Mechanisms underlying glycosylation-mediated loss of ecotropic receptor function in murine MDTF cells and...

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Ecotropic Moloney murine leukemia virus (Mo-MLV) uses cationic amino acid transporter type 1 (CAT1) as a receptor.
• Mus dunni tail fibroblast (MDTF) cells are resistant to Mo-MLV, unlike NIH 3T3 cells.
• CAT1 receptors possess N-linked glycosylation sites in the third extracellular loop (ECL3), a critical region for Mo-MLV binding.

Purpose of the Study:

• To identify the specific amino acid residues in CAT1 responsible for differential Mo-MLV receptor function.
• To elucidate the role of N-linked glycosylation in Mo-MLV binding and infection.
• To understand the molecular basis of virus-host co-evolution.

Main Methods:

• Construction and analysis of mutant mCAT1 receptors with specific amino acid substitutions and insertions in ECL3.
• Cell-based assays to measure Mo-MLV infection and transduction efficiency.
• Treatment with tunicamycin, an N-linked glycosylation inhibitor, to assess the impact of glycosylation status.
• Mo-MLV binding assays to quantify viral attachment to cells expressing different CAT1 variants.

Main Results:

• A Gly insertion at position 236 in mCAT1 (mCAT1/insG) significantly reduced Mo-MLV receptor function and viral binding.
• Inhibition of N-linked glycosylation of mCAT1/insG-expressing cells increased Mo-MLV transduction titers.
• The Ile-to-Val substitution at position 214 in dCAT1 was also investigated for its role in receptor function.

Conclusions:

• A single amino acid insertion (Gly at position 236) in the ECL3 of mCAT1 is sufficient to confer the Mo-MLV-resistant phenotype observed in dCAT1.
• N-linked glycosylation of CAT1 plays a crucial role in modulating Mo-MLV infection efficiency.
• These findings provide significant insights into the molecular mechanisms underlying virus-host interactions and co-evolution.