INTRODUCTION
The genome of influenza A virus is segmented into eight single-stranded negative-sense RNAs. While this genome segmentation provides influenza viruses with a considerable evolutionary advantage through gene reassortment, it complicates the genome packaging of the progeny virions. For decades, the genome-packaging mechanism had been controversial (
1,
2); however, recent studies clearly favor the selective packaging model in which influenza virions selectively incorporate a complete set of eight distinct vRNAs. The selective packaging model is experimentally supported by (i) electron microscopic studies showing that eight ribonucleoprotein complexes (RNPs), which are composed of each vRNA, multiple copies of nucleoprotein (NP), and an RNA-dependent RNA polymerase complex, are packaged into progeny virions in a characteristic arrangement (
3–6), (ii) a single-molecule fluorescent
in situ hybridization analysis and an analysis of using artificial vRNAs encoding different fluorescent reporter proteins showing that most virions contain only one copy of each vRNA (
7,
8), and (iii) reverse genetics studies demonstrating that segment-specific packaging signal sequences are necessary for efficient packaging of vRNAs into progeny virions (
9–14). It is now widely accepted that the majority of each influenza A virion incorporates one copy of each of the eight different vRNAs (
15).
Genome packaging signals have been found in all eight influenza A virus vRNAs (
1,
9–16). Each packaging signal is bipartite, being located in the noncoding and terminal coding regions of both the 3′ and 5′ ends of each vRNA. Each packaging signal is unique to each vRNA, and it is this uniqueness that probably leads to the discriminate incorporation of the individual vRNAs into progeny virions. The introduction of mutations into the genome-packaging signal markedly reduces the packaging efficiency of the mutated vRNA into progeny virions (
16–24), as well as that of the other vRNAs (
14,
18,
21), demonstrating the significance of the genome-packaging signal for vRNA incorporation into virions. Recent electron microscopic and biochemical studies suggest that there should be interactions among the vRNAs, most likely in the form of RNPs (
5,
6,
25); however, it remains unclear how these packaging signals function during vRNA incorporation into virions. Here, we used reverse genetics to clarify the role of the packaging signals. We found that the so-called genome-packaging signal serves two distinct functions: the virion incorporation of RNP and the bundling of multiple RNPs.
MATERIALS AND METHODS
Cells and virus.
Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco modified Eagle medium containing 10% fetal calf serum (FCS). Madin-Darby canine kidney (MDCK) cells were maintained in Eagle minimal essential medium (MEM) containing 5% newborn calf serum. All cells were maintained at 37°C under 5% CO2. Influenza A/WSN/33 (H1N1; referred to as WSN) was propagated in MDCK cells.
Plasmid construction.
pPolI NP(0)GFP(0) and pPoII NP(120)GFP(120) were described previously (
15). To generate pPolI N0-FLAG NP, the open reading frame (ORF) of pPolI-WSN-NP was replaced with the NFLAG NP gene from pCAGGS NFLAG NP. Briefly, the NP ORF in pPolI-WSN-NP was eliminated by back-to-back PCR with primers containing BsmBI sites. Using the BsmBI sites, the NFALG NP gene containing BsmBI sites at both ends, which was generated by PCR, was cloned. To generate pPolI N49ATC-FLAG NP, three ATG codons in the 49 nucleotides at the 5′ end of the NP cDNA were changed by using site-directed mutagenesis. The remaining region from the 50th nucleotide to the end of the NP ORF was replaced with the NFLAG NP gene as described above. pPolI N49ATCinv-FLAG NP was generated by back-to-back PCR using pPolI N49ATC-FLAG NP as a template, which was digested with BsmBI and self-ligated. The primers used were as follows: N49ATCinvF (5′-CACACACGTCTCTCTAGCAAACCACGGAAACCAGCGGCGATGGACTACAAGGACGACGACGACAAG-3′) and N49ATCinvR (5′-CACACACGTCTCGCTAGAATGCTTGTCTAGCTCTGACTAGGATTTCGATGTCACTCTGTGAGTGATTATCCACACACGTCTCGCTAGAATGCTTGTCTAGCTCTGACTAGCACACACGTCTCTCTAGCAAACCACGGAAACCAGCGGCG-3′). All constructs were sequenced to confirm the absence of undesired substitutions.
Reverse genetics.
