Recombinant Friend spleen focus-forming virus Glycoprotein 55 (env)

What is the molecular structure of Friend SFFV glycoprotein 55?

The Friend SFFV envelope glycoprotein, gp55, is a 409-amino-acid glycoprotein with an apparent molecular weight of 55,000 Da . It is encoded by the env gene of SFFV and represents a modified recombinant protein with domains related to envelope glycoproteins of endogenously inherited polytropic murine leukemia viruses (MuLVs) and ecotropic MuLVs . Structurally, gp55 contains an amino-terminal polytropic domain, a proline-rich linker, and a carboxyl-terminal region related to the envelope glycoproteins encoded by ecotropic host range MuLVs . A hallmark feature of pathogenic SFFVs is that their env genes contain a 585-base deletion and a single-base insertion in the ecotropic region, causing a translational frameshift and premature termination of the encoded protein .

What is the evolutionary origin of SFFV gp55?

SFFV is a recombinant between ecotropic murine type C virus (specifically Friend MuLV) and the env gene region of xenotropic type C virus . Molecular hybridization studies have demonstrated that SFFV acquired specific xenotropic viral genetic information not present in the Friend helper virus . Using cDNA probes that detect xenotropic sequences contained in SFFV, researchers have mapped these acquired sequences to the env gene region of xenotropic virus . This recombination event was critical in conferring the unique pathogenic properties of SFFV, allowing the resulting gp55 protein to interact with erythropoietin receptors and induce erythroblastosis. Similar recombination patterns have been observed in other acute transforming RNA tumor viruses, suggesting a common mechanism for their formation .

How is gp55 post-translationally modified?

Upon synthesis, gp55 undergoes several post-translational modifications that impact its function. The protein is primarily retained in the endoplasmic reticulum, with only a small fraction being processed to the cell surface . The cell surface form of gp55 exists as a disulfide-bonded dimer, which appears necessary for activation of the erythropoietin receptor (EPO-R) . The interaction between gp55 and EPO-R occurs predominantly in the endoplasmic reticulum, as evidenced by the finding that most EPO-R associated with gp55 is endoglycosidase H-sensitive . This interaction stabilizes EPO-R, causing it to remain within the rough endoplasmic reticulum for longer periods compared to cells without gp55 .

What is the molecular mechanism of gp55-induced EPO-R activation?

The molecular mechanism by which gp55 causes erythroleukemia begins with its direct binding to the erythropoietin receptor (EPO-R), mimicking the action of erythropoietin . Unlike the natural ligand erythropoietin, which induces transient receptor activation, gp55 binding leads to constitutive activation of EPO-R signaling pathways . This persistent activation results in continuous proliferation signals to erythroid progenitor cells, eventually leading to erythroblastosis and, ultimately, erythroleukemia .

The interaction occurs at two cellular locations with potentially different signaling outcomes:

• At the cell surface: A small fraction of gp55 is processed to the cell surface as disulfide-bonded dimers, which can interact with surface-expressed EPO-R .

• In the endoplasmic reticulum: The majority of gp55-EPO-R interactions occur within the endoplasmic reticulum, where gp55 binds to and stabilizes the endoglycosidase H-sensitive form of EPO-R . These intracellular complexes may send growth-promoting signals to the cell through mechanisms distinct from surface receptor activation .

Experimental evidence for this mechanism comes from co-expression studies showing that when interleukin-3 (IL-3)-dependent lymphoid cells are co-infected with SFFV and a virus carrying the EPO-R gene, the cells become growth factor-independent . Similarly, cells transfected with both EPO-R and gp55 cDNAs grow in the absence of erythropoietin .

How do different forms of EPO-R interact with gp55?

The erythropoietin receptor exists in multiple glycosylated forms, each with distinct properties and potential for interaction with gp55. Research has identified three major forms of EPO-R in cells transfected with EPO-R cDNA :

Most of the EPO-R that associates with gp55 is endoglycosidase H-sensitive (64-kDa form), suggesting that interactions primarily occur in the endoplasmic reticulum . The interaction with gp55 appears to increase the stability of the endoglycosidase H-sensitive EPO-R, suggesting that gp55 binding prevents normal receptor trafficking and degradation . Very little EPO-R is expressed on the cell surface under normal conditions, and all three forms are degraded rapidly in the absence of gp55 .

What structural features of gp55 are essential for EPO-R binding and activation?

Several structural features of gp55 are critical for its ability to bind and activate EPO-R:

What cell culture systems are optimal for studying gp55-EPO-R interactions?

Several cell culture systems have proven valuable for studying gp55-EPO-R interactions:

• Interleukin-3 (IL-3)-dependent lymphoid cell lines: When transfected with EPO-R cDNA, these cells become responsive to erythropoietin . Co-expression of gp55 in these cells renders them growth factor-independent, providing a clean system to study the functional consequences of gp55-EPO-R interaction .

• Fibroblast cell lines: Co-expression of gp55 and murine EPO-R in fibroblast cell lines has been used to demonstrate direct binding between these proteins . These cells do not normally express EPO-R or respond to erythropoietin, making them useful for isolating the specific effects of gp55-EPO-R interaction.

