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NF90 binds the dengue virus RNA 3' terminus and is a positive regulator of dengue virus replication.

Gomila RC, Martin GW, Gehrke L - PLoS ONE (2011)

Bottom Line: Viral RNA translation and replication are regulated by sequence and structural elements in the 5' and 3' untranslated regions (UTR) and by host cell and/or viral proteins that bind them.NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny.NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.

View Article: PubMed Central - PubMed

Affiliation: Division of Health Sciences and Technology and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Viral RNA translation and replication are regulated by sequence and structural elements in the 5' and 3' untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5' m7GpppG cap, and a conserved 3'-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3' terminus.

Methodology/principal findings: Proteins eluted from a dengue 3' SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny.

Conclusions/significance: The results indicate that NF90 interacts with the 3' SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.

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Related in: MedlinePlus

Binding specificity assessed by competition.(A) Electrophoretic mobility shift assay of a competitive RNA binding analysis. All reactions contained the same amount of radiolabeled dengue 3′ SL RNA and included the same volume of 500 mM affinity column chromatography eluate. Lanes 1 and 5 represent dengue 3′SL RNA plus protein extract, showing RNP2 without added competitor RNA. Lanes 2–4 represent competitor dengue 3′SL RNA added at 5, 15, and 30-fold molar excesses. Lanes 6–8 represent addition of competitor AMV 3′UTR RNA at a molar excesses of 5, 15 and 30 fold. (B) Quantification of labeled RNA in the bound fraction (RNP 2) from panel A normalized relative to the total amount of RNP2 in the absence of competitor (lanes 1 and 5, respectively). Upper trace: AMV 3′UTR RNA competitor; lower trace: dengue 3′ SL RNA competitor.
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pone-0016687-g002: Binding specificity assessed by competition.(A) Electrophoretic mobility shift assay of a competitive RNA binding analysis. All reactions contained the same amount of radiolabeled dengue 3′ SL RNA and included the same volume of 500 mM affinity column chromatography eluate. Lanes 1 and 5 represent dengue 3′SL RNA plus protein extract, showing RNP2 without added competitor RNA. Lanes 2–4 represent competitor dengue 3′SL RNA added at 5, 15, and 30-fold molar excesses. Lanes 6–8 represent addition of competitor AMV 3′UTR RNA at a molar excesses of 5, 15 and 30 fold. (B) Quantification of labeled RNA in the bound fraction (RNP 2) from panel A normalized relative to the total amount of RNP2 in the absence of competitor (lanes 1 and 5, respectively). Upper trace: AMV 3′UTR RNA competitor; lower trace: dengue 3′ SL RNA competitor.

Mentions: To extend the analysis, competitive binding assays were conducted to assess binding specificity. The dengue 3′ SL has significant secondary structure [4], [5], therefore the structured alfalfa mosaic virus (AMV) 3′ untranslated region (UTR) RNA [20] was used for comparative competition experiments. Radiolabeled dengue 3′SL RNA was added to the 500 mM affinity column eluates along with competitor RNA, represented by either unlabeled cognate dengue 3′SL RNA, or AMV 3′UTR RNA. The RNP2 band was detected in the absence of competitor RNA (Figure 2A, lanes 1 and 5). In the presence of a thirty-fold molar excess of dengue 3′SL competitor RNA, the amount of labeled RNA in the RNP2 complex was reduced by more than 80% (Figure 2A, lane 4; Figure 2B, lower trace), demonstrating effective competition. By comparison, a thirty-fold molar excess of the AMV 3′UTR was much less effective, reducing the amount of shifted RNP2 by only about 30% (Figure 2A, lane 8; Figure 2B, upper trace). The four-fold binding differential is evidence that proteins in the 500 mM eluate form specific complexes with the dengue 3′ SL RNA. Taken together, the data presented in Figures 1 and 2 demonstrate that dengue 3′ SL RNA interacting proteins were enriched by RNA affinity chromatography.


