Disruption of influenza virus packaging signals results in various misassembled genome complexes

The eight different genomic viral RNAs (vRNAs) of influenza A virus are typically packaged into virions as a (7 + 1) complex of seven viral ribonucleoproteins (vRNPs) around a central vRNP. Mutations in the packaging signals of these vRNAs are predicted to result in virions lacking specific vRNPs. However, whether these vRNPs are then replaced by vRNP-like host RNAs is an open question. Here, we report that mutations in two packaging signals of influenza A/SC35M virus, which cause the near-complete loss of four vRNAs from the total virion population, result in several distinct virion subpopulations with unusual vRNP content. Half of the virions analyzed are empty or contain incomplete genome complexes with one to four vRNPs. Unexpectedly, most other virions contain atypical (7 + 1) complexes consisting of multiple copies of the four efficiently packaged vRNAs, but no host RNAs. The reported set of misassembled genome complexes suggests that packaging signals coordinate a modular genome assembly process. IMPORTANCE The influenza A virus genome consists of eight distinct viral RNAs (vRNAs) that are typically packaged into a single virion as an octameric complex. How this genome complex is assembled and incorporated into the virion is poorly understood, but previous research suggests a coordinative role for packaging signals present in all vRNAs. Here, we show that disruption of two packaging signals in a model H7N7 influenza A virus results in a mixture of virions with unusual vRNA content, including empty virions, virions with one to four vRNAs, and virions with octameric complexes composed of vRNA duplicates. Our results suggest that (i) the assembly of error-free octameric complexes proceeds through a series of defined vRNA sub-complexes and (ii) virions can bud without incorporating complete octameric complexes.