• BaF3/EpoR cells: This erythropoietin-dependent cell line has been used to test the ability of different SFFV variants to convert cells to factor independence . The development of factor-independent derivatives upon infection with SFFV provides a functional readout of gp55 activity.

• Bone marrow cultures: These primary cultures more closely mimic the natural target cells of SFFV and have been used to study erythroblast proliferation induced by gp55 .

When designing experiments with these systems, researchers should consider the expression levels of both gp55 and EPO-R, as well as the glycosylation status of EPO-R, which can influence interaction outcomes.

What techniques are used to detect and characterize gp55-EPO-R complexes?

Multiple biochemical and molecular techniques have been employed to detect and characterize gp55-EPO-R complexes:

• Co-immunoprecipitation: This technique can demonstrate physical association between gp55 and EPO-R in cells expressing both proteins .

• Western blot analysis: Western blotting with antisera that react with murine retroviral envelope glycoproteins has been used to detect viral components capable of activating EPO-R . This technique can also identify different forms of EPO-R based on their molecular weights and glycosylation patterns .

• Endoglycosidase H sensitivity assays: Treatment of cell lysates with endoglycosidase H can distinguish between high-mannose (ER-resident) and complex (post-Golgi) glycosylated forms of proteins, helping to determine the subcellular location of gp55-EPO-R interactions .

• Growth factor independence assays: Functional assays measuring cell proliferation in the absence of erythropoietin or other growth factors provide evidence of successful gp55-EPO-R interaction and activation .

• DNA sequence analysis: Sequencing viral genomes from different factor-independent cell clones has revealed the specific env gene sequences required for EPO-R activation .

How can recombinant gp55 be produced for structural and functional studies?

Production of recombinant gp55 for research purposes can be achieved through several approaches:

How can studying gp55-EPO-R interactions inform our understanding of cytokine receptor activation?

The study of gp55-EPO-R interactions provides unique insights into cytokine receptor activation mechanisms:

• Ligand-independent activation: gp55 represents a naturally evolved viral protein capable of activating a cytokine receptor without mimicking the structure of its natural ligand . This demonstrates alternative mechanisms for receptor activation that could inform therapeutic approaches.

• Intracellular receptor activation: The finding that gp55-EPO-R complexes within the endoplasmic reticulum can send growth-promoting signals challenges the conventional view that cytokine receptors signal exclusively from the cell surface . This suggests potential novel signaling mechanisms that could be exploited for therapeutic purposes.

• Structural requirements for activation: By analyzing which domains of gp55 are essential for EPO-R binding and activation, researchers can gain insights into the key structural features required for cytokine receptor activation in general .

• Pathological receptor activation: The study of gp55-induced transformation provides a model for understanding how aberrant cytokine receptor activation contributes to disease processes, particularly leukemia development .

What can novel SFFV variants tell us about the functional domains of gp55?

Analysis of novel SFFV variants has provided valuable information about the functional domains of gp55:

• An array of novel murine SFFVs with different env gene structures has been identified through in vivo selection during serial passages in mice . These evolutionary intermediates culminated in the formation of novel SFFVs capable of converting erythropoietin-dependent cells to factor independence .

• DNA sequence analysis of viral genomes cloned from different factor-independent cell clones revealed env genes with open reading frames encoding proteins of varying lengths (644, 449, and 187 amino acids) . Despite their differences, all three env genes contained 3' regions identical to that of SFFV, including a 6-bp duplication and a single-base insertion shown to be critical for pathogenesis .

• Interestingly, these novel env gene sequences did not contain polytropic sequences typically found in classical SFFV strains and were divergent in their 5' regions . This suggests they originated by recombination and partial deletions of endogenously inherited MuLV env sequences .

• These findings indicate that the requirements for EPO-R activation by SFFV-related viruses depend primarily on sequences at the 3' end of the env gene rather than on the polytropic regions or the 585-base deletions common among classical SFFV strains .

• Sequence analysis of different recombinants and deletion mutants revealed that short direct and indirect repeat sequences frequently flanked the deletions, suggesting a reverse transcriptase template jumping mechanism for this rapid retroviral diversification .

How might understanding gp55 function contribute to therapeutic strategies for erythroleukemia?

Understanding the molecular mechanisms of gp55-induced erythroleukemia could inform novel therapeutic approaches:

• Targeted inhibition of gp55-EPO-R interaction: Knowledge of the specific binding interfaces between gp55 and EPO-R could enable the design of small molecule inhibitors or peptide mimetics that block this interaction, potentially arresting the growth of SFFV-transformed cells .

• Disruption of intracellular signaling: Since gp55-EPO-R complexes within the endoplasmic reticulum appear to send growth-promoting signals, interventions targeting these intracellular signaling pathways might provide therapeutic benefits .

• Exploiting receptor internalization and degradation pathways: Understanding how gp55 stabilizes EPO-R and prevents its normal degradation could lead to strategies that restore receptor turnover in transformed cells .

• Immunotherapeutic approaches: As a viral protein, gp55 represents a tumor-specific antigen that could be targeted by immunotherapeutic strategies, including CAR-T cells or therapeutic vaccines .

• Model system for testing broader leukemia therapies: The well-characterized molecular mechanism of gp55-induced transformation provides a valuable model system for testing novel therapeutic approaches that might have applicability to other forms of leukemia with aberrant cytokine receptor signaling .