Viruses and virus-like particles (VLPs) were generated as described previously (
15). Briefly, 293T cells (10
6 cells per 3.5-cm culture dish) were transfected with a cocktail of 18 plasmids (0.1 μg of each of 8 pPolI plasmids for vRNA generation, 1 μg of PB1, PB2, PA, NP, HA, NA, NS1, and NS2 expression plasmids, 2 μg of M1 expression plasmid, and 0.1 μg of M2 expression plasmid) by using TransIT293 (PanVera). To generate VLPs with a single vRNA, we used 0.8 μg of a single pPolI plasmid instead of the 8 pPolI plasmids. At 24 h posttransfection, the supernatants containing viruses or VLPs were harvested after a 30-min incubation at 37°C with TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-trypsin (final concentration, 0.5 μg/ml). Cell debris was removed by centrifugation.
Western blot analysis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots were performed according to standard procedures. Plasmid-transfected 293T cells were lysed in SDS sample buffer, separated on a 4 to 20% gradient Tris-glycine gel, and then transferred to a polyvinylidene difluoride membrane. Blots were probed with a mouse anti-FLAG M2 monoclonal antibody (MAb; Sigma), rabbit anti-WSN R309 serum, or a mouse anti-β actin MAb (Sigma), followed by horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG or HRP-conjugated donkey anti-rabbit IgG (GE Healthcare). They were then visualized by using ECL Plus Western blotting detection reagents (GE Healthcare) according to the manufacturer's instructions.
Flow cytometry.
MDCK cells were infected with VLPs containing NP(0)GFP(0) vRNA or NP(120)GFP(120) vRNA, together with a helper A/WSN/33 virus to supply intact NP protein in trans. At 8 h postinfection, the cells were rinsed with phosphate-buffered saline (PBS) containing 0.02% EDTA and trypsinized to prepare a single cell suspension, which contained 2% FCS and 0.01% sodium azide. Green fluorescent protein (GFP) expression was analyzed in an FL1 detector of FACSCalibur (Becton Dickinson).
Plaque assay.
MDCK cells infected with viruses were cultured in MEM containing 0.3% bovine serum albumin, 0.5 μg of TPCK-trypsin/ml, and 1% agarose for 48 h. Plaques were counted after crystal violet staining.
VLP infection assay.
MDCK cells were infected with viruses or VLPs, together with a helper WSN virus. At 7 h postinfection, the cells were fixed with 4% paraformaldehyde in phosphate buffer for 15 min at room temperature, permeabilized with PBS containing 0.2% Triton X-100 for 10 min, and incubated with an anti-FLAG M2 MAb or rabbit anti-WSN R309 serum. The cells were detected by using a Vectastain Elite ABC kit (Vector Laboratories) and 3,3′-diaminobenzidine (DAB) staining.
DISCUSSION
Although the selective packaging model for the influenza virus genome in which genome-packaging signals play a major role is now widely accepted, it has remained unclear how the packaging signals ensure that the eight different vRNAs are packaged. Here we used reverse genetics to show that the noncoding regions of the NP vRNA are sufficient for its incorporation into virions and that the packaging signal that includes both coding and noncoding elements of the NP vRNA is essential for the incorporation of a complete set of eight vRNAs into virions. We therefore propose that the noncoding regions of vRNAs serve as a virion incorporation signal and that the coding regions serve as a bundling signal that ensures the recruitment of the full set of vRNAs.
Consistent with previous reports (
26,
27), the noncoding regions of NP vRNA were sufficient to pack the NP(0)GFP(0) vRNA into virus particles (
Fig. 1). Interestingly, the terminal coding sequences adjacent to the noncoding regions did not increase the packaging efficiency of the NP(120)GFP(120) vRNA in the absence of the other vRNAs (
Fig. 1), suggesting that the 3′ and 5′ noncoding regions
per se are sufficient to signal the incorporation of that vRNA. In contrast, in the presence of the other seven vRNAs, the incorporation efficiency of the NP(120)GFP(120) vRNA was increased (
Fig. 1), implying that the other vRNAs play a critical role in augmenting the packaging efficiency of the recombinant NP vRNA itself, possibly by direct or indirect interactions with the packaging signal of the recombinant NP vRNA (
5,
6,
25).
