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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

RNA binding activities eluted from the dengue 3′SL RNA columns.S10 extract from K562 cells was prepared and chromatographed as described in the Materials and Methods section. In both panels, lane 1 represents the dengue 3′SL RNA only. The second lane shows the binding properties of proteins that washed through the column in the flow through (FT) fraction. The five column washes are analyzed in lanes 3–7. Bound proteins were step-eluted with 250 mM, 500 mM, 1 M and 2 M NaCl (lanes 8–11 respectively). (A) The S10 extract was pre-cleared by passing it over the control (lacking bound RNA) column. (B) The pre-cleared extract was chromatographed on the dengue 3′SL affinity column. The EMSA using the fractions eluted from the RNA affinity column shows three binding activities eluted from the column, RNP1, RNP2, and RNP3 (lanes 9–11).
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pone-0016687-g001: RNA binding activities eluted from the dengue 3′SL RNA columns.S10 extract from K562 cells was prepared and chromatographed as described in the Materials and Methods section. In both panels, lane 1 represents the dengue 3′SL RNA only. The second lane shows the binding properties of proteins that washed through the column in the flow through (FT) fraction. The five column washes are analyzed in lanes 3–7. Bound proteins were step-eluted with 250 mM, 500 mM, 1 M and 2 M NaCl (lanes 8–11 respectively). (A) The S10 extract was pre-cleared by passing it over the control (lacking bound RNA) column. (B) The pre-cleared extract was chromatographed on the dengue 3′SL affinity column. The EMSA using the fractions eluted from the RNA affinity column shows three binding activities eluted from the column, RNP1, RNP2, and RNP3 (lanes 9–11).

Mentions: We used RNA affinity column chromatography [18], [19] to identify proteins in complex with the dengue 3′SL RNA (Figure 1). After passing pre-cleared cell extracts over the RNA-coupled affinity column, a stepwise elution was performed using buffers containing increasing NaCl concentrations to distinguish low affinity from higher affinity interactors. RNA binding activity in the fractions was assessed by electrophoretic mobility shift assay (EMSA). For the control column (matrix only), RNA-protein complexes were detected only in the flow-through or initial wash fractions (Figure 1A, lanes 2 and 3). The absence of any significant shifted bands in further washes or NaCl elution fractions (Figure 1A, lanes 3–11) indicates that the matrix has minimal non-specific binding activity.


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)

RNA binding activities eluted from the dengue 3′SL RNA columns.S10 extract from K562 cells was prepared and chromatographed as described in the Materials and Methods section. In both panels, lane 1 represents the dengue 3′SL RNA only. The second lane shows the binding properties of proteins that washed through the column in the flow through (FT) fraction. The five column washes are analyzed in lanes 3–7. Bound proteins were step-eluted with 250 mM, 500 mM, 1 M and 2 M NaCl (lanes 8–11 respectively). (A) The S10 extract was pre-cleared by passing it over the control (lacking bound RNA) column. (B) The pre-cleared extract was chromatographed on the dengue 3′SL affinity column. The EMSA using the fractions eluted from the RNA affinity column shows three binding activities eluted from the column, RNP1, RNP2, and RNP3 (lanes 9–11).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016687-g001: RNA binding activities eluted from the dengue 3′SL RNA columns.S10 extract from K562 cells was prepared and chromatographed as described in the Materials and Methods section. In both panels, lane 1 represents the dengue 3′SL RNA only. The second lane shows the binding properties of proteins that washed through the column in the flow through (FT) fraction. The five column washes are analyzed in lanes 3–7. Bound proteins were step-eluted with 250 mM, 500 mM, 1 M and 2 M NaCl (lanes 8–11 respectively). (A) The S10 extract was pre-cleared by passing it over the control (lacking bound RNA) column. (B) The pre-cleared extract was chromatographed on the dengue 3′SL affinity column. The EMSA using the fractions eluted from the RNA affinity column shows three binding activities eluted from the column, RNP1, RNP2, and RNP3 (lanes 9–11).
Mentions: We used RNA affinity column chromatography [18], [19] to identify proteins in complex with the dengue 3′SL RNA (Figure 1). After passing pre-cleared cell extracts over the RNA-coupled affinity column, a stepwise elution was performed using buffers containing increasing NaCl concentrations to distinguish low affinity from higher affinity interactors. RNA binding activity in the fractions was assessed by electrophoretic mobility shift assay (EMSA). For the control column (matrix only), RNA-protein complexes were detected only in the flow-through or initial wash fractions (Figure 1A, lanes 2 and 3). The absence of any significant shifted bands in further washes or NaCl elution fractions (Figure 1A, lanes 3–11) indicates that the matrix has minimal non-specific binding activity.

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