Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses.

The fragmented nature of the influenza A genome allows the exchange of gene segments when two or more influenza viruses infect the same cell, but little is known about the rules underlying this process. Here, we studied genetic reassortment between the A/Moscow/10/99 (H3N2, MO) virus originally isolated from human and the avian A/Finch/England/2051/91 (H5N2, EN) virus and found that this process is strongly biased. Importantly, the avian HA segment never entered the MO genetic background alone but always was accompanied by the avian PA and M fragments. Introduction of the 5' and 3' packaging sequences of HA(MO) into an otherwise HA(EN) backbone allowed efficient incorporation of the chimerical viral RNA (vRNA) into the MO genetic background. Furthermore, forcing the incorporation of the avian M segment or introducing five silent mutations into the human M segment was sufficient to drive coincorporation of the avian HA segment into the MO genetic background. These silent mutations also strongly affected the genotype of reassortant viruses. Taken together, our results indicate that packaging signals are crucial for genetic reassortment and that suboptimal compatibility between the vRNA packaging signals, which are detected only when vRNAs compete for packaging, limit this process.

Interaction network linking the human H3N2 influenza A virus genomic RNA segments.

The genome of influenza A viruses is comprised of eight negative-sense viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). In order to be infectious, an influenza A viral particle must encapsidate at least one copy of each of the vRNAs. Thus, even though genome segmentation is evolutionary advantageous, it undeniably complicates viral assembly, which is believed to occur through a selective mechanism that still remains to be understood. Using electron tomography 3D-reconstructions, we show that the eight vRNPs of an influenza A Moscow/10/99 (H3N2) virus are interconnected within a star-like structure as they emerge from a unique "transition zone" at the budding tip of the virions. Notably, this "transition zone" is thick enough to accommodate all described packaging signals. We also report that, in vitro, each vRNA segment is involved in a direct contact with at least one other vRNA partner, in a single network of intermolecular interactions. We show that in several cases, the regions involved in vRNA/vRNA interactions overlap with previously identified packaging signals. Our results thus provide support for the involvement of RNA/RNA interactions in the selection and specific packaging of influenza A genomic RNAs, which appear embedded into an organised supramolecular complex likely held together by direct base-pairings between packaging signals.

A supramolecular assembly formed by influenza A virus genomic RNA segments.

The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common 'transition zone' located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.