cellular microrna and p bodies -...

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

4

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.




5. Whap happen after HIV‐1 mRNA have been transported into P body?

‐‐ Host miRNAs are involved in negatively controlling HIV‐1 replication


‐‐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







?

‐‐miR‐29a down regulates HIV‐1 infectivity and productivity

miR‐29a2. Which miRNA could be involved in HIV‐1 mRNA expression modulation?


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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








‐‐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?







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?







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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