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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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Ectopic expression of KUN-miR-1. (A) Diagram representing the RNA transcript of the ectopically expressed pre-KUN-miR-1 in pIZ vector. MCS, multi-cloning site. (B) Northern blot of RNA derived from C6/36 cells transfected with plasmid DNA pIZ/pre-KmiR-1 (48 h after transfection) encoding WNVKUN pre-miRNA using 3′ probe specific for KUN-miR-1. pIZ, empty vector. tRNA is shown as loading control. (C) Schematic diagram of Semliki Forest virus (SFV) replicon constructs expressing WNVKUN sfRNA and pre-KUN-miR-1. sfRNA and pre-KUN-miR-1 were inserted in the 3′NCR of the SFV replicon downstream of the GFP gene. SFV replicon RNA is first transcribed into the complementary (−) strand from which subgenomic RNA encoding GFP gene and 3′NCR is produced in large excess to the (+) strand genomic RNA. Both WNVKUN RNAs are produced primarily from this subgenomic RNA. (D) Northern blot hybridization with KUN-miR-1-specific probe of C6/36 cells infected with VRPs containing encapsidated GFP-expressing SFV replicon RNAs encoding pre-KUN-miR-1 RNA or sfRNA. RNA was isolated at 4 days post-infection. The faint upper band is the intermediate band also observed in mosquito virus-infected (Figure 1B) or plasmid-transfected cells (Figure 3B). M, mock-infected cells; GFP, SFV1-GFP VRP-infected cells; sfRNA, SFV1-GFP-sfRNA VRP-infected cells; pre-KUN-miR-1, SFV1-GFP-pre-KUN-miR1 VRP-infected cells.
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gkr848-F3: Ectopic expression of KUN-miR-1. (A) Diagram representing the RNA transcript of the ectopically expressed pre-KUN-miR-1 in pIZ vector. MCS, multi-cloning site. (B) Northern blot of RNA derived from C6/36 cells transfected with plasmid DNA pIZ/pre-KmiR-1 (48 h after transfection) encoding WNVKUN pre-miRNA using 3′ probe specific for KUN-miR-1. pIZ, empty vector. tRNA is shown as loading control. (C) Schematic diagram of Semliki Forest virus (SFV) replicon constructs expressing WNVKUN sfRNA and pre-KUN-miR-1. sfRNA and pre-KUN-miR-1 were inserted in the 3′NCR of the SFV replicon downstream of the GFP gene. SFV replicon RNA is first transcribed into the complementary (−) strand from which subgenomic RNA encoding GFP gene and 3′NCR is produced in large excess to the (+) strand genomic RNA. Both WNVKUN RNAs are produced primarily from this subgenomic RNA. (D) Northern blot hybridization with KUN-miR-1-specific probe of C6/36 cells infected with VRPs containing encapsidated GFP-expressing SFV replicon RNAs encoding pre-KUN-miR-1 RNA or sfRNA. RNA was isolated at 4 days post-infection. The faint upper band is the intermediate band also observed in mosquito virus-infected (Figure 1B) or plasmid-transfected cells (Figure 3B). M, mock-infected cells; GFP, SFV1-GFP VRP-infected cells; sfRNA, SFV1-GFP-sfRNA VRP-infected cells; pre-KUN-miR-1, SFV1-GFP-pre-KUN-miR1 VRP-infected cells.

Mentions: To determine whether KUN-miR-1 can be generated from the pre-miRNA in isolation from other viral or virus-induced host factors and is not a degradation product from the end of the viral genome, we cloned the pre-KUN-miR-1 sequence in the insect expression plasmid vector pIZ/V5-His, transfected the resulting pIZ/pre-KmiR-1 plasmid into C6/36 cells and analysed RNA from transfected cells by northern blot with the 3′ probe. It is worth mentioning that the transcript produced from this plasmid contained a 5′-UTR from the vector (53 nt) followed by 79 nt pre-KUN-miR-1 sequence, and followed by another 428 nt of vector sequences comprising parts of the multi-cloning site, V5 epitope, 6×His tag and 3′-UTR containing a polyA signal (Figure 3A); therefore, the pre-miRNA SL was not located at the very end of the transcript but flanked by vector sequences. A 21-nt RNA representing KUN-miR-1 was clearly detected (Figure 3B), thus confirming that KUN-miR-1 is indeed produced from the pre-miRNA sequence and is not an artefact of degradation from the end of the viral genome. An RNA band of 40 nt between mature miRNA and pre-miRNA, which was also detected in WNVKUN-infected mosquito cells (Figure 1C), was visible in pIZ/pre-KmiR-1-transfected cells. Intermediate fragments are frequently observed for other miRNAs [e.g. (25,45)].Figure 3.


