Cellular MicroRNA and P BodiesModulate Host-HIV-1 Interactions
指導教授: 張麗冠 博士
演講者: 黃柄翰
Date: 2009/10/19
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MicroRNAbiogenesis
Nat Rev Genet. 2009Feb;10(2):94‐108. Review.
6. miRISC:mature miRNA + RISC
miRISC
1. Pri‐miRNA:primary – miRNA
2. Drosha:RNaseIII
3. DCR‐1:Dicer 1 – RNaseIII
4. RISC: RNA‐induced silencing complex
5. RISC proteins:Argonaute 1、Argonaute 2 、Dicer
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Mature miRNA seed region
Bases 2~ 8 ,numered from the 5’ end of miRNA, called miRNA seed, by using seed region miRNA can bind to target sequence.
Cell. 2003 Dec 26;115(7):787‐98.3
miRNA binding to partially complementary sites in mRNA 3’ UTRs is considered to be the halmark of animal miRNA regulation.
How miRNAs find out theis targets?
How do miRNAs work?
EMBO J. 2008 Feb 6;27(3):471‐81.
1. miRNPs= miRISCs
1. Blockingtranslation initination
2. Blocking translationelongation
3. Deadenylation
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Processing body ( p-body )
Nat Rev Mol Cell Biol. 2007 Jan;8(1):23‐36.
1. mRNPs aggregation
2. For mRNAstorage or mRNAdegradation
3. Compose of Dcp1、
Dcp2、RCK/p54
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Human immunodeficiency virus – 1 (HIV-1)
Baltimore Classification — VIRNA
IcosahedralEnveloped
(+)ssRNA 2copies
Tropism : T cell and marcophage(CD4+,CXCR4)
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HIV-1 life cycle
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Specific aims
Whether HIV‐1 mRNA expression is regulated by miRISC and P body?
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Framework
1. Make sure that HIV‐1 regulated bymiRNA regulation machinery.
2. Which miRNA could be involved in HIV‐1 mRNA expression modulation?
3. Whether miR‐29a directly targets HIV‐1 mRNA?
4. miR‐29a enchances HIV‐1 mRNA interactionwith RISC and P body
5. Whap happen after HIV‐1 mRNA have been transported into P body? 9
Silencing Dicer Enhances HIV-1 Replication
HIV‐1 expression vector : pNL4‐3 LucR‐E‐
Cell line : 293T10
Silencing Drosha Enhances HIV-1 Replication
HIV‐1 expression vector : pNL4‐3 LucR‐E‐
Cell line : 293T11
Framework
1. Make sure that HIV‐1 regulated by miRNA regulation machinery.
2. Which miRNA could be involved in HIV‐1 mRNA expression modulation?
3. Whether miR‐29a directly targets HIV‐1 mRNA?
4. miR‐29a enchances HIV‐1 mRNA interactionwith RISC and P body
5. Whap happen after HIV‐1 mRNA have been transported into P body?
‐‐ Host miRNAs are involved in negatively controlling HIV‐1 replication
1. Make sure that HIV‐1 regulated by miRNA regulation machinery.
‐‐miRNAs are involved in negatively controling HIV‐1 replication
?
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Predict targets of human miRNA in HIV-1 3’ UTR
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Predicted miR-29a target site in HIV-1 3’ UTR
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HIV-1 transcripts
HIV‐1 has 9 genes those genes can transcribe 12 proteins.
All the transcripts have the same 3’ UTR.
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Inhibiting miR-29a increases HIV-1 infectivity and production
Cell line : 293T
HIV‐1 expression vector :pNL4‐3 LucR‐E‐
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Overexpression miR-29a mimic decrease HIV-1 infectivity and production
Cell line: 293T Cell line : T Cell H9
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HIV‐1 expression vector :pNL4‐3 LucR‐E‐ HIV‐1 expression vector :pNL4‐3 LucR‐E‐
Framework
2. Which miRNA could be involved in HIV‐1 mRNA expression modulation?
3. Whether miR‐29a directly targets HIV‐1 mRNA?
4. miR‐29a enchances HIV‐1 mRNA interactionwith RISC and P body
5. Whap happen after HIV‐1 mRNA have been transported into P body?
1. Make sure that HIV‐1 regulated by miRNA regulation machinery.
‐‐miRNAs are involved in negatively controling HIV‐1 replication
?
‐‐miR‐29a down regulates HIV‐1 infectivity and productivity
miR‐29a2. Which miRNA could be involved in HIV‐1 mRNA expression modulation?
