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West Nile virus encodes a microRNA-like small RNA in the 3' untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells.

Hussain M, Torres S, Schnettler E, Funk A, Grundhoff A, Pijlman GP, Khromykh AA, Asgari S - Nucleic Acids Res. (2011)

Bottom Line: Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels.Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced.KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.

ABSTRACT
West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3' untranslated region (3'-UTR) of the flavivirus genome, in particular the terminal 3' stem-loop (3'SL) fulfils multiple functions in virus replication and virus-host interactions. Using the Kunjin strain of WNV (WNV(KUN)), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3'SL. Transcription of WNV(KUN) pre-miRNA (3'SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNV(KUN) virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication.

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KUN-miR-1 mimic increases virus RNA replication of IRAΔCS3 mutant WNVKUN virus defective in generation of sfRNA. RT–qPCR results of RNA samples from Aag2 cells infected for 72 h with IRAΔCS3 mutant WNVKUN virus, which were previously mock-transfected or transfected with KUN-miR-1 mimic, control mimic or mut1 mimic for 48 h. To quantify viral gRNA, specific primers to the virus capsid region were used. Data were first normalized against RPS17 gene transcript levels and then a fold change in gRNA was calculated against mock-infected values. Error bars indicate standard deviations of averages from two biological and three technical replicates.
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gkr848-F7: KUN-miR-1 mimic increases virus RNA replication of IRAΔCS3 mutant WNVKUN virus defective in generation of sfRNA. RT–qPCR results of RNA samples from Aag2 cells infected for 72 h with IRAΔCS3 mutant WNVKUN virus, which were previously mock-transfected or transfected with KUN-miR-1 mimic, control mimic or mut1 mimic for 48 h. To quantify viral gRNA, specific primers to the virus capsid region were used. Data were first normalized against RPS17 gene transcript levels and then a fold change in gRNA was calculated against mock-infected values. Error bars indicate standard deviations of averages from two biological and three technical replicates.

Mentions: To further confirm the role for KUN-miR-1 and induction of GATA4 in viral replication, we transfected Aag2 cells with KUN-miR-1 mimic, which was shown to induce expression of GATA4 (Figure 6D), and then infected the cells with inefficiently replicating IRAΔCS3 mutant WNVKUN virus largely defective in KUN-miR-1 production (Figure 1C). RT–qPCR results demonstrated that virus genomic RNA replication was increased by ~3.5 fold compared with cells either mock-transfected or transfected with a control mimic (Figure 7; P < 0.001). There was also a slight increase in viral genomic RNA replication in mut1 mimic RNA-transfected cells (~1.7-fold), but the difference was not statistically significant (P > 0.05). The results show that KUN-miR-1 mimic, which induces GATA4, is able to enhance replication of the mutant virus that is largely deficient in miRNA production.Figure 7.


West Nile virus encodes a microRNA-like small RNA in the 3' untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells.

Hussain M, Torres S, Schnettler E, Funk A, Grundhoff A, Pijlman GP, Khromykh AA, Asgari S - Nucleic Acids Res. (2011)

KUN-miR-1 mimic increases virus RNA replication of IRAΔCS3 mutant WNVKUN virus defective in generation of sfRNA. RT–qPCR results of RNA samples from Aag2 cells infected for 72 h with IRAΔCS3 mutant WNVKUN virus, which were previously mock-transfected or transfected with KUN-miR-1 mimic, control mimic or mut1 mimic for 48 h. To quantify viral gRNA, specific primers to the virus capsid region were used. Data were first normalized against RPS17 gene transcript levels and then a fold change in gRNA was calculated against mock-infected values. Error bars indicate standard deviations of averages from two biological and three technical replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300009&req=5

gkr848-F7: KUN-miR-1 mimic increases virus RNA replication of IRAΔCS3 mutant WNVKUN virus defective in generation of sfRNA. RT–qPCR results of RNA samples from Aag2 cells infected for 72 h with IRAΔCS3 mutant WNVKUN virus, which were previously mock-transfected or transfected with KUN-miR-1 mimic, control mimic or mut1 mimic for 48 h. To quantify viral gRNA, specific primers to the virus capsid region were used. Data were first normalized against RPS17 gene transcript levels and then a fold change in gRNA was calculated against mock-infected values. Error bars indicate standard deviations of averages from two biological and three technical replicates.
Mentions: To further confirm the role for KUN-miR-1 and induction of GATA4 in viral replication, we transfected Aag2 cells with KUN-miR-1 mimic, which was shown to induce expression of GATA4 (Figure 6D), and then infected the cells with inefficiently replicating IRAΔCS3 mutant WNVKUN virus largely defective in KUN-miR-1 production (Figure 1C). RT–qPCR results demonstrated that virus genomic RNA replication was increased by ~3.5 fold compared with cells either mock-transfected or transfected with a control mimic (Figure 7; P < 0.001). There was also a slight increase in viral genomic RNA replication in mut1 mimic RNA-transfected cells (~1.7-fold), but the difference was not statistically significant (P > 0.05). The results show that KUN-miR-1 mimic, which induces GATA4, is able to enhance replication of the mutant virus that is largely deficient in miRNA production.Figure 7.

Bottom Line: Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels.Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced.KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.

ABSTRACT
West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3' untranslated region (3'-UTR) of the flavivirus genome, in particular the terminal 3' stem-loop (3'SL) fulfils multiple functions in virus replication and virus-host interactions. Using the Kunjin strain of WNV (WNV(KUN)), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3'SL. Transcription of WNV(KUN) pre-miRNA (3'SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNV(KUN) virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication.

Show MeSH
Related in: MedlinePlus