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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

NF90 depletion reduces production of infectious dengue virus.Supernatants from WT and NF90-depleted cells infected with dengue virus (multiplicity of infection: 0.5) were used to infect DC-SIGN-expressing K562 cells (12, 46). At 14 hours post-infection, cells were harvested and analyzed for viral infection by flow cytometry using antibodies against dengue virus prM. Panels A-D are representative dot plots from the assay of a single set of supernatants from WT (MDA-MB-435-GFP) and KD (shDRBP76-GFP cells), FSC: forward scattering. (E) Bar graph summarizing the results of three independent experiments. Asterisks represent statistical significance at P<0.02.
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pone-0016687-g007: NF90 depletion reduces production of infectious dengue virus.Supernatants from WT and NF90-depleted cells infected with dengue virus (multiplicity of infection: 0.5) were used to infect DC-SIGN-expressing K562 cells (12, 46). At 14 hours post-infection, cells were harvested and analyzed for viral infection by flow cytometry using antibodies against dengue virus prM. Panels A-D are representative dot plots from the assay of a single set of supernatants from WT (MDA-MB-435-GFP) and KD (shDRBP76-GFP cells), FSC: forward scattering. (E) Bar graph summarizing the results of three independent experiments. Asterisks represent statistical significance at P<0.02.

Mentions: To extend the analysis, we used a flow cytometry-based assay to quantify infectious dengue particles released by MDA-MB-435-GFP and shDRBP76-GFP cells at 24, 36, and 48 hours after infection with dengue virus. Supernatants from infected cells were used to infect K562 cells that express the lectin DC-SIGN, which is important for dengue virus adherence and entry [27]. After a 14-hour incubation period, flow cytometry was used to score the number of infected K562 cells, identified by positive staining for dengue prM protein [28] (Figure 7). Figure 7A serves as a representative negative control and demonstrates the sensitivity limit. The amounts of virus in the supernatants were not significantly different in MDA-MB-435-GFP vs. shDRBP76-GFP cells at 24 hours post infection in three independent experiments, when only about 5–7% of cells were infected (Figures 7B and 7E). The quantified differences between infectious particles produced by dengue-infected control and NF90 knockdown cells was statistically significant at the 36 and 48-hour time points post-infection, with the NF90-depleted cells lagging the control cells by approximately 39% and 70%, respectively. Taken together, the data presented in Figures 6 and 7 suggest that NF90 has a positive functional role in the life cycle of dengue virus.


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)

NF90 depletion reduces production of infectious dengue virus.Supernatants from WT and NF90-depleted cells infected with dengue virus (multiplicity of infection: 0.5) were used to infect DC-SIGN-expressing K562 cells (12, 46). At 14 hours post-infection, cells were harvested and analyzed for viral infection by flow cytometry using antibodies against dengue virus prM. Panels A-D are representative dot plots from the assay of a single set of supernatants from WT (MDA-MB-435-GFP) and KD (shDRBP76-GFP cells), FSC: forward scattering. (E) Bar graph summarizing the results of three independent experiments. Asterisks represent statistical significance at P<0.02.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046124&req=5

pone-0016687-g007: NF90 depletion reduces production of infectious dengue virus.Supernatants from WT and NF90-depleted cells infected with dengue virus (multiplicity of infection: 0.5) were used to infect DC-SIGN-expressing K562 cells (12, 46). At 14 hours post-infection, cells were harvested and analyzed for viral infection by flow cytometry using antibodies against dengue virus prM. Panels A-D are representative dot plots from the assay of a single set of supernatants from WT (MDA-MB-435-GFP) and KD (shDRBP76-GFP cells), FSC: forward scattering. (E) Bar graph summarizing the results of three independent experiments. Asterisks represent statistical significance at P<0.02.
Mentions: To extend the analysis, we used a flow cytometry-based assay to quantify infectious dengue particles released by MDA-MB-435-GFP and shDRBP76-GFP cells at 24, 36, and 48 hours after infection with dengue virus. Supernatants from infected cells were used to infect K562 cells that express the lectin DC-SIGN, which is important for dengue virus adherence and entry [27]. After a 14-hour incubation period, flow cytometry was used to score the number of infected K562 cells, identified by positive staining for dengue prM protein [28] (Figure 7). Figure 7A serves as a representative negative control and demonstrates the sensitivity limit. The amounts of virus in the supernatants were not significantly different in MDA-MB-435-GFP vs. shDRBP76-GFP cells at 24 hours post infection in three independent experiments, when only about 5–7% of cells were infected (Figures 7B and 7E). The quantified differences between infectious particles produced by dengue-infected control and NF90 knockdown cells was statistically significant at the 36 and 48-hour time points post-infection, with the NF90-depleted cells lagging the control cells by approximately 39% and 70%, respectively. Taken together, the data presented in Figures 6 and 7 suggest that NF90 has a positive functional role in the life cycle of dengue virus.

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