‐‐miR‐29a down regulates HIV‐1 infectivity and productivity
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Double mutation of HIV-1 and miR-29a
miR
Sequence
???? miR
Sequence
miR
Sequence
cis – element effect :miRNA and mRNA 3’UTR
Normal reporter gene
Modefied reporter gene Target mRNA 3’UTR
Rna Pol II
Rna Pol IImiRNA
Cell line: 293T
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miR
Sequence
???? miR
Sequence
miR
Sequence
Virus production was suppressed by miR-29a / miR-29a-mt
miR-29a don’t inhibit HIV-1 mutantCell line: 293T
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Overexpression miR‐29a
miR-29a don’t inhibit HIV-1 mutantCell line: T cell H9
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Overexpression miR‐29a
Dicer depleting enhances HIV-1 production
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Framework
3. Whether miR‐29a directly targets HIV‐1 mRNA?
4. miR‐29a enchances HIV‐1 mRNA interactionwith RISC and P body
5. Whap happen after HIV‐1 mRNA have been transported into P body?
1. Make sure that HIV‐1 regulated by miRNA regulation machinery.
‐‐miRNAs are involved in negatively controling HIV‐1 replication
2. Which miRNA could be involved in HIV‐1 mRNA expression modulation?
‐‐miR‐29a down regulates HIV‐1 infectivity and productivity
‐‐miR‐29a directly targets to HIV‐1 mRNA 3’ UTR.
3. Whether miR‐29a directly targets HIV‐1 mRNA?
‐‐miR‐29a directly targets to HIV‐1 mRNA 3’ UTR.
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miR-29a enhances HIV-1 mRNA interaction with RISC
Ago2
Ago1
G7 cap AAAAA
Protein A/G
PCR 26
Co‐Immunoprecipitation
miR‐29a enhances HIV‐1 mRNA interaction with P body
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RCK/p54
G7 cap AAAAA
Protein A/G
PCR
HIV-1 mRNA co localizes with P body
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A3G:APOBEC3G P body marker
fluorescence in situhybridization (FISH)
‐‐Locked Nucleic Acidprobe – Cy3
P body proteins
Depleting P body proteins enhance HIV-1 productivity and infectivity
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P body proteins silencing enhances HIV‐1 productivity and infectivity
Framework
4. miR‐29a enchances HIV‐1 Mrnainteraction with RISC and P body.
5. What happen after HIV‐1 mRNA have been transported into P body?
1. Make sure that HIV‐1 regulated by miRNA regulation machinery.
‐‐miRNAs are involved in negatively controling HIV‐1 replication
2. Which miRNA could be involved in HIV‐1 mRNA expression modulation?
‐‐miR‐29a down regulates HIV‐1 infectivity and productivity
3. Whether miR‐29a directly targets HIV‐1 mRNA?
‐‐miR‐29a directly targets to HIV‐1 mRNA 3’ UTR.
4. miR‐29a enchances HIV‐1 mRNA interaction with RISC and P body.
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P body depletion increases viral protein synthesis without affecting its mRNA level
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The proposed model of HIV-1 mRNAbe regulated by P body
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Framework
5. What happen after HIV‐1 mRNA have been transported into P body?
1. Make sure that HIV‐1 regulated by miRNA regulation machinery.
‐‐miRNAs are involved in negatively controling HIV‐1 replication
2. Which miRNA could be involved in HIV‐1 mRNA expression modulation?
‐‐miR‐29a down regulates HIV‐1 infectivity and productivity
3. Whether miR‐29a directly targets HIV‐1 mRNA?
‐‐miR‐29a directly targets to HIV‐1 mRNA 3’ UTR.
4. miR‐29a enchances HIV‐1 mRNA interaction with RISC and P body.
‐‐miR‐29a facilitates suppression of HIV‐1 translation by enhancing the association of viral mRNA with P body
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Framework
1. miRNAs are involved in negatively controling HIV‐1 replication
2.miR‐29a down regulates HIV‐1 infectivity and productivity
3. miR‐29a directly targets to HIV‐1 mRNA 3’ UTR.
4. miR‐29a enchances HIV‐1 mRNAinteraction with RISC and P body.
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5. miR‐29a facilitates suppression of HIV‐1 translation by enhancing the association of viral mRNA with P body
Conclusions
1. Authors provide an example of a single host miRNA regulating HIV‐1 production and infectivity.
2. miR‐29a and P bodies can modulate virus mRNA expression.
3. miRNA‐mediated antiviral mechanism.
4. HIV‐1 has evolved a mechanism utilizing miR‐29a to modulate its own life cycle.
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Conclusions
1. Authors provide an example of a single host miRNA regulating HIV‐1 production and infectivity.
2. miR‐29a and P bodies can modulate virus mRNA expression.
3. miRNA‐mediated antiviral mechanism.
4. HIV‐1 has evolved a mechanism utilizing miR‐29a to modulate its own life cycle.
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HIV strains
Divided by env gene similarity.
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M
O
Thanks for your attention
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