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. 2006 Mar;80(5):2318-25.
doi: 10.1128/JVI.80.5.2318-2325.2006.

Hierarchy among viral RNA (vRNA) segments in their role in vRNA incorporation into influenza A virions

Yukiko Muramoto  1 , Ayato Takada, Ken Fujii, Takeshi Noda, Kiyoko Iwatsuki-Horimoto, Shinji Watanabe, Taisuke Horimoto, Hiroshi Kida, Yoshihiro Kawaoka
Affiliations

Hierarchy among viral RNA (vRNA) segments in their role in vRNA incorporation into influenza A virions

Yukiko Muramoto et al. J Virol. 2006 Mar.

Abstract

The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of the system used to determine infectious virion formation and the incorporation efficiencies of test vRNA segments. 293T cells were transfected with plasmids for the production of WSN VLPs. Forty-eight hours later, the supernatants were mixed with helper PR8 virus and used to infect MDCK cells. At 12 h postinfection, the numbers of GFP- or WSN HA-positive cells were counted by flow cytometry; alternatively, the numbers of cells expressing WSN NP were determined. The number of cells expressing HA or NP was used to determine the number of infectious VLPs in the experiments presented in Table 1. The number of cells expressing GFP reflects the number of VLPs possessing a test vRNA. The incorporation efficiency of a test vRNA was determined by dividing the number of GFP-expressing cells by the sum of the number of cells expressing WSN HA and the number of GFP-expressing cells.
FIG. 2.
FIG. 2.
Schematic diagram of mutant PB2 vRNAs and their efficiencies in virion formation and virion incorporation. The numbers of VLPs and the virion incorporation efficiencies of mutant PB2 vRNAs were determined by using the numbers of WSN HA- and GFP-expressing cells as a denominator. All mutants are shown in the negative-sense orientation. Each mutant contains the GFP reading frame (green bar); 27 and 34 nucleotides of the 3′ and 5′ noncoding regions, respectively (gray bars); and coding regions of various lengths (black bars). The dotted lines represent deleted sequences of the PB2 coding region. PB2(−) indicates the omission of this vRNA (i.e., VLPs were generated using only seven vRNA segments). The results shown are representative data from six independent experiments.
FIG. 3.
FIG. 3.
Schematic diagram of mutant PB1 vRNAs and their efficiencies in virion formation and virion incorporation. Experiments were performed as outlined in the legend to Fig. 2. All mutants are shown in the negative-sense orientation. Each mutant contains the GFP sequence (green bar) flanked by 24 and 43 nucleotides of the 3′ and 5′ noncoding regions, respectively (gray bars), and coding regions of various lengths (black bars). PB1(−) indicates the omission of this vRNA. The results shown are representative data from three independent experiments.
FIG. 4.
FIG. 4.
Schematic diagram of mutant PA vRNAs and their efficiencies in virion formation and virion incorporation. Experiments were performed as outlined in the legend to Fig. 2. All mutants are shown in the negative-sense orientation. Each mutant contains the GFP sequence (green bar) flanked by 24 and 58 nucleotides of the 3′ and 5′ noncoding regions, respectively (gray bars), and coding regions of various lengths (black bars). PA(−) indicates that this vRNA was omitted. The results shown are representative data from six independent experiments.
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