Figure 7.

NS reporter influenza A viruses: Schematic representation of the NS segment from WT (A) and reporter (B–E) influenza A viruses as described in Figure 1. Influenza A WT NS gene segment encodes for NS1 and NEP via alternative splicing. Nucleotide lengths for the NCR, ψ, and NS segment are indicated; (B) Multicistronic transcription using a caspase recognition site: Multicistronic NS reporter influenza A viruses were generated by insertion of a caspase recognition site (CRS) after the NS1 ORF; (C) Multicistronic transcription using stop-start sequence: Multicistronic NS reporter influenza A viruses were generated by insertion of a stop/start transcription site after the NS1 ORF for independent translation of NS1, reporter gene, and NEP; (D) NS1 fusion protein: Reporter genes were fused to native NS1 ORF with a short linker (SL). A splice acceptor mutation (SAM; *) inhibits NEP alternative splicing. NEP expression occurs after 2A cleavage. The 5′ ψ are duplicated and contain NEP N-terminal amino acid codons; (E) Tricistronic transcription of the NS segment: Reporter gene and NEP expression occurs after two 2A cleavage sites. The 5′ ψ are duplicated and contain NEP N-terminal amino acid codons. An HA tag and a heterologous dimerization domain (Dcm) were added after the NS1 ORF.