NF90 binds the dengue virus RNA 3' terminus and is a positive regulator of dengue virus replication.

Gomila RC, Martin GW, Gehrke L - PLoS ONE (2011)

Binding specificity assessed by competition.(A) Electrophoretic mobility shift assay of a competitive RNA binding analysis. All reactions contained the same amount of radiolabeled dengue 3′ SL RNA and included the same volume of 500 mM affinity column chromatography eluate. Lanes 1 and 5 represent dengue 3′SL RNA plus protein extract, showing RNP2 without added competitor RNA. Lanes 2–4 represent competitor dengue 3′SL RNA added at 5, 15, and 30-fold molar excesses. Lanes 6–8 represent addition of competitor AMV 3′UTR RNA at a molar excesses of 5, 15 and 30 fold. (B) Quantification of labeled RNA in the bound fraction (RNP 2) from panel A normalized relative to the total amount of RNP2 in the absence of competitor (lanes 1 and 5, respectively). Upper trace: AMV 3′UTR RNA competitor; lower trace: dengue 3′ SL RNA competitor.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046124&req=5

pone-0016687-g002: Binding specificity assessed by competition.(A) Electrophoretic mobility shift assay of a competitive RNA binding analysis. All reactions contained the same amount of radiolabeled dengue 3′ SL RNA and included the same volume of 500 mM affinity column chromatography eluate. Lanes 1 and 5 represent dengue 3′SL RNA plus protein extract, showing RNP2 without added competitor RNA. Lanes 2–4 represent competitor dengue 3′SL RNA added at 5, 15, and 30-fold molar excesses. Lanes 6–8 represent addition of competitor AMV 3′UTR RNA at a molar excesses of 5, 15 and 30 fold. (B) Quantification of labeled RNA in the bound fraction (RNP 2) from panel A normalized relative to the total amount of RNP2 in the absence of competitor (lanes 1 and 5, respectively). Upper trace: AMV 3′UTR RNA competitor; lower trace: dengue 3′ SL RNA competitor.
Mentions: To extend the analysis, competitive binding assays were conducted to assess binding specificity. The dengue 3′ SL has significant secondary structure [4], [5], therefore the structured alfalfa mosaic virus (AMV) 3′ untranslated region (UTR) RNA [20] was used for comparative competition experiments. Radiolabeled dengue 3′SL RNA was added to the 500 mM affinity column eluates along with competitor RNA, represented by either unlabeled cognate dengue 3′SL RNA, or AMV 3′UTR RNA. The RNP2 band was detected in the absence of competitor RNA (Figure 2A, lanes 1 and 5). In the presence of a thirty-fold molar excess of dengue 3′SL competitor RNA, the amount of labeled RNA in the RNP2 complex was reduced by more than 80% (Figure 2A, lane 4; Figure 2B, lower trace), demonstrating effective competition. By comparison, a thirty-fold molar excess of the AMV 3′UTR was much less effective, reducing the amount of shifted RNP2 by only about 30% (Figure 2A, lane 8; Figure 2B, upper trace). The four-fold binding differential is evidence that proteins in the 500 mM eluate form specific complexes with the dengue 3′ SL RNA. Taken together, the data presented in Figures 1 and 2 demonstrate that dengue 3′ SL RNA interacting proteins were enriched by RNA affinity chromatography.

Bottom Line: Viral RNA translation and replication are regulated by sequence and structural elements in the 5' and 3' untranslated regions (UTR) and by host cell and/or viral proteins that bind them.NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny.NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.

View Article: PubMed Central - PubMed

Affiliation: Division of Health Sciences and Technology and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Viral RNA translation and replication are regulated by sequence and structural elements in the 5' and 3' untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5' m7GpppG cap, and a conserved 3'-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3' terminus.

Methodology/principal findings: Proteins eluted from a dengue 3' SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny.

Conclusions/significance: The results indicate that NF90 interacts with the 3' SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.

Show MeSH
Related in: MedlinePlus