Complete and Incomplete Genome Packaging of Influenza A and B Viruses

Abstract

The genomes of influenza A and B viruses comprise eight segmented, single-stranded, negative-sense viral RNAs (vRNAs). Although segmentation of the virus genome complicates the packaging of infectious progeny into virions, it provides an evolutionary benefit in that it allows viruses to exchange vRNAs with other strains. Influenza A viruses are believed to package their eight different vRNAs in a specific manner. However, several studies have shown that many viruses are noninfectious and fail to package at least one vRNA. Therefore, the genome-packaging mechanism is not fully understood. In this study, we used electron microscopy to count the number of ribonucleoproteins (RNPs) inside the virions of different influenza A and B virus strains. All eight strains examined displayed eight RNPs arranged in a “7+1” configuration in which a central RNP was surrounded by seven RNPs. Three-dimensional analysis of the virions showed that at least 80% of the virions packaged all eight RNPs; however, some virions packaged only five to seven RNPs, with the exact proportion depending on the strain examined. These results directly demonstrate that most viruses package eight RNPs, but some do indeed package fewer. Our findings support the selective genome-packaging model and demonstrate the variability in the number of RNPs incorporated by virions, suggesting that the genome-packaging mechanism of influenza viruses is more flexible than previously thought.

Incomplete influenza A virus genomes are abundant but readily complemented during spatially structured viral spread

“…While this failure of protein expression could be explained by a failure in transcription or translation, the results of our single-cell sorting assay indicate that the vRNA segments themselves are absent, as they should be amplified by the helper virus polymerase even if they do not encode functional proteins. As in Brooke et al, our method does not discriminate between the alternative possibilities that segments are absent from virions themselves or are lost within the cell, but published results suggest that a single virion usually contains a full genome 22, 23 . Importantly, our single cell assay quantifies the frequencies of all eight segments, rather than only those that can be detected indirectly by staining for protein expression, and therefore allows for analysis of the associations between segments.…”

“…Replication and expression of only a subset of the genome may be explained by two potential mechanisms: either the majority of particles lack one or more genome segments, or segments are readily lost in the process of infection before they can be replicated. Electron microscopy has shown that most particles contain eight distinct RNA segments 22 , and FiSH-based detection of viral RNAs indicated that a virion tends to contain one copy of each segment 23 , suggesting that most particles contain full genomes. Regardless of the molecular mechanisms that lead to the phenomenon of incomplete IAV genomes, their frequent occurrence suggests that complementation by co-infection at the cellular level is an underappreciated aspect of the viral life cycle.…”

Establishment of a Reverse Genetics System for Influenza D Virus

“…Previous studies support that IAV most likely contains a selective packaging mechanism (39–42). IAV and IBV incorporate eight RNPs arranged in a specific pattern “1+7”, in which seven RNPs surround the central one (40, 43, 44). Surprisingly, artificially generated 7-segmented IAV, which lacks an HA segment, incorporated rRNA as the eighth RNP instead of an HA segment (37).…”

Learning the sequence of influenza A genome assembly during viral replication using point process models and fluorescence in situ hybridization

“…The NP and viral RNA associate in a non-uniform manner, where regions of the RNA are unbound by NP and capable of forming complex structures [5]. Macro-organization of vRNPs within the viral capsid show tight packaging, organized in a ‘7+1’ orientation, a single vRNP center shaft surrounded by seven others (4–6).…”