CTL escape viral variants. I. Generation and molecular characterization

Area of Science:

• Immunology
• Virology
• Structural Biology

Background:

• Cytotoxic T lymphocytes (CTLs) are crucial for controlling viral infections.
• CTL recognition of infected cells depends on viral peptides presented by MHC class I molecules, such as H-2Db.
• The lymphocytic choriomeningitis virus (LCMV) has three known H-2Db-restricted CTL epitopes derived from its glycoproteins (GP1, GP2) and nucleoprotein (NP).

Purpose of the Study:

• To investigate how mutations in LCMV viral peptides affect recognition by H-2Db-restricted CTLs.
• To understand the structural basis of CTL escape mutations.
• To assess the likelihood of CTL escape variant emergence in vivo.

Main Methods:

• Selection of viral variants with mutations in known CTL epitopes.
• Nucleic acid sequencing to identify mutations.
• Competitive inhibition binding assays to assess peptide-MHC binding affinity.
• CTL recognition and lysis assays using peptide-coated target cells.
• Computer graphic analysis of peptide-MHC-TCR interactions.
• In vivo CTL response assessment after inoculation with mutated viruses.

Main Results:

• Single mutations in GP1, GP2, or NP peptides allowed viral variants to escape CTL recognition.
• No significant differences in H-2Db binding affinity were observed between wild-type and variant peptides.
• Mutated peptides failed to be recognized by specific CTL clones, indicating altered T cell receptor interaction.
• Structural analysis revealed mutated residues are surface-exposed and accessible to the T cell receptor.
• In vivo, escape from CTL response required mutations in multiple epitopes, a low-probability event.

Conclusions:

• Mutations in viral epitopes can mediate CTL escape by altering T cell receptor binding, not MHC binding.
• CTL escape variants may require multiple mutations to emerge effectively in vivo.
• Understanding CTL-epitope interactions is vital for designing effective antiviral strategies.