV infections:

• ELISA-based assays: The purified recombinant nucleoprotein can be used to coat microtitre plates for direct or competitive ELISA (c-ELISA) formats. The high purity levels attainable for histidine-tagged recombinant proteins make them suitable assay reagents with improved reliability and reproducibility .

• Western blot analysis: Recombinant protein can serve as positive controls for Western blot detection of HPWMoV in plant samples.

• Production of specific antibodies: The purified recombinant protein can be used to generate polyclonal or monoclonal antibodies for various immunodetection methods.

• Lateral flow devices: Development of field-applicable rapid tests using the recombinant protein as a capture or detection reagent.

For HPWMoV detection, ELISA methods have been successfully employed using polyclonal antibodies in coated plates, with alkaline phosphatase-conjugated antibodies and p-nitrophenyl phosphate substrate for detection .

What strategies can address cross-reactivity issues in serological detection using recombinant HPWMoV capsid protein?

To address cross-reactivity issues in serological detection using recombinant HPWMoV capsid protein, researchers can implement several strategies:

• Competitive ELISA (c-ELISA) formats: These can improve specificity by using virus-specific monoclonal antibodies that compete with sample antibodies for binding to the recombinant protein.

• Epitope mapping and engineering: Identifying unique epitopes on the HPWMoV nucleoprotein that are not conserved in related viruses and potentially engineering recombinant proteins focusing on these unique regions.

• Careful selection of cutoff thresholds: Establishing appropriate cutoff thresholds for positivity in assays. For instance, in similar virus detection systems, heterologous cross-reactivity was detected but remained significantly below the cutoff threshold of 50 percent inhibition (PI) .

• Pre-absorption with heterologous antigens: Treating test sera with heterologous antigens to remove cross-reactive antibodies before testing with HPWMoV antigens.

• Multiple antigenic marker approach: Using a combination of different viral proteins or epitopes for confirmation of positive results.

The high purity levels attainable for recombinant protein preparations also contribute to improved specificity by eliminating potential interfering components .

How can recombinant HPWMoV capsid protein be utilized in studying virus-host interactions?

Recombinant HPWMoV capsid protein can serve as a valuable tool for studying virus-host interactions through various approaches:

• Protein-protein interaction studies: Using techniques such as co-immunoprecipitation, yeast two-hybrid, or pull-down assays to identify host proteins that interact with the viral nucleoprotein.

• Localization studies: Utilizing fluorescently tagged recombinant nucleoprotein to track its localization within plant cells and tissues during infection.

• RNA binding specificity analysis: Determining the RNA sequences or structures preferentially bound by the nucleoprotein, which may reveal mechanisms of viral RNA selection and packaging.

• Immunoprecipitation followed by RNA sequencing (RIP-Seq): Identifying viral and host RNAs that associate with the nucleoprotein in vivo.

• Production of virus-like particles (VLPs): Generating non-infectious VLPs to study virus assembly, structure, and entry mechanisms.

• Host immune response studies: Investigating how the nucleoprotein triggers or evades host immune responses, potentially identifying pathways for resistance breeding.

These applications can provide insights into the molecular mechanisms of HPWMoV pathogenesis and host range determination .

What are the key challenges in producing structural stable recombinant HPWMoV capsid protein for crystallography studies?

Producing structurally stable recombinant HPWMoV capsid protein for crystallography studies presents several challenges:

• Protein solubility: Viral capsid proteins often have hydrophobic regions that promote self-association, potentially leading to aggregation during expression and purification.

• Conformational homogeneity: Ensuring that the recombinant protein adopts a single, native-like conformation rather than multiple states that would interfere with crystal formation.

• Post-translational modifications: If the native nucleoprotein undergoes post-translational modifications, recombinant expression systems may not replicate these exactly, affecting structure.

• RNA binding: The HPWMoV nucleoprotein naturally binds RNA, and contaminating nucleic acids might need to be removed for crystallization.

• Self-aggregation: As noted for other reovirus capsid proteins, these types of proteins have a natural ability to assemble into virus-like structures under suitable environmental conditions, which may complicate crystallization efforts .

To address these challenges, researchers might need to explore different expression constructs (including truncated versions), purification conditions, and crystallization additives to obtain diffracting crystals.

How does the presence of viral RNA affect the structural properties of HPWMoV capsid protein, and how can this be studied?

The presence of viral RNA likely influences the structural properties of HPWMoV capsid protein in several ways:

• Conformational changes: RNA binding may induce conformational changes in the nucleoprotein structure to accommodate the nucleic acid.

• Oligomerization: RNA binding could promote nucleoprotein oligomerization, essential for nucleocapsid assembly.

• Stability: The nucleoprotein-RNA complex might exhibit different stability compared to the free protein.

