Selective incorporation of influenza virus RNA segments into virions

Abstract

The genome of influenza A virus is comprised of eight viral RNA (vRNA) segments. Although the products of all eight vRNA segments must be present for viral replication, little is known about the mechanism(s) responsible for incorporation of these segments into virions. Two models have been proposed for the generation of infectious virions containing eight vRNA segments. The random-incorporation model assumes a common structural feature in all the vRNAs, enabling any combination of vRNAs to be incorporated randomly into virions. The selective-incorporation model predicts the presence of specific structures in each vRNA segment, leading to the incorporation of a set of eight vRNA segments into virions. Here we demonstrate that eight different vRNA segments must be present for efficient virion formation and that sequences within the coding region of (and thus unique to) the neuraminidase vRNA possess a signal that drives incorporation of this segment into virions. These findings indicate a unique contribution from individual vRNA segments and thus suggest a selective (rather than random) mechanism of vRNA recruitment into virions. The neuraminidase vRNA incorporation signal and others yet to be identified should provide attractive targets for the attenuation of influenza viruses in vaccine production and the design of new antiviral drugs.

Figure 1

Effect of the number of RNA segments on the efficiency of virion production. 293T cells were transfected with protein expression plasmids for nine structural proteins and plasmids for the production of eight, seven, or six different vRNA segments. The plasmid for NS vRNA production possesses two mutations to eliminate NS2 protein production (12) such that the resultant virus does not undergo multiple cycles of replication. The plasmids for HA and NA vRNA production contain mutations that eliminate protein production, thus avoiding the effects of these proteins expressed from vRNA. To produce a virus with seven segments, we eliminated the plasmid for HA (−HA) or NA (−NA) vRNA production, whereas for the production of a six-segment virus we omitted the plasmids for both the HA and NA vRNA. Released virions were harvested at 24 (black bar) or 48 h (white bar) posttransfection and titrated by immunostaining virus-infected MDCK cells with antiserum to the influenza WSN strain.

Figure 2

Schematic diagram of mutant vRNAs. NAFLAG RNA contains the 3′ noncoding region of NA vRNA (19 nucleotides), 153 nucleotides of the NA coding regions, corresponding to the cytoplasmic tail (6 aa), the transmembrane (29 aa) and stalk (16 aa) regions, and nucleotides for the FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) in the negative sense, two sequential stop codons (TAA TAG) in the negative sense, 185 nucleotides corresponding to the NA C terminus, and the 5′ noncoding region of NA vRNA. NAFLAGMet(−) RNA has changes in both the NA start codon and a downstream ATG codon (the 15th codon) in frame with the NA reading frame from ATG to GCG (in the positive sense) such that a protein is not produced from the reading frame, as indicated by a box with dashed lines. Both of these RNAs are shown in the negative-sense orientation. The horizontal broken line indicates a deletion. The lengths of the regions are not to scale. Predicted gene products from individual RNA segments are shown on the right.

Figure 3

Confirmation of virus production with truncated NA RNA segments. (A) MDCK cells were infected with NA(−) (a and d), NAFLAG (b and e), or NAFLAGMet(−) (c and f) viruses and overlaid with 0.6% agarose. After incubation for 48 h at 37°C, the cells were fixed and permeated with 0.1% Triton X-100 in 3% formaldehyde solution. The viral proteins or FLAG epitope were detected by immunostaining with antiserum to influenza WSN strain (a–c) or anti-FLAG monoclonal antibody (d–f) as the primary antibody and biotinylated secondary antibody with the VECTASTAIN ABC kit (Vector Laboratories). (B) MDCK cells infected with NA(−), NAFLAG, or NAFLAGMet(−) viruses were incubated, fixed, and permeated as described above. The FLAG sequence in mRNA was detected by in situ hybridization with a digoxigenin-labeled probe specific for the sequence and visualized with the DIG nucleic acid-detection kit (Roche).

Figure 4

Effect of truncated NA vRNA on efficiency of viral growth. Three hundred plaque-forming units of NAFLAG (○) or NAFLAGMet(−) (●) virus were mixed with 3 × 10⁴ plaque-forming units of NA(−) virus, used to infect subconfluent MDCK cells (multiplicity of infection, 0.01), and incubated for 72 h at 37°C in the presence of V. cholerae sialidase. The virus in the supernatant was used to infect MDCK cells. This procedure was repeated four more times. In each passage, virus in the supernatant was used to perform plaque assays and determine the percentage of plaques positive for anti-FLAG epitope by immunostaining (NAFLAG) with anti-FLAG monoclonal antibody or by in situ hybridization [NAFLAGMet(−)] for the FLAG sequence with a probe specific for this sequence.

Figure 5

The coding region is important for NA vRNA virion incorporation. MDCK cells were infected with NA(183)GFP(157) (A) or NA(0)GFP(0) (B) virus and overlaid with 0.6% agarose. The infected cells were incubated for 48 h at 37°C, and the plaques were photographed under fluorescent light together with limited normal light to identify plaques. The lengths of the regions in the RNA constructs are not to scale. Note the limited number of GFP-positive cells in the plaque produced by the NA(0)GFP(0) mutant contrasted with the abundance of GFP-expressing cells in plaques produced by NA(183)GFP(157).

Figure 6

Identification of the region critical for vRNA incorporation into virions. Each mutant contains the GFP ORF (inserted in frame with the NA ORF) flanked by stop codons, 19 nucleotides of the 3′ noncoding region, and 28 nucleotides of the 5′ noncoding region of NA vRNA. The NA coding regions are shown as beige bars. The virion incorporation efficiency of each NA-GFP RNA was calculated as the percentage of GFP-positive plaques among total plaques.