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, 75 (10), 4912-7

Influenza A Virus NEP (NS2 Protein) Downregulates RNA Synthesis of Model Template RNAs

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Influenza A Virus NEP (NS2 Protein) Downregulates RNA Synthesis of Model Template RNAs

R Bullido et al. J Virol.

Abstract

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.

Figures

FIG. 1
FIG. 1
Plasmid pGEM-NS2 inhibits expression of a model CAT RNA. (A) COS-1 cell cultures (106 cells) were infected with vaccinia virus vTF7–3 and transfected with a DNA mixture containing liposomes and plasmids pGEM-NP (2 μg), pGEM-PB1 (0.6 μg), pGEM-PB2 (0.6 μg), pGEM-PA (0.1 μg), and pCATCA-18 (0.5 μg) (see text for details) and the indicated amounts (expressed in micrograms) of plasmid pGEM-NS2, following the protocol detailed previously (21). After 24 h of incubation, cell extracts were prepared and tested for CAT activity with [14C]chloramphenicol and thin-layer chromatography as described by Mena et al. (21). CAT activity values were expressed as a percentage of the activity obtained in the sample that was not transfected with plasmid pGEM-NS2. (B). Extracts from the cultures transfected with different amounts of plasmid pGEM-NS2 and from MDCK cells that had been either mock infected (M) or infected with the influenza virus A/Victoria/3/75 (Flu) (multiplicity of infection of 5) for 24 h were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a serum raised against a bacterially expressed, purified His-tagged virus strain, A/Victoria/3/75 NEP. As indicated in the figure, two doses (2 and 10 μl) of each of the cell extracts were loaded into the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel.
FIG. 2
FIG. 2
Schematic representation of the plasmids containing NEP gene sequences. Plasmids pGEM-NS2 and pGEM-NS2Δ have been described previously (9, 21). For these two plasmids, the 5′-end influenza virus noncoding nucleotide sequences that are located upstream of the NEP initiation codon are shown. Plasmid pGEM-NS2ΔATG was obtained by removing the nucleotide sequences (69 bp) between the EcoRI and MunI restriction sites present in plasmid pGEM-NS2Δ. Plasmid pGEM-NS2Δ was linearized with restriction enzyme MunI, treated with mung bean nuclease, and religated to yield plasmid pGEM-NS2ΔFAS, which lacks 4 bp compared to plasmid pGEM-NS2Δ. Open arrow, T7 RNA polymerase promoter; ▪, nontranslated influenza virus-derived sequences at the 5′ ends of the NEP mRNA; □, nucleotide sequences coding for the NEP gene; , pGEM-3 derived sequences; ATG, NEP initiation codon.
FIG. 3
FIG. 3
Effect of the pGEM-NS2-derived plasmids on CAT expression mediated by the influenza A and B virus recombinant polymerases. In section FLU A, COS-1 cell cultures were infected with vTF7–3 and transfected with mixtures including the plasmids encoding the influenza A virus RNP protein components, plasmid pCATCA18, and the indicated pGEM-NS2-derived plasmid (at a concentration of 2 μg) as described in the legend to Fig. 1. The sample “−” was transfected only with the plasmids encoding the four RNP protein components and the CAT RNA. The cell cultures were collected and tested for CAT activity as indicated in Fig. 1, and the CAT activity values obtained were expressed as a percentage of the activity obtained with the sample “−.” The lower panels show a Western blotting analysis of the corresponding cell extracts probed with antibodies that recognized the NP (α-NP) or NEP (α-NEP) of the virus strain A/Victoria/3/75. In section FLU B, COS- 1 cell extracts were processed as indicated above for the FLU A cultures, except that the cells expressed a type B influenza virus-like CAT RNA and the RNP components of virus strain B/Panamá/45/90. The plasmids used to produce the type B RNP components were pribo-NSBCAT (0.5 μg), pGB-NP-7 (2 μg), pGB-PB1–89.1 (0.6 μg), pGB-PB2–2 (0.6 μg), and pGB-PA-4 (0.1 μg) (described in reference 16). The lower panels show a Western blotting analysis of the corresponding cell extracts probed with antibodies that recognized influenza B virus NP (α-NP) or A/Victoria/3/75 NEP (α-NEP).
FIG. 4
FIG. 4
Accumulation of virus-specific RNA products in cells expressing the recombinant influenza A virus polymerase. COS-1 cells were infected with vTF7–3 and transfected with different DNA mixtures as described in the legend to Fig. 1. The DNA mixtures contained plasmids encoding the influenza A virus NP, PB1, PB2, and PA proteins (samples 2, 3, and 4), or the mixture used lacked the plasmid encoding PA (sample 1). In addition, the DNA mixtures transfected in samples 2 and 3 contained the plasmids pGEM-NS2Δ and pGEM-NS2ΔFAS, respectively. All transfection mixtures also contained a plasmid analogous to plasmid pCATCA18 that directed the synthesis of either a negative-sense (pT7vNSΔCAT-RT, 313 nt) (A) or a positive-sense (pT7cNSΔCAT-RT, 306 nt) (B) model RNA template. At 24 h postinfection with vTF7–3, total RNA was obtained following cell lysis with TRIZOL reagent (Gibco-BRL) and fractionated into poly(A)+ and poly(A) RNAs by oligo(dT)cellulose chromatography. Aliquots of the poly(A)+ fractions were then analyzed for the presence of mRNAs derived from the synthetic model RNA by the RNase protection assay (with the RPA III kit from Ambion) with a negative-sense labeled probe (mRNA panels). The poly(A) fractions were self-annealed and treated with RNase A to select for vRNA-cRNA hybrids (see reference 27) and were then analyzed for the presence of cRNA (A [cRNA]) or vRNA (B [vRNA]) by the RNase A protection assay by using 32P-labeled probes with predetermined polarity (28). The protected, labeled fragments were visualized after electrophoresis in a 4% sequencing gel and autoradiography. In all panels, the mobility of the slowest-migrating band was that expected for the different RNA products, as determined by comparison to DNA markers included in the gel (not shown).

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