Recombinant Coturnix coturnix japonica Subgroup A Rous sarcoma virus receptor pg950, partial

What is the Rous sarcoma virus receptor pg950 in Japanese quail?

The pg950 receptor in Japanese quail (Coturnix coturnix japonica) is also known as the Subgroup A Rous sarcoma virus receptor. This protein is alternatively named the Low density lipoprotein receptor-related protein, suggesting its evolutionary relationship to the LDL receptor family . The receptor serves as the cellular binding site that enables Rous sarcoma virus (RSV) attachment and entry into quail cells. Research has shown that Japanese quail are susceptible to various strains of Rous sarcoma virus, with specific strains like Bryan "high-titer" and Harris strains demonstrating the ability to be serially passaged with cell-free tumor extracts .

How does strain-specific interaction occur between RSV and host receptors?

Different strains of Rous sarcoma virus demonstrate varied infectivity patterns in Japanese quail cells. While the Bryan "high-titer," Carr-Zilber, Harris, Prague, Schmidt-Ruppin, and "29" strains all produce tumors upon primary inoculation, only the Bryan "high-titer" and Harris strains can be effectively serially passaged using cell-free tumor extracts . This selective response suggests strain-specific interactions with the pg950 receptor, possibly indicating differences in binding affinity, receptor recognition domains, or post-binding events in the viral entry process. Analysis using end-point dilution technique and interference testing has confirmed that tumor extracts from the Bryan "high-titer" strain passaged ten times in quail contained a Rous-associated virus .

What experimental systems are available for studying pg950 receptor function?

Current research methods for studying the pg950 receptor include recombinant protein expression systems utilizing baculovirus vectors, which enable the production of the partial receptor protein with high purity (>85% by SDS-PAGE) . These recombinant proteins can be used in binding studies, structural analyses, and functional assays. In vivo systems utilizing Japanese quail have been established for tumor induction studies with various RSV strains . Additionally, cell culture systems derived from Japanese quail have been developed, as referenced in related research articles such as the "Pharaoh" line culture of Japanese quail cells that provides a leukosis-free system for virus reproduction .

What are the optimal storage and handling conditions for recombinant pg950?

The recombinant pg950 protein should be stored according to strict protocols to maintain its biological activity. For liquid formulations, the shelf life is generally 6 months when stored at -20°C/-80°C. Lyophilized forms demonstrate greater stability, with a shelf life of 12 months at -20°C/-80°C . Repeated freezing and thawing should be avoided as this can compromise protein integrity. For short-term use, working aliquots can be stored at 4°C for up to one week . When handling the protein, it is recommended to briefly centrifuge the vial prior to opening to ensure the contents are at the bottom of the tube.

How should recombinant pg950 be reconstituted for experimental applications?

For optimal reconstitution of the recombinant protein, it should be dissolved in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . To enhance stability during storage, it is recommended to add glycerol to a final concentration of 5-50%, with 50% being the standard default concentration used by manufacturers . This glycerol addition helps prevent protein denaturation during freeze-thaw cycles and extends the usable life of the reconstituted protein. After reconstitution, the solution should be aliquoted for long-term storage at -20°C/-80°C to minimize the number of freeze-thaw cycles.

What expression systems yield functional recombinant pg950 protein?

The baculovirus expression system has been successfully employed to produce functional recombinant pg950 protein . This system utilizes insect cells infected with recombinant baculovirus carrying the pg950 gene, allowing for proper eukaryotic post-translational modifications. The baculovirus system is particularly advantageous for complex mammalian proteins as it facilitates proper folding and often yields higher amounts of functional protein compared to bacterial expression systems . This approach aligns with broader recombinant protein production strategies that have revolutionized the field of drug metabolism and pharmacokinetics research .

How can recombinant pg950 be used to study viral evolution and adaptation?

Recombinant pg950 provides a valuable tool for investigating the evolutionary adaptations of Rous sarcoma virus. By conducting binding assays with different RSV strains and the recombinant receptor, researchers can identify specific viral mutations that enhance receptor recognition. Similar approaches have been used in coronavirus research to understand receptor-binding domain adaptations across species barriers . In experimental settings, researchers could generate RSV variants through serial passage in quail cells, then assess changes in binding affinity to recombinant pg950 to identify adaptive mutations. This approach would parallel studies showing that only specific RSV strains (Bryan "high-titer" and Harris) could be serially passaged in quail .

What insights can pg950 receptor studies provide for cross-species viral transmission?

Understanding the interactions between the pg950 receptor and RSV provides a model system for studying species barriers in viral transmission. Research has shown that Japanese quail demonstrate a selective response to different RSV strains, suggesting that receptor recognition plays a crucial role in determining host range . By comparing the structure and binding properties of pg950 with related receptors in other avian species, researchers can identify key determinants of host specificity. This approach is conceptually similar to studies of SARS-CoV receptor recognition, where differences in ACE2 receptors across species present barriers for viral infection, and specific mutations in the viral receptor-binding domain enable cross-species jumps .

How does the structural biology of pg950 inform its function?

While the complete structural characterization of pg950 is still evolving, its alternative name as a low-density lipoprotein receptor-related protein suggests structural homology to the LDL receptor family . These receptors typically contain multiple functional domains including ligand-binding repeats, EGF-like domains, and transmembrane regions. Structural studies combining X-ray crystallography, cryo-electron microscopy, and molecular dynamics simulations would provide insights into how pg950 recognizes and binds to RSV. Such structural information could reveal binding interfaces and conformational changes that occur during virus-receptor interactions, similar to the detailed structural analyses that have elucidated coronavirus-receptor interactions .