• Protection from degradation: RNA binding may protect both the RNA and protein from degradation.

These RNA-induced effects can be studied through multiple approaches:

• Cryo-electron microscopy: Comparing structures of free nucleoprotein versus nucleoprotein-RNA complexes.

• Circular dichroism spectroscopy: Measuring changes in secondary structure upon RNA binding.

• Limited proteolysis: Identifying regions protected from proteolysis in the presence of RNA.

• Hydrogen-deuterium exchange mass spectrometry: Mapping regions with altered solvent accessibility upon RNA binding.

• Nuclear magnetic resonance (NMR): Investigating structural dynamics of the protein in free and RNA-bound states.

The observation that HPWMoV forms thread-like ribonucleoproteins indicates strong structural interplay between the nucleoprotein and viral RNA .

What approaches can be used to study the molecular determinants of HPWMoV capsid protein involvement in viral transmission by wheat curl mites?

Understanding the molecular determinants of HPWMoV capsid protein involvement in mite transmission requires specialized approaches:

• Recombinant protein binding assays: Testing whether purified nucleoprotein can bind to wheat curl mite tissues or extracts.

• Mutagenesis studies: Creating systematic mutations in the nucleoprotein to identify regions critical for transmission.

• Comparative analysis: Comparing amino acid sequences and structures of nucleoproteins from efficiently transmitted versus poorly transmitted isolates.

• Competition assays: Determining if purified recombinant nucleoprotein can block transmission by competing for binding sites in the vector.

• Immunolocalization: Using antibodies against the nucleoprotein to track its location within the mite vector.

• Transcriptomics and proteomics: Identifying mite proteins that interact with the viral nucleoprotein.

While the nucleoprotein primarily functions in encapsidating viral RNA, these studies could reveal additional roles in transmission. It's worth noting that in related viruses like tospoviruses, glycoproteins rather than nucleoproteins have been implicated in vector transmission, suggesting the need to study multiple viral proteins in transmission contexts .

What PCR-based detection methods are most sensitive for HPWMoV, and how can they be optimized when using recombinant capsid protein as a positive control?

For PCR-based detection of HPWMoV, researchers have developed specific primers targeting the nucleoprotein gene. According to the search results, primers targeting the nucleoprotein region (forward: TTT ATG GCT CTT TGT ATT GG, reverse: TAT GTT TCC CCT CTT TGT G) can amplify a 339 bp product . To optimize these methods:

• Positive control development: Purified recombinant HPWMoV capsid protein can be used to generate synthetic control templates by reverse-transcribing the corresponding gene segment and cloning it into a plasmid vector.

• Sensitivity enhancement: Implementing nested PCR or real-time PCR with probes designed based on the nucleoprotein sequence can improve detection sensitivity.

• RNA extraction optimization: Testing different RNA extraction methods to maximize viral RNA recovery from plant samples.

• Internal controls: Including plant housekeeping gene primers as internal controls to verify extraction quality and absence of PCR inhibitors.

• Multiplexing: Developing multiplex PCR protocols to simultaneously detect HPWMoV and other wheat viruses, such as wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV), which often co-infect wheat .

The recombinant capsid protein can serve as a valuable positive control for both immunological and molecular detection methods, ensuring assay reliability and providing a standard for quantification.

How can researchers optimize storage conditions to maintain the stability and antigenicity of recombinant HPWMoV capsid protein preparations?

To optimize storage conditions for maintaining the stability and antigenicity of recombinant HPWMoV capsid protein preparations, researchers should consider:

• Buffer composition: Testing various buffer systems, pH values, and salt concentrations to identify conditions that maximize protein stability.

• Cryoprotectants: Adding stabilizers such as glycerol, sucrose, or trehalose to prevent freeze-thaw damage.

• Storage temperature: Evaluating stability at different temperatures (-80°C, -20°C, 4°C) for both short-term and long-term storage.

• Aliquoting: Dividing the purified protein into small aliquots to avoid repeated freeze-thaw cycles, which can lead to denaturation.

• Lyophilization: Testing whether freeze-drying preserves antigenicity better than liquid storage.

• Protein concentration: Determining optimal concentration ranges that prevent aggregation while maintaining sufficient activity.

Based on experience with similar recombinant viral proteins, highly purified preparations tend to be stable after numerous freeze/thaw cycles compared to unpurified recombinant preparations, which might be less stable due to the presence of extraneous proteins with enzymatic activity or tendency for uncontrolled aggregation .

What are the critical parameters for developing a quantitative ELISA using recombinant HPWMoV capsid protein, and how can assay performance be validated?

For developing a quantitative ELISA using recombinant HPWMoV capsid protein, several critical parameters should be optimized:

• Coating concentration: Determining the optimal concentration of recombinant protein for plate coating through checkerboard titration.