How do the packaging signals ensure that all eight different vRNAs are incorporated? As shown in
Fig. 3 and
4, N0-FLAG NP vRNA and N49ATCinve-FLAG NP vRNA did not support the production of replication-competent virions, although N0-FLAG NP vRNA and N49ATCinve-FLAG NP vRNA could be incorporated into virus particles. These findings suggest that the replication-deficient virions produced with N0-FLAG NP vRNA or N49ATCinve-FLAG NP vRNA lacked the complete set of eight vRNAs. Given that the packaging efficiency of the mutant NP vRNAs used here was similar in the absence of the other seven vRNAs (
Fig. 5), it appears that the packaging signal of NP vRNA plays an important role in recruiting the other vRNAs into the virion during the genome-packaging process. Thus, it may be that the eight individual vRNAs mutually recruit the other vRNAs to ensure that the full complement of vRNAs is incorporated into the virions.
On the basis of our findings, we propose that the traditional packaging signal, which includes the terminal coding region and the noncoding regions at the 3′ and 5′ ends of each vRNA, can be divided into two separate signals: the incorporation signal and the bundling signal (
Fig. 6). The incorporation signal, defined as the sequence required for the incorporation of the vRNA in which it resides, is located in the 3′ and 5′ noncoding regions. A previous study showed that a recombinant vRNA composed of the antisense-oriented chloramphenicol acetyltransferase gene flanked by the 3′ and 5′ noncoding regions (i.e., the recombinant vRNA possessed only the incorporation signal) was sufficient for the packaging of that vRNA (
27). However, the virus lost the recombinant vRNA after several passages, indicating that this incorporation signal sequence is not sufficient for the segment to be maintained stably within the virions over multiple replication cycles (
27). Another report supports this notion; when reporter vRNAs possessing the antisense-oriented green or yellow fluorescent protein gene flanked by the 3′ and 5′ noncoding regions were used for virus production in a reverse genetics system (i.e., the reporter vRNA possessed only the incorporation signal), 3 to 5% of the virus particles carried at least two copies of the reporter gene (
28), suggesting that these vRNAs with only the incorporation signal were incorporated into virions in a somewhat nonspecific manner, where interactions between RNP and M1 may also be involved (
29). Such nonspecific packaging of the vRNAs may allow virions to inadvertently carry more than eight vRNAs (
30,
31).
The bundling signal, which is defined as the sequence that signals the incorporation of the other vRNAs, should correspond to the terminal coding regions of the vRNA. In the absence of the other vRNAs, the bundling signal is not involved in the efficient virion incorporation of the vRNA in which it resides (
Fig. 1 and
5). However, in the presence of the other vRNAs, the bundling signal increases both the incorporation efficiency of the vRNA in which it resides (
Fig. 1) and that of the other vRNAs (
Fig. 1 and
3). This is likely because the bundling signal of one vRNA may interact with that of another vRNA (
5,
6,
25), and vice versa, to mutually increase the incorporation efficiencies of the vRNAs. Previous studies have shown that a recombinant vRNA that possesses only the incorporation signal does not specifically compete with the other vRNAs in the genome-packaging process (
8,
28), whereas a defective-interference (DI) vRNA, which typically contains the bundling signal, competes with its parental vRNA specifically (
32,
33). Thus, it is possible that the bundling signal not only increases the incorporation efficiencies of vRNAs but also confers the selective mechanism upon the genome-packaging process.
The bundling signal appears to possess another important feature. Previous studies have shown that vRNA containing a reporter gene flanked by its 3′ and 5′ noncoding regions (i.e., only the strict packaging signal) is not maintained stably during multiple replication cycles (
9,
27). However, a DI vRNA containing the 3′ and 5′ noncoding regions, as well as the additional coding sequences (i.e., the bundling signal), is stably maintained during virus replication (
32,
33). These reports suggest that the bundling signal is required for the virus to stably maintain the vRNAs in a specific manner and therefore is responsible for the selectivity of vRNA packaging.
In conclusion, by using a reverse genetics system, we demonstrated that the traditional packaging signal consists of a virion incorporation signal and a bundling signal. This finding provides important insights into how influenza viruses ensure the integrity of their genome. Further studies are needed to better understanding the selective genome-packaging mechanism of influenza viruses, which could yield excellent targets for the development of anti-influenza drugs that inhibit the assemblage and incorporation of the viral genome.