Abstract
Influenza A virus vRNA segments contain specific packaging signals at their termini that overlap the coding regions. To further characterise segment 5 packaging signals, we introduced synonymous mutations into the terminal coding regions of the vRNA and characterised the replicative fitness of the resulting viruses. Most mutations tested were well-tolerated, but a virus with alterations to NP codons 464-466, near the 5′-end of the vRNA, produced small plaques and replicated to around one-tenth of the level of wild type virus. The mutant virus supported normal levels of NP and segment 5 vRNA synthesis but packaged reduced levels of both segment 5 and segment 3 into virus particles. This suggests an interaction between segments 3 and 5 during influenza A virus assembly.
Highlights
The negative-sense RNA of the influenza A virus genome is divided into eight segments, which are packaged into new virions as they assemble at the plasma membrane of infected cells
We showed that the introduction of synonymous mutations into normally highly conserved codons in the terminal regions of segments 1, 6 and 7 caused defects in virion RNA (vRNA) incorporation into virus particles, consistent with the presence of cis-acting packaging signals [14,18]
The structure of a defective interfering (DI) RNA derived from segment 5 [29], our previous bioinformatics analysis [14], as well as the minimal segment 5-derived sequences required to package a reporter gene [12] indicated that, to the other segments of the influenza A genome, the terminal regions of segment 5 vRNA contained packaging signals (Fig. 1A)
Summary
The negative-sense RNA of the influenza A virus genome is divided into eight segments, which are packaged into new virions as they assemble at the plasma membrane of infected cells. The virus has evolved a mechanism to help ensure each of its eight segments is selectively packaged [6,7]. This selective packaging mechanism utilises cis-acting RNA packaging signals in each of the eight segments, the location of which has been inferred from the structure of defective interfering (DI) RNAs [6,8], the packaging of recombinant virion RNA (vRNA) molecules [7,9,10,11,12,13] and the conservation of primary nucleotide sequences [14,15,16].
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