• Blocking agents: Testing different blocking agents (BSA, casein, non-fat dry milk) to minimize background while maintaining specific binding.

• Antibody dilutions: Optimizing primary and secondary antibody dilutions to maximize signal-to-noise ratio.

• Incubation conditions: Establishing optimal temperature and time for each incubation step.

• Substrate development: Determining optimal substrate concentration and development time.

• Standard curve preparation: Creating a reliable standard curve using purified recombinant protein at known concentrations.

To validate assay performance, researchers should assess:

• Analytical sensitivity: Determining the limit of detection and limit of quantification.

• Analytical specificity: Testing cross-reactivity with related viruses and proteins.

• Precision: Measuring intra-assay and inter-assay coefficient of variation.

• Accuracy: Comparing results with established reference methods.

• Linearity: Ensuring proportionality between concentration and signal across the measurement range.

• Robustness: Evaluating assay performance under varying conditions.

For HPWMoV detection, ELISA methods have been implemented using virus-specific polyclonal antibodies in carbonate buffer (pH 9.6) for coating, with alkaline phosphatase-conjugated antibodies and p-nitrophenyl phosphate substrate for detection .

What are the most promising future applications of recombinant HPWMoV capsid protein in agricultural biotechnology?

Recombinant HPWMoV capsid protein holds significant potential for various applications in agricultural biotechnology:

• Development of highly sensitive and specific diagnostic assays for early detection of HPWMoV infections in the field, enabling timely management decisions.

• Production of virus-like particles (VLPs) as vaccine candidates to induce resistance in plants through systemic acquired resistance mechanisms.

• Generation of transgenic plants expressing the nucleoprotein in modified forms that interfere with virus replication through pathogen-derived resistance.

• Creation of antibody-based detection systems for high-throughput screening of germplasm for resistance.

• Development of novel plant protection strategies based on understanding the interaction between the nucleoprotein and host defense mechanisms.

• Improvement of epidemiological models by enabling large-scale surveillance using standardized detection methods based on the recombinant protein.

• Using the nucleoprotein as a carrier or fusion partner for displaying antigens against other plant pathogens, creating multivalent detection or protection systems.

These applications could significantly contribute to improved management of HPWMoV and related diseases in wheat and corn production systems.

How might structural studies of HPWMoV capsid protein inform development of antiviral strategies?

Structural studies of HPWMoV capsid protein could inform antiviral strategies through several mechanisms:

• Identification of critical functional domains: Determining regions essential for RNA binding, protein-protein interactions, or assembly would reveal potential targets for intervention.

• Rational design of inhibitors: Structure-based drug design could yield small molecules that bind specifically to the nucleoprotein and disrupt its function.

• Peptide inhibitors: Designing peptides that mimic interaction interfaces and competitively inhibit essential virus-host protein interactions.

• Understanding resistance mechanisms: Comparing structures of nucleoproteins from resistant and susceptible plant varieties might reveal natural defense mechanisms.

• Epitope mapping: Identifying immunodominant regions could guide development of antibody-based therapeutics or diagnostics.

• Protein engineering: Creating modified versions of the nucleoprotein that retain antigenic properties but interfere with virus assembly when expressed in transgenic plants.

• Vector transmission disruption: Identifying structural features involved in mite transmission could lead to strategies for blocking transmission.

These structural insights could ultimately contribute to developing targeted approaches to control HPWMoV infections in crops .

What interdisciplinary approaches might advance understanding of HPWMoV capsid protein function in viral pathogenesis?

Advancing understanding of HPWMoV capsid protein function in viral pathogenesis requires interdisciplinary approaches combining:

• Structural biology and biophysics: Using techniques like X-ray crystallography, cryo-electron microscopy, and NMR to determine the three-dimensional structure and dynamics of the nucleoprotein alone and in complexes.

• Genomics and transcriptomics: Studying host gene expression changes in response to the nucleoprotein to identify pathways involved in pathogenesis.

• Proteomics: Identifying host proteins that interact with the nucleoprotein through techniques like mass spectrometry-based interactomics.

• Computational biology: Employing molecular dynamics simulations to model nucleoprotein-RNA interactions and predict effects of mutations.

• Plant pathology and entomology: Investigating the role of the nucleoprotein in virus-plant-vector interactions through field and greenhouse studies.

• Synthetic biology: Creating artificial systems to study nucleoprotein function outside the context of viral infection.

• Immunology: Examining the role of the nucleoprotein in triggering or evading plant immune responses.

• Agricultural engineering: Developing novel delivery methods for nucleoprotein-based diagnostics or interventions in field settings.

This interdisciplinary approach would provide comprehensive insights into how the HPWMoV nucleoprotein contributes to the virus life cycle, host range, and disease development, potentially leading to innovative control strategies .