Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions

Efficiency of incorporation of recombinant NS segments. Incorporation efficiencies of recombinant NS segments with different lengths of NS coding regions were determined as described in the legend to Fig. 1. The noncoding and coding regions of the NS vRNA are represented by grey and white bars, respectively, while the green bars represent the coding region of the GFP gene. The dotted line represents deleted sequences of the NS coding region. The results shown are representative data from three independent experiments.

Efficiencies of incorporation of NS segments with nucleotide substitutions in the NS coding regions. The nucleotide sequences at the 3′ (positions 27 to 56) or 5′ (positions 832 to 864) end of the NS coding region are shown. The silent mutations introduced are shown by asterisks. The coding regions of the NS vRNA and GFP are represented by white and green bars, respectively. The results shown are representative data for three independent experiments. (A) Efficiencies of incorporation of the NS-GFP segment with nucleotide substitutions in the NS coding regions. Incorporation efficiencies were determined as described in the legend to Fig. 1. (B) Efficiencies of incorporation of the NS segment with nucleotide substitutions in the NS coding regions. To determine the incorporation efficiency of mutant NS vRNA segments, NS2- and NP-expressing cells were counted at 24 h postinfection.

Growth properties of mutant viruses containing silent mutations in the coding regions of the NS segment. MDCK cells were infected with virus at an multiplicity of infection of 0.001. At the indicated times after infection, the virus titer in the supernatant was determined. The values are means and standard deviations from triplicate experiments. •, m1; ○, m2; ▪, m3; □, wild type.

Importance of NS segment-specific noncoding regions for incorporation of recombinant NS segments. (A) Schematic diagram of the NS vRNA. Segment-specific complementary sequences are underlined. The poly(U) sequence for the addition of the poly(A) tail to the mRNA is shown in italics. The grey boxes represent U12 and U13. (B) Efficiencies of incorporation of recombinant NS segments with deletions in the NS segment-specific noncoding regions. U12 and U13 are represented by grey boxes. The coding regions of the NS vRNA and GFP gene are represented by white and green bars, respectively. The dotted lines represent sequences deleted from the NS segment. The results shown are representative data from three independent experiments.

Comparison of test NS vRNA amounts and GFP expression levels in plasmid-transfected cells. (A) Quantitative analysis of NS(0)GFP(0), NSΔNTR, and NS(150)GFP(150) vRNAs produced from PolI plasmids in 293T cells by real-time quantitative reverse transcription-PCR. 293T cells (10⁶ cells) were transfected with 18 plasmids. At 48 h posttransfection, total RNA was extracted from 293T cells and quantified by real-time PCR with GFP and NP sequence-specific primers and probes as described in Materials and Methods. The primers and probes for the NS-GFP segments detect NS-GFP but not the authentic NS segment. Experiments were performed in the presence (+) or absence (−) of reverse transcriptase (RT) to demonstrate that the results are not affected by the plasmids used for the experiments. NS2KO represents NS2 knockout virus, which does not produce NS2(NEP) protein and does not undergo multiple cycles of replication (27). It therefore serves as a negative control for detection of NS-GFP and a positive control for the NP segment. The results shown are representative data from three independent experiments. Error bars indicate standard deviations. (B) Comparison of GFP expression levels in PolI plasmid-transfected 293T cells by fluorescence microscopy. At 48 h after transfection, the cells were observed by fluorescence microscopy.