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. 2013 Jun;87(12):6582-8.
doi: 10.1128/JVI.02495-12. Epub 2013 Mar 20.

Downregulation of Nipah virus N mRNA occurs through interaction between its 3' untranslated region and hnRNP D

Kimihiro Hino  1 , Hiroki Sato, Akihiro Sugai, Masahiko Kato, Misako Yoneda, Chieko Kai
Affiliations

Downregulation of Nipah virus N mRNA occurs through interaction between its 3' untranslated region and hnRNP D

Kimihiro Hino et al. J Virol. 2013 Jun.

Abstract

Nipah virus (NiV) is a nonsegmented, single-stranded, negative-sense RNA virus belonging to the genus Henipavirus, family Paramyxoviridae. NiV causes acute encephalitis and respiratory disease in humans, is associated with high mortality, and poses a threat in southern Asia. The genomes of henipaviruses are about 18,246 nucleotides (nt) long, which is longer than those of other paramyxoviruses (around 15,384 nt). This difference is caused by the noncoding RNA region, particularly the 3' untranslated region (UTR), which occupies more than half of the noncoding RNA region. To determine the function(s) of the NiV noncoding RNA region, we investigated the effects of NiV 3' UTRs on reporter gene expression. The NiV N 3' UTR (nt 1 to 100) demonstrated strong repressor activity associated with hnRNP D protein binding to that region. Mutation of the hnRNP D binding site or knockdown of hnRNP D resulted in increased expression of the NiV N 3' UTR reporter. Our findings suggest that NiV N expression is repressed by hnRNP D through the NiV N 3' UTR and demonstrate the involvement of posttranscriptional regulation in the NiV life cycle. To the best of our knowledge, this provides the first report of the functions of the NiV noncoding RNA region.

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Figures

Fig 1
Fig 1
Effects of NiV 3′ UTRs on reporter gene expression. (A) UTR reporters were constructed by insertion of NiV 3′ UTRs downstream of the luciferase gene in pGL3control. Luciferase activities were measured at 24 h posttransfection and normalized to Renilla luciferase activities. The value of pGL3control was set to 100. The error bars indicate standard deviations. (B) Downregulation by the NiV N UTR resulted in a decrease in NiV N protein levels. Proteins were detected by Western blotting. (C) Reporter RNAs were detected by Northern blotting. For endogenous controls, 18S rRNA was stained with methylene blue.
Fig 2
Fig 2
Deletion analysis of the NiV N 3′ UTR. (Left) cis-Acting elements in the NiV N 3′ UTR were identified using luciferase reporters attached to NiV N 3′ UTR fragments. (Right) Luciferase activities were measured at 24 h posttransfection and normalized to Renilla luciferase activities. The value of the pGL3control was set to 100. The 7th and 14th graph bars from the top are identical. Also, the 1st and 7th graph bars from the bottom are identical. The error bars indicate standard deviations.
Fig 3
Fig 3
Identification of NiV N 3′ UTR binding proteins. (A) Binding proteins were identified using RNA pulldown assays with 3′-end biotin-labeled RNAs corresponding to the full-length NiV N 3′ UTR (586 nt), followed by silver staining. Candidate bands are indicated by arrowheads. (B) Both hnRNP A/B and D localized in the nucleus and cytoplasm. (C) Endogenous hnRNP A/B and D bound to the NiV N 3′ UTR. The bands indicated by arrowheads are hnRNP A/B or D.
Fig 4
Fig 4
Identification of the hnRNP binding site in the NiV N 3′ UTR. The hnRNP binding site was identified using RNA pulldown assays in the presence of competitors of different lengths. RNAs corresponding to the full-length NiV N 3′ UTR (586 nt) were labeled at the 3′ end with biotin and used as RNA probes. hnRNP protein was detected by Western blotting.
Fig 5
Fig 5
Mutations in the hnRNP binding site impaired downregulation by the NiV N 3′ UTR. (A) Wild-type (wt) (above) and mutated (mut) (below) sequences around the hnRNP binding site. Underlining indicates the region in which nucleotides are changed. (B) Mutations in the hnRNP binding site impaired the binding of hnRNP proteins to the RNA probe. pIpC was used as a nonspecific RNA to standardize the amount of total RNAs in the reaction complex. EMSA, electrophoretic mobility shift assay. (C) The 100-nt NiV N 3′ UTR region with or without mutations at nt 41 to 60 was transfected into HeLa cells. Luciferase activities were measured at 24 h posttransfection and normalized to Renilla luciferase activities. The value of pGL3control was set to 100. (D) RT-PCR was used to detect mRNAs that attached to the 100-nt wild-type or mutated NiV N UTR. 18S rRNAs were detected as endogenous controls. The half-lives were calculated from the intensities of the bands.
Fig 6
Fig 6
Expression of NiV N 3′ UTR reporters was increased by knockdown of hnRNP D. (A) siRNAs designed to knock down hnRNP A/B, hnRNP D, or EGFP (designated sihnRNP A/B, sihnRNP D, or siEGFP) were transfected into HeLa cells. The membranes were cut at the 50-kDa marker before addition of antibody. (B) Expression levels of NiV N 3′ UTR reporters when hnRNP A/B or D was repressed. Luciferase activities were measured and normalized to Renilla luciferase activities. The value of siEGFP-transfected cells was set to 100. The error bars indicate standard deviations. (C) Expression levels of six NiV UTR reporters when hnRNP D was repressed. The six NiV 3′ UTR reporters were transfected into hnRNP D KD cells. Luciferase activities were measured and normalized to Renilla luciferase activities. The value of siEGFP-transfected cells was set to 100.
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