Attenuation of Influenza A Virus mRNA Levels by Promoter Mutations
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Fig. 1.Representation of the conserved sequences of influenza A virus vRNA in the panhandle/RNA-fork conformation (8, 14). Conserved base pairs in the double-stranded region of the RNA-fork, involving both the 5′ and 3′ ends of the RNA segment, are boxed. Numbering of residues starts from the 3′ end and from the 5′ end. The 5′-end numbers are distinguished by primes. (A) Base pairs of D1, D2, D3, and D1/2 transfectant viruses in the conserved double-stranded region. Changed base pairs are highlighted. (B) Sequences of single-nucleotide mutants.
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Fig. 2.Growth curves of transfectant influenza viruses on MDBK cells. Confluent MDBK cells in 35-mm-diameter dishes were infected with wild-type (WT) influenza A/WSN/33 virus and with the transfectant D1, D2, D3, and D1/2 viruses at an MOI of 0.01. At the indicated time points, infectious particles present in the media were titrated by plaque assay in MDBK cells. The presented values are averages from duplicate experiments.
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Fig. 3.
NA protein levels in transfectant viruses. Wild-type (WT) influenza A/WSN/33 virus and transfectant D1, D2, and D3 (A) or D1/2 (B) virus were grown in MDBK cells and purified through 30 to 60% sucrose gradients. Proteins of purified viruses, untreated (−) or treated with PNGase F (+), were separated by SDS-PAGE (12% gel) and stained with Coomassie brilliant blue. Positions of untreated proteins are shown on the left; positions of proteins treated with PNGase F are indicated on the right. The position of PNGase F is shown on the right. Arrowheads indicate positions of the NA treated with PNGase F (17).
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Fig. 4.vRNA of purified transfectant D1, D2, D3, and D1/2 viruses. RNAs extracted from purified wild-type (WT) A/WSN/33 and transfectant D1, D2, D3, and D1/2 viruses were separated in a 2.8% polyacrylamide gel containing 7 M urea and visualized by silver staining. The positions of RNAs that encode the polymerase proteins (Ps), HA, NP, NA, M1 and M2 proteins (M), and NS1 and NEP (26) proteins (NS) are indicated. The position of 18S RNA, a contaminating cellular RNA often present in viral RNA preparations, is also indicated. Arrowheads indicate the position of the NA-specific vRNA segments. The origin of the faint band in lanes D1, D2, and D3, moving slightly faster than the NP, is unknown.
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Fig. 5.NA-specific vRNA levels in purified transfectant D1, D2, D3, and D1/2 viruses (A) and in cells infected with transfectants D2 and D1/2 (B). (A) Viral RNA was extracted from wild-type (WT) A/WSN/33 virus and the D1, D2, D3, or D1/2 transfectant purified through a 30% sucrose cushion. (B) Total RNAs from MDBK cells infected at an MOI of 2 with wild-type (WT) A/WSN/33 virus and the D2 or D1/2 transfectant were extracted at the indicated time points postinfection (p.i.). Both viral and total RNAs were subjected to primer extension analysis, using primers specific for the NA and NS vRNAs, and the primer extension products were analyzed on a 5% polyacrylamide gel containing 7 M urea. The expected length of the primer extension products for the NA segment is 259 nt; that for the control NS segment is 196 nt. Size markers in nucleotides are shown on the left.
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Fig. 6.NA-specific mRNA and cRNA levels in cells infected with transfectant D2 or D1/2. Total RNAs from MDBK cells infected at an MOI of 2 with wild-type (WT) A/WSN/33 virus or the D2 or D1/2 transfectant were extracted at the indicated time points postinfection (p.i.) and subjected to primer extension analysis, using primers specific for the positive-sense NA and HA RNAs. Primer extension products were analyzed on a 5% polyacrylamide gel containing 7 M urea. The expected length of the primer extension products for the NA cRNA is 125 nt; that for the NA mRNA is about 135 to 140 nt due to the presence of host cell mRNA-derived heterogeneous capped RNA primers at the 5′ end of viral mRNAs. The expected length of HA-specific extension products is 94 nt for the cRNA and about 104 to 109 nt for the mRNA. Size markers in nucleotides are shown on the left.
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Fig. 7.In vitro transcription of NA-specific RNP complexes and analysis of the transcription products on oligo(dT)-cellulose. In vitro transcription reactions with RNPs isolated from wild-type (WT) A/WSN/33 virus and D2 or D1/2 transfectant virus were performed with globin mRNA as primer (for details see Materials and Methods). One third of the transcription products was directly analyzed (A) on a 3% polyacrylamide gel in 7 M urea. Two-thirds of the transcription products were separated on oligo(dT)-cellulose, and fractions depleted of (B) and enriched in (C) poly(A)-containing molecules were analyzed on the same gel. A longer exposure of the D1/2 products enriched in poly(A)-containing molecules is shown. The positions of the NP- and NA-specific transcription products are indicated.
- Copyright © 1998 American Society for Microbiology