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)

Ectopic expression of KUN-miR-1. (A) Diagram representing the RNA transcript of the ectopically expressed pre-KUN-miR-1 in pIZ vector. MCS, multi-cloning site. (B) Northern blot of RNA derived from C6/36 cells transfected with plasmid DNA pIZ/pre-KmiR-1 (48 h after transfection) encoding WNVKUN pre-miRNA using 3′ probe specific for KUN-miR-1. pIZ, empty vector. tRNA is shown as loading control. (C) Schematic diagram of Semliki Forest virus (SFV) replicon constructs expressing WNVKUN sfRNA and pre-KUN-miR-1. sfRNA and pre-KUN-miR-1 were inserted in the 3′NCR of the SFV replicon downstream of the GFP gene. SFV replicon RNA is first transcribed into the complementary (−) strand from which subgenomic RNA encoding GFP gene and 3′NCR is produced in large excess to the (+) strand genomic RNA. Both WNVKUN RNAs are produced primarily from this subgenomic RNA. (D) Northern blot hybridization with KUN-miR-1-specific probe of C6/36 cells infected with VRPs containing encapsidated GFP-expressing SFV replicon RNAs encoding pre-KUN-miR-1 RNA or sfRNA. RNA was isolated at 4 days post-infection. The faint upper band is the intermediate band also observed in mosquito virus-infected (Figure 1B) or plasmid-transfected cells (Figure 3B). M, mock-infected cells; GFP, SFV1-GFP VRP-infected cells; sfRNA, SFV1-GFP-sfRNA VRP-infected cells; pre-KUN-miR-1, SFV1-GFP-pre-KUN-miR1 VRP-infected cells.
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gkr848-F3: Ectopic expression of KUN-miR-1. (A) Diagram representing the RNA transcript of the ectopically expressed pre-KUN-miR-1 in pIZ vector. MCS, multi-cloning site. (B) Northern blot of RNA derived from C6/36 cells transfected with plasmid DNA pIZ/pre-KmiR-1 (48 h after transfection) encoding WNVKUN pre-miRNA using 3′ probe specific for KUN-miR-1. pIZ, empty vector. tRNA is shown as loading control. (C) Schematic diagram of Semliki Forest virus (SFV) replicon constructs expressing WNVKUN sfRNA and pre-KUN-miR-1. sfRNA and pre-KUN-miR-1 were inserted in the 3′NCR of the SFV replicon downstream of the GFP gene. SFV replicon RNA is first transcribed into the complementary (−) strand from which subgenomic RNA encoding GFP gene and 3′NCR is produced in large excess to the (+) strand genomic RNA. Both WNVKUN RNAs are produced primarily from this subgenomic RNA. (D) Northern blot hybridization with KUN-miR-1-specific probe of C6/36 cells infected with VRPs containing encapsidated GFP-expressing SFV replicon RNAs encoding pre-KUN-miR-1 RNA or sfRNA. RNA was isolated at 4 days post-infection. The faint upper band is the intermediate band also observed in mosquito virus-infected (Figure 1B) or plasmid-transfected cells (Figure 3B). M, mock-infected cells; GFP, SFV1-GFP VRP-infected cells; sfRNA, SFV1-GFP-sfRNA VRP-infected cells; pre-KUN-miR-1, SFV1-GFP-pre-KUN-miR1 VRP-infected cells.
Mentions: To determine whether KUN-miR-1 can be generated from the pre-miRNA in isolation from other viral or virus-induced host factors and is not a degradation product from the end of the viral genome, we cloned the pre-KUN-miR-1 sequence in the insect expression plasmid vector pIZ/V5-His, transfected the resulting pIZ/pre-KmiR-1 plasmid into C6/36 cells and analysed RNA from transfected cells by northern blot with the 3′ probe. It is worth mentioning that the transcript produced from this plasmid contained a 5′-UTR from the vector (53 nt) followed by 79 nt pre-KUN-miR-1 sequence, and followed by another 428 nt of vector sequences comprising parts of the multi-cloning site, V5 epitope, 6×His tag and 3′-UTR containing a polyA signal (Figure 3A); therefore, the pre-miRNA SL was not located at the very end of the transcript but flanked by vector sequences. A 21-nt RNA representing KUN-miR-1 was clearly detected (Figure 3B), thus confirming that KUN-miR-1 is indeed produced from the pre-miRNA sequence and is not an artefact of degradation from the end of the viral genome. An RNA band of 40 nt between mature miRNA and pre-miRNA, which was also detected in WNVKUN-infected mosquito cells (Figure 1C), was visible in pIZ/pre-KmiR-1-transfected cells. Intermediate fragments are frequently observed for other miRNAs [e.g. (25,45)].Figure 3.

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