Competitive Incorporation of Homologous Gene Segments of Influenza A Virus into Virions

The influenza A virus genome is segmented into eight negative-sense RNAs. Although this segmented genome allows viruses to evolve rapidly through gene reassortment, all of the eight viral RNA (vRNA) segments need to be introduced into a cell for the viruses to be infectious (18). In virions, eight kinds of RNA molecules were detected at a comparable molar ratio in purified viruses (17). Previously, we used electron microscopy to show that eight rods, which were most likely vRNA-associated ribonucleoprotein complexes (RNPs), were packed in budding virions in an orderly fashion (12). Further, the three-dimensional structure of the RNPs revealed that the eight rod-like structures were of different lengths and well organized but asymmetrically arranged in progeny virions (2, 13). These findings suggest that the individual influenza virions incorporate eight vRNA segments; however, it remains unclear whether the eight rods correspond to the eight genetically distinct vRNA segments (i.e., the eight distinct vRNA segments are present in individual virions) or some rods are genetically identical (i.e., multiple copies of homologous vRNA segments are packaged in individual virions and the eight vRNA segments are maintained as virus populations).

We therefore attempted to assess the incorporation efficiency of two reporter protein-encoding virus-like RNAs derived from identical vRNA segments. We (3, 4, 10, 13–15) and others (1, 5–9) have reported that the noncoding and coding sequences at the 3′ and 5′ ends of each vRNA segment are essential for efficient segment incorporation into virions. According to these findings, we constructed a plasmid encoding two RNA polymerase I promoter-driven transcription cassettes for virus-like RNA expression (Fig. 1A to C). One transcription cassette produces negative-sense RNA containing the 3′ noncoding sequence of the neuraminidase (NA) vRNA segment, 183 nucleotides of the 3′ coding sequence of the NA vRNA segment, the open reading frame of green fluorescent protein (GFP; Clontech), 157 nucleotides of the 5′ coding sequence of the NA vRNA segment, and the 5′ noncoding sequence of the NA vRNA segment (Fig. 1A). Another cassette produces the same virus-like RNA except that the GFP reporter gene was replaced with the gene encoding DsRed-monomer fluorescent protein (DsRed; Clontech) (Fig. 1B). The length of the coding sequence at each end is required for efficient incorporation of the NA vRNA segment (4). To ensure that the two virus-like vRNAs encoding different reporter proteins were produced in the same cells, we constructed plasmids containing the two transcription cassettes. Further, to eliminate any effect of gene order in the plasmid on the expression level of the virus-like RNAs, we prepared two types of plasmids containing the two transcription cassettes in a different order (Fig. 1C).

To confirm that the resultant “tandem” reporter plasmids indeed produced both GFP- and DsRed-encoding virus-like RNAs in cells, 293T cells were transfected with the plasmids together with four protein plasmids for the expression of the A/WSN/33 (H1N1, WSN) viral polymerase subunits (PB2, PB1, and PA) and nucleoprotein NP, which are required for vRNA transcription and replication (18). At 24 h posttransfection, GFP- and/or DsRed-expressing cells were counted under a fluorescence microscope. Approximately half (45.7%) of the fluorescent protein-positive cells expressed both GFP and DsRed (Fig. 1D).

Next, to assess the incorporation efficiency of the two virus-like NA vRNA segments encoding the reporter proteins into single progeny virions, we generated WSN-based NA-knockout recombinant influenza viruses by using reverse genetics (11) with the tandem reporter plasmids instead of a plasmid for the expression of the intact NA vRNA. At 24 h posttransfection, culture supernatants were clarified and subjected to plaque assays in Madin-Darby canine kidney (MDCK) cells. These NA-knockout viruses formed small plaques in MDCK cells as described previously (4); most of the plaques expressed fluorescent proteins at 24 to 48 h postinfection (data not shown). We then counted the GFP- and/or DsRed-positive plaques. Most of the plaques expressed only either GFP (59.3%) or DsRed (39.1%) (Fig. 1E); only a small portion (1.6%) of the plaques was positive for both GFP and DsRed (Fig. 1E). These results suggest that the two reporter virus-like RNAs competed for incorporation into individual virions.

The competitive effect of the two reporter virus-like RNAs was also assessed by using PB2 vRNA segment-based virus-like RNAs. We constructed tandem reporter plasmids for the recombinant PB2 vRNA segment (Fig. 2A to C). These plasmids encoded the 3′ and 5′ ends of the PB2 vRNA segment including 120 nucleotides of each coding sequence, which were required for efficient incorporation of the PB2 vRNA segment (15). The expression of both GFP- and DsRed-encoding virus-like RNAs in cells transfected with the tandem reporter plasmids was confirmed as described above. Similar to the results with the NA vRNA segment-based plasmids, approximately half (53.5%) of the transfected cells expressed GFP and DsRed (Fig. 2D). By using these tandem reporter plasmids instead of an intact PB2 vRNA-expressing plasmid, we generated PB2-knockout viruses by means of reverse genetics. We then counted the GFP- and/or DsRed-positive plaques formed by the transfectant viruses in PB2 protein-expressing MDCK cells that were established by using a retrovirus vector as described previously (16). Like the NA-knockout viruses, these PB2-knockout viruses mainly formed plaques expressing only either GFP (75.6%) or DsRed (23.8%); only a limited portion (0.6%) of the plaques expressed both GFP and DsRed (Fig. 2E). These results indicate that, as with the NA segment, only a single PB2 segment is incorporated into virions.

Here, we demonstrated that two virus-like RNAs derived from identical vRNA segments competed for incorporation into progeny viruses. Although half of the cells transfected with plasmids for virus generation were positive for two reporter proteins (Fig. 1D and 2D), most of the plaques formed by the transfectant viruses were positive for only one or the other protein (Fig. 1E and 2E). These findings suggest that individual influenza virions incorporate single, not multiple, copies of homologous vRNA segments, that is, the eight distinct vRNA segments required for virus replication are present in individual virions.

We thank Susan Watson for editing the manuscript.

This work was supported by ERATO (Japan Science and Technology Agency), by a grant-in-aid for Specially Promoted Research from the Ministries of Education, Culture, Sport, Science, and Technology, by a grant-in-aid from Health, Labor, and Welfare of Japan, by a Contract Research Fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, and by National Institute of Allergy and Infectious Diseases Public Health Service research grants.