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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

Protein composition and RNA binding activity of eluted fractions.Proteins in the column fractions were analyzed by SDS-PAGE followed by silver stain. (A) Lanes 1, 3 and 5 represent eluted fractions from the matrix-only column (-), while lanes 2, 4 and 6 represent fractions eluted from the RNA affinity column (+). Two stained bands were differentially present in the 500 mM sample eluted from the RNA column (lane 4, asterisks), and excised for MALDI-TOF MS analysis. (B) The 500 mM and 1 M column eluates were analyzed by northwestern blotting, using the dengue 3′ SL RNA as a probe. Left lane: 500 mM eluate; right lane: 1 M eluate. (C) Northwestern blot comparison of the 500 mM eluates from the control (-) and RNA-coupled (+) affinity columns. Left lane: 500 mM fraction from the control Sepharose column. Right lane: 500 mM fraction from the dengue 3′SL RNA column.
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pone-0016687-g003: Protein composition and RNA binding activity of eluted fractions.Proteins in the column fractions were analyzed by SDS-PAGE followed by silver stain. (A) Lanes 1, 3 and 5 represent eluted fractions from the matrix-only column (-), while lanes 2, 4 and 6 represent fractions eluted from the RNA affinity column (+). Two stained bands were differentially present in the 500 mM sample eluted from the RNA column (lane 4, asterisks), and excised for MALDI-TOF MS analysis. (B) The 500 mM and 1 M column eluates were analyzed by northwestern blotting, using the dengue 3′ SL RNA as a probe. Left lane: 500 mM eluate; right lane: 1 M eluate. (C) Northwestern blot comparison of the 500 mM eluates from the control (-) and RNA-coupled (+) affinity columns. Left lane: 500 mM fraction from the control Sepharose column. Right lane: 500 mM fraction from the dengue 3′SL RNA column.

Mentions: SDS-PAGE and silver staining, coupled with northwestern blotting, were used to characterize proteins eluted in the 500 mM RNA affinity chromatography eluate. By comparing the stain patterns from the control (no RNA) and dengue 3′ SL RNA columns, we observed that two distinct protein bands with approximate molecular weights of 140 kDa and 90 kDa were enriched in the dengue 3′SL RNA affinity column eluate (Figure 3A, compare lanes 3 and 4; asterisks). To determine if the stained 140 kDa and 90 kDa bands correlated with direct dengue 3′ SL RNA binding potential, a northwestern blot assay, using a radiolabeled dengue 3′ SL RNA probe, was performed. The data (Figure 3B) demonstrate a prominent signal in the 90 kDa region of the gel, along with lower intensity signals at 140 kDa and 50 kDa. Relatively weak signal was observed in the 50 KDa region of the 1M eluate separation (Figure 3B, right lane). As a specificity control, we compared 500 mM NaCl eluates from the control column (lacking bound RNA) and the dengue 3′ SL affinity column in the northwestern blot assay. The results demonstrate that the probe bound to the 140 kDa and 90 kDa bands in the RNA affinity column eluate (Figure 3C, right lane 2); however, no signal was present in the control column eluate (Figure 3C, left lane). The 140 kDa and 90 kDa bands seen in lane 1 of the northwestern analysis (Figure 3B) correlate with two bands of similar molecular weight observed in the silver stained gel (Figure 3A, lane 4), suggesting that proteins of these molecular weights bind the dengue 3′ SL RNA directly. These data are evidence that the 140 kDa and 90 kDa proteins bind directly to the dengue virus 3′ SL RNA.


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)

Protein composition and RNA binding activity of eluted fractions.Proteins in the column fractions were analyzed by SDS-PAGE followed by silver stain. (A) Lanes 1, 3 and 5 represent eluted fractions from the matrix-only column (-), while lanes 2, 4 and 6 represent fractions eluted from the RNA affinity column (+). Two stained bands were differentially present in the 500 mM sample eluted from the RNA column (lane 4, asterisks), and excised for MALDI-TOF MS analysis. (B) The 500 mM and 1 M column eluates were analyzed by northwestern blotting, using the dengue 3′ SL RNA as a probe. Left lane: 500 mM eluate; right lane: 1 M eluate. (C) Northwestern blot comparison of the 500 mM eluates from the control (-) and RNA-coupled (+) affinity columns. Left lane: 500 mM fraction from the control Sepharose column. Right lane: 500 mM fraction from the dengue 3′SL RNA column.
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Related In: Results  -  Collection

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

pone-0016687-g003: Protein composition and RNA binding activity of eluted fractions.Proteins in the column fractions were analyzed by SDS-PAGE followed by silver stain. (A) Lanes 1, 3 and 5 represent eluted fractions from the matrix-only column (-), while lanes 2, 4 and 6 represent fractions eluted from the RNA affinity column (+). Two stained bands were differentially present in the 500 mM sample eluted from the RNA column (lane 4, asterisks), and excised for MALDI-TOF MS analysis. (B) The 500 mM and 1 M column eluates were analyzed by northwestern blotting, using the dengue 3′ SL RNA as a probe. Left lane: 500 mM eluate; right lane: 1 M eluate. (C) Northwestern blot comparison of the 500 mM eluates from the control (-) and RNA-coupled (+) affinity columns. Left lane: 500 mM fraction from the control Sepharose column. Right lane: 500 mM fraction from the dengue 3′SL RNA column.
Mentions: SDS-PAGE and silver staining, coupled with northwestern blotting, were used to characterize proteins eluted in the 500 mM RNA affinity chromatography eluate. By comparing the stain patterns from the control (no RNA) and dengue 3′ SL RNA columns, we observed that two distinct protein bands with approximate molecular weights of 140 kDa and 90 kDa were enriched in the dengue 3′SL RNA affinity column eluate (Figure 3A, compare lanes 3 and 4; asterisks). To determine if the stained 140 kDa and 90 kDa bands correlated with direct dengue 3′ SL RNA binding potential, a northwestern blot assay, using a radiolabeled dengue 3′ SL RNA probe, was performed. The data (Figure 3B) demonstrate a prominent signal in the 90 kDa region of the gel, along with lower intensity signals at 140 kDa and 50 kDa. Relatively weak signal was observed in the 50 KDa region of the 1M eluate separation (Figure 3B, right lane). As a specificity control, we compared 500 mM NaCl eluates from the control column (lacking bound RNA) and the dengue 3′ SL affinity column in the northwestern blot assay. The results demonstrate that the probe bound to the 140 kDa and 90 kDa bands in the RNA affinity column eluate (Figure 3C, right lane 2); however, no signal was present in the control column eluate (Figure 3C, left lane). The 140 kDa and 90 kDa bands seen in lane 1 of the northwestern analysis (Figure 3B) correlate with two bands of similar molecular weight observed in the silver stained gel (Figure 3A, lane 4), suggesting that proteins of these molecular weights bind the dengue 3′ SL RNA directly. These data are evidence that the 140 kDa and 90 kDa proteins bind directly to the dengue virus 3′ SL RNA.

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