Identification of RNA motif for segmented genome packaging of influenza virus

The influenza A virus genome consists of eight single-stranded negative-sense RNA segments (vRNA). Recent studies favor the selective packaging model in which influenza virion selectively incorporates a set of eight distinct vRNAs. The terminal sequences conserved in all segments are the minimal RNA sequences required for packaging of vRNAs. Moreover, each vRNA segment contains a unique packaging signal located in the terminal coding regions for efficient packaging. However, these RNA motifs are poorly defined. Here, we try to identify the RNA motifs for efficient viral genome packaging by using recombinant viruses that possess random sequences in the terminal coding region of segment 4 (HA segment) and segment 7 (M segment). We made recombinant virus pool that did not express M2 by introducing premature stop codons and possess a random sequence region at the downstream of the stop codons. To identify the RNA motifs, the recombinant virus was passaged using MDCK-M2 and the sequence of the inserted region was determined using next-generation sequencer after 1, 3, 5 passages.