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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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Validation of KUN-miR-1 functionality by Firefly luciferase-based sensor constructs. (A) Schematic representation of the WNV 3′SL. A1A2 and C1C2 sequences for tandem repeat cloning into miRNA sensor constructs are indicated in black and grey highlights, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in bold. The arrows indicate KUN-miR-1. (B) Schematic representation of miRNA sensor constructs for expression in insect (pMT-Fluc) cells. A1A2 and C1C2 tandem repeats and their reverse complements (rc) are indicated in black and grey highlights, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in bold. Fluc, Firely luciferase; MT, metallothionein promoter; pA, polyadenylation signal; Xb, XbaI; Xh, XhoI restriction sites. (C) Silencing of miRNA sensor constructs by sfRNA expression in Drosophila S2 cells. Cells were co-transfected with pMT-Renilla, pMT-Fluc-(sensor constructs) and either pIB-sfRNA or pIB-MBP (negative control). After induction, the relative luciferase expression (Firefly/Renilla) was determined 24 h post-induction (hpi) and the mean of two independent experiments in duplicate is shown with standard error.
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gkr848-F4: Validation of KUN-miR-1 functionality by Firefly luciferase-based sensor constructs. (A) Schematic representation of the WNV 3′SL. A1A2 and C1C2 sequences for tandem repeat cloning into miRNA sensor constructs are indicated in black and grey highlights, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in bold. The arrows indicate KUN-miR-1. (B) Schematic representation of miRNA sensor constructs for expression in insect (pMT-Fluc) cells. A1A2 and C1C2 tandem repeats and their reverse complements (rc) are indicated in black and grey highlights, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in bold. Fluc, Firely luciferase; MT, metallothionein promoter; pA, polyadenylation signal; Xb, XbaI; Xh, XhoI restriction sites. (C) Silencing of miRNA sensor constructs by sfRNA expression in Drosophila S2 cells. Cells were co-transfected with pMT-Renilla, pMT-Fluc-(sensor constructs) and either pIB-sfRNA or pIB-MBP (negative control). After induction, the relative luciferase expression (Firefly/Renilla) was determined 24 h post-induction (hpi) and the mean of two independent experiments in duplicate is shown with standard error.

Mentions: The miRNA sensor constructs contain repeats of (complementary) 5′ and 3′ regions (harbouring KUN-miR-1) of the WNV 3′SL, designated A1A2 and C1C2 (Figure 4A). The WNV miRNA sensor constructs were made by tandem repetition PCR using 40 nt long primers WNV-miRNA-A1A2, -B1B2, -C1C2 and -D1D2 (Table 1). In this PCR, the last 20 nt of primers WNV-miRNA-A1A2 or -C1C2 are complementary to the last 20 nt of the WNV-miRNA-B1B2 or -D1D2 primers, respectively. A PCR yields a ladder of PCR products consisting of A1A2 (or C1C2) repeat elements. PCR products of ~500 nt (~12 repeats) were isolated, cloned into pJET1.2 plasmid (Fermentas) and sequenced (Eurofins Operon, Germany). A short phosphorylated XbaI linker (NotI-XbaI linker-Phos, Table 1) was inserted into the NotI site of these plasmids and the A1A2 and C1C2 repeat regions were cloned in both orientations as XbaI fragments downstream of Fluc genes in the insect expression vector pMT-Fluc (39). The first set of sensor constructs contains 12 repeats of the A1A2 sequence, and the other set contains 13 repeats of the C1C2 sequence (Figure 4B).Table 1.


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)

Validation of KUN-miR-1 functionality by Firefly luciferase-based sensor constructs. (A) Schematic representation of the WNV 3′SL. A1A2 and C1C2 sequences for tandem repeat cloning into miRNA sensor constructs are indicated in black and grey highlights, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in bold. The arrows indicate KUN-miR-1. (B) Schematic representation of miRNA sensor constructs for expression in insect (pMT-Fluc) cells. A1A2 and C1C2 tandem repeats and their reverse complements (rc) are indicated in black and grey highlights, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in bold. Fluc, Firely luciferase; MT, metallothionein promoter; pA, polyadenylation signal; Xb, XbaI; Xh, XhoI restriction sites. (C) Silencing of miRNA sensor constructs by sfRNA expression in Drosophila S2 cells. Cells were co-transfected with pMT-Renilla, pMT-Fluc-(sensor constructs) and either pIB-sfRNA or pIB-MBP (negative control). After induction, the relative luciferase expression (Firefly/Renilla) was determined 24 h post-induction (hpi) and the mean of two independent experiments in duplicate is shown with standard error.
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Related In: Results  -  Collection

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gkr848-F4: Validation of KUN-miR-1 functionality by Firefly luciferase-based sensor constructs. (A) Schematic representation of the WNV 3′SL. A1A2 and C1C2 sequences for tandem repeat cloning into miRNA sensor constructs are indicated in black and grey highlights, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in bold. The arrows indicate KUN-miR-1. (B) Schematic representation of miRNA sensor constructs for expression in insect (pMT-Fluc) cells. A1A2 and C1C2 tandem repeats and their reverse complements (rc) are indicated in black and grey highlights, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in bold. Fluc, Firely luciferase; MT, metallothionein promoter; pA, polyadenylation signal; Xb, XbaI; Xh, XhoI restriction sites. (C) Silencing of miRNA sensor constructs by sfRNA expression in Drosophila S2 cells. Cells were co-transfected with pMT-Renilla, pMT-Fluc-(sensor constructs) and either pIB-sfRNA or pIB-MBP (negative control). After induction, the relative luciferase expression (Firefly/Renilla) was determined 24 h post-induction (hpi) and the mean of two independent experiments in duplicate is shown with standard error.
Mentions: The miRNA sensor constructs contain repeats of (complementary) 5′ and 3′ regions (harbouring KUN-miR-1) of the WNV 3′SL, designated A1A2 and C1C2 (Figure 4A). The WNV miRNA sensor constructs were made by tandem repetition PCR using 40 nt long primers WNV-miRNA-A1A2, -B1B2, -C1C2 and -D1D2 (Table 1). In this PCR, the last 20 nt of primers WNV-miRNA-A1A2 or -C1C2 are complementary to the last 20 nt of the WNV-miRNA-B1B2 or -D1D2 primers, respectively. A PCR yields a ladder of PCR products consisting of A1A2 (or C1C2) repeat elements. PCR products of ~500 nt (~12 repeats) were isolated, cloned into pJET1.2 plasmid (Fermentas) and sequenced (Eurofins Operon, Germany). A short phosphorylated XbaI linker (NotI-XbaI linker-Phos, Table 1) was inserted into the NotI site of these plasmids and the A1A2 and C1C2 repeat regions were cloned in both orientations as XbaI fragments downstream of Fluc genes in the insect expression vector pMT-Fluc (39). The first set of sensor constructs contains 12 repeats of the A1A2 sequence, and the other set contains 13 repeats of the C1C2 sequence (Figure 4B).Table 1.

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