What control samples should be included when studying pg950 receptor functions?

Robust experimental design for pg950 receptor studies should include several key controls. Negative controls should utilize cells lacking the pg950 receptor or with blocked receptors using specific antibodies. Positive controls should include cell lines known to express the receptor and demonstrate susceptibility to RSV infection. When using recombinant pg950, denatured protein samples can serve as negative controls to confirm that biological activity depends on proper protein folding. For virus-receptor binding assays, competitive inhibition using soluble receptor fragments or antibodies targeting the virus attachment protein can validate specificity. These approaches would build upon the experimental methods used in studies of RSV strain specificity in Japanese quail .

What techniques are effective for quantifying pg950-virus interactions?

Several biophysical and biochemical techniques can effectively quantify interactions between recombinant pg950 and RSV. Surface plasmon resonance (SPR) allows real-time measurement of binding kinetics and affinity constants between immobilized receptor and viral particles or purified viral attachment proteins. Enzyme-linked immunosorbent assays (ELISA) using recombinant pg950 can measure binding of viral components. Flow cytometry with fluorescently labeled viruses can assess binding to cells expressing pg950. Additionally, biolayer interferometry offers another label-free approach for measuring binding kinetics. These quantitative approaches would complement the end-point dilution technique and interference tests previously used to analyze RSV in Japanese quail .

How can researchers distinguish between receptor binding and post-binding events?

Differentiating between receptor binding and post-binding events in RSV infection requires specific experimental approaches. Time-of-addition experiments using inhibitors that target different stages of viral entry can identify when particular steps occur. Temperature-shift experiments exploit the fact that receptor binding often occurs at low temperatures (4°C) while fusion and internalization require physiological temperatures (37°C). Confocal microscopy with differentially labeled virus and cellular components can track the progression of viral particles through the entry process. Additionally, cell-cell fusion assays using cells expressing viral envelope proteins and target cells expressing pg950 can isolate the fusion step from the binding step. These approaches would help explain the observed differences in infectivity among RSV strains in Japanese quail .

What is the relationship between pg950 polymorphisms and disease susceptibility?

Genetic variations in the pg950 receptor likely influence susceptibility to RSV infection and tumor development in Japanese quail populations. Researchers investigating this relationship should sequence the pg950 gene from different quail lineages and correlate polymorphisms with susceptibility to different RSV strains. Site-directed mutagenesis of recombinant pg950 could identify critical residues for virus binding. Similar approaches have been used to study how changes in coronavirus receptors across species affect binding affinity and infection . These studies would provide insights into the molecular basis of host range restriction and potentially identify genetic markers for breeding programs focused on disease resistance.

Can the pg950-RSV model inform broader understanding of retroviral pathogenesis?

The pg950-RSV interaction in Japanese quail represents a valuable model for studying fundamental aspects of retroviral pathogenesis. The selective response of quail to different RSV strains provides an opportunity to identify viral determinants of host range and pathogenicity . Comparative studies between avian species with different susceptibilities to RSV could identify critical differences in receptor structure and expression patterns. Additionally, the quail model allows for the study of oncogenesis in the context of natural host-pathogen interactions. These findings could inform broader understanding of retroviral evolution, host adaptation, and disease mechanisms applicable to other retroviral infections across different host species.

How might CRISPR-Cas9 technology enhance pg950 receptor studies?

CRISPR-Cas9 gene editing offers powerful approaches for advancing pg950 receptor research. Researchers could generate quail cell lines with pg950 knockout to confirm receptor specificity, create cell lines expressing modified receptors to identify critical functional domains, and develop transgenic quail models with altered pg950 expression or structure. Additionally, CRISPR activation or interference systems could modulate pg950 expression levels to assess dose-dependent effects on viral susceptibility. These genetic manipulation approaches would build upon the foundation of earlier RSV studies in Japanese quail and leverage the advanced recombinant DNA technologies that have transformed drug metabolism and pharmacokinetics research .

What comparative genomic approaches could reveal about pg950 evolution?

Comparative genomics of pg950 across avian species could provide insights into evolutionary pressures from retroviral infections. Researchers should sequence and compare pg950 homologs across diverse avian lineages, construct phylogenetic trees to map receptor evolution in relation to species divergence, and conduct selection analyses to identify residues under positive selection that might represent virus-resistance adaptations. Similar approaches have revealed how coronavirus receptors have evolved under selective pressure across mammalian species . These analyses would contextualize the unique properties of the Japanese quail pg950 receptor that enable selective responses to different RSV strains .

How can systems biology integrate pg950 receptor function into broader cellular networks?

Systems biology approaches can place pg950 receptor function within broader cellular signaling and regulatory networks. Researchers should conduct transcriptomic and proteomic analyses of quail cells before and after RSV infection to identify co-regulated genes and proteins, map signaling pathways activated upon receptor engagement, and develop computational models of virus-receptor interactions and downstream effects. Integration of these data with physiological outcomes in infected birds would provide a multi-scale understanding of RSV pathogenesis. These approaches would extend the historical studies of RSV in Japanese quail using modern techniques similar to those that have transformed recombinant protein research in pharmacology .