Abstract
For each influenza virus genome segment, the coding sequence is flanked by non-coding (NC) regions comprising shared, conserved sequences and specific, non-conserved sequences. The latter and adjacent parts of the coding sequence are involved in genome packaging, but the precise role of the non-conserved NC sequences is still unclear. The aim of this study is to better understand the role of the non-conserved non-coding sequences in the incorporation of the viral segments into virions. The NA-segment NC sequences were systematically replaced by those of the seven other segments. Recombinant viruses harbouring two segments with identical NC sequences were successfully rescued. Virus growth kinetics and serial passages were performed, and incorporation of the viral segments was tested by real-time RT-PCR. An initial virus growth deficiency correlated to a specific defect in NA segment incorporation. Upon serial passages, growth properties were restored. Sequencing revealed that the replacing 5′NC sequence length drove the type of mutations obtained. With sequences longer than the original, point mutations in the coding region with or without substitutions in the 3′NC region were detected. With shorter sequences, insertions were observed in the 5′NC region. Restoration of viral fitness was linked to restoration of the NA segment incorporation.
Highlights
In humans, influenza A viruses are responsible for yearly epidemics of respiratory tract infections that represent a constant burden for national health services worldwide[1]
In agreement with the fact that transcription and replication of the X-NA-X segments was similar to wild-type, no major differences in expression levels of the NA as analysed by western-blot were observed between the different constructs tested and wild-type, except for PB1-NA-PB1 and HA-NA-HA for which expression of the NA was reduced (Supplementary Figure S8a)
It was proposed that the NC signals drive the incorporation of the viral RNA (vRNA) into newly formed viral particles, while sequences in the coding regions serve as a bundling signal to ensure that a complete set of the different genomic viral ribonucleoproteins (vRNP) is packaged[6,23]
Summary
Influenza A viruses are responsible for yearly epidemics of respiratory tract infections that represent a constant burden for national health services worldwide[1]. Numerous studies have demonstrated that packaging signals of all eight segments encompass sequences from both the 3′and 5′NC regions and from the adjacent coding regions in the vRNA6,15–22. These studies revealed that mutations in the packaging signals of a given segment could impact the incorporation of other segments. We and others have recently shown that the sequence of the non-conserved NC regions is not critical for efficient rescue of infectious viruses, suggesting the existence of some degree of flexibility in the compatibility between the packaging signals in the NC and coding regions[28,29,30]. Upon serial passages in cell culture, we found that the length of the heterologous 5′NC region was key to the type of changes required for virus fitness recovery and restoration of incorporation of the NA segment
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