Influenza A Virus Negative Strand RNA Is Translated for CD8⁺ T Cell Immunosurveillance

Area of Science:

• Virology
• Immunology
• Molecular Biology

Background:

• Influenza A virus (IAV) genomic RNA is typically considered noncoding.
• Segment 8 of IAV contains a conserved open reading frame for a potential protein, NEG8.
• The expression of NEG8 from IAV has not been previously demonstrated.

Purpose of the Study:

• To investigate the potential for translation from the negative strand of IAV.
• To explore the role of such translation in CD8+ T cell immunosurveillance.
• To determine if genetically engineered IAV can present viral peptides for immune recognition.

Main Methods:

• Genetic insertion of the SIINFEKL peptide into IAV negative-strand gene segments.
• Generation of recombinant IAV encoding SIINFEKL within the NEG8 open reading frame or neuraminidase-stalk sequence.
• Assessment of K^b-SIINFEKL complex formation on cell surfaces.
• In vitro and in vivo activation assays using OT-I T cells.

Main Results:

• SI SIINFEKL peptide appended to the predicted C terminus of NEG8 resulted in cell surface K^b-SIINFEKL complex generation.
• Activation of OT-I T cells was observed both in vitro and in vivo.
• Recombinant IAV with SIINFEKL embedded in the neuraminidase-stalk sequence also activated OT-I T cells in mice.
• Direct detection of NEG8 protein expression in infected mouse cells was unsuccessful.

Conclusions:

• Sequences on the negative strand of single-stranded RNA viruses can be translated.
• This translation process is relevant for antiviral immunosurveillance.
• Engineered IAV can present viral peptides, stimulating adaptive immune responses.