Cellular MicroRNA and P Bodies Modulate 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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1 Cellular MicroRNA and P Bodies Modulate Host-HIV-1 Interactions 指導教授 : 張麗冠博士 演講者 : 黃柄翰 Date: 2009/10/19 1

2 MicroRNA biogenesis 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 mirisc 6. mirisc: mature mirna + RISC Nat Rev Genet Feb;10(2): Review. 2

3 Mature mirna seed region How mirnas find out theis targets? Bases 2~ 8,numered from the 5 end of mirna, called mirna seed, by using seed region mirna can bind to target sequence. mirna binding to partially complementary sites in mrna 3 UTRs is considered to be the halmark of animal mirna regulation. 3 Cell Dec 26;115(7):

4 How do mirnas work? 1. Blocking translation initination 2. Blocking translation elongation 1. mirnps = miriscs 3. Deadenylation EMBO J Feb 6;27(3):

5 Processing body ( p-body ) 1. mrnps aggregation 2. For mrna storage or mrna degradation 3. Compose of Dcp1 Dcp2 RCK/p54 Nat Rev Mol Cell Biol Jan;8(1):

6 Human immunodeficiency virus 1 (HIV-1) Baltimore Classification VI RNA Icosahedral Enveloped (+)ssrna 2copies Tropism : T cell and marcophage (CD4+,CXCR4) 6

7 HIV-1 life cycle 7

8 Specific aims Whether HIV 1 mrna expression is regulated by mirisc and P body? 8

9 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 interaction with RISC and P body 5. Whap happen after HIV 1 mrna have been transported into P body? 9

10 Silencing Dicer Enhances HIV-1 Replication HIV 1 expression vector : pnl4 3 LucR E Cell line : 293T 10

11 Silencing Drosha Enhances HIV-1 Replication HIV 1 expression vector : pnl4 3 LucR E Cell line : 293T 11

12 Framework 1. Make sure that HIV 1 regulated by mirna regulation machinery. mirnas are involved in negatively controling HIV 1 replication? 1. Make sure that HIV 1 regulated by mirna regulation machinery. Host mirnas are involved in negatively controlling HIV 1 replication 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 interaction with RISC and P body 5. Whap happen after HIV 1 mrna have been transported into P body? 12

13 Predict targets of human mirna in HIV- 1 3 UTR 13

14 Predicted mir-29a target site in HIV-1 3 UTR 14

15 HIV-1 transcripts HIV 1 has 9 genes those genes can transcribe 12 proteins. All the transcripts have the same 3 UTR. 15

16 Inhibiting mir-29a increases HIV-1 infectivity and production Cell line : 293T HIV 1 expression vector :pnl4 3 LucR E 16

17 Overexpression mir-29a mimic decrease HIV-1 infectivity and production Cell line: 293T HIV 1 expression vector :pnl4 3 LucR E Cell line : T Cell H9 HIV 1 expression vector :pnl4 3 LucR E 17

18 Framework 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? 2. Which mir 29a mirna down regulates could HIV 1 be involved in infectivity and productivity HIV 1 mrna expression modulation? mir 29a down regulates HIV 1 infectivity and productivity 3. Whether mir 29a directly targets HIV 1 mrna? 4. mir 29a enchances HIV 1 mrna interaction with RISC and P body 5. Whap happen after HIV 1 mrna have been transported into P body? 18

19 Double mutation of HIV-1 and mir-29a mir Sequence???? mir Sequence mir Sequence

20 cis element effect : mirna and mrna 3 UTR Rna Pol II Normal reporter gene Rna Pol II mirna Modefied reporter gene Target mrna 3 UTR

21 Virus production was suppressed by mir-29a / mir-29a-mt Cell line: 293T mir???? mir mir Sequence Sequence Sequence 21

22 mir-29a don t inhibit HIV-1 mutant Cell line: 293T Overexpression mir 29a 22

23 mir-29a don t inhibit HIV-1 mutant Cell line: T cell H9 Overexpression mir 29a 23

24 Dicer depleting enhances HIV-1 production 24

25 Framework 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. 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 5. Whap happen after HIV 1 mrna have been transported into P body? 25

26 mir-29a enhances HIV-1 mrna interaction with RISC Co Immunoprecipitation Ago1 Protein A/G G7 cap PCR AAAAA Ago2 26

27 mir 29a enhances HIV 1 mrna interaction with P body RCK/p54 Protein A/G G7 cap PCR AAAAA 27

28 HIV-1 mrna co localizes with P body fluorescence in situ hybridization (FISH) Locked Nucleic Acid probe Cy3 P body proteins A3G: APOBEC3G P body marker 28

29 Depleting P body proteins enhance HIV-1 productivity and infectivity P body proteins silencing enhances HIV 1 productivity and infectivity 29

30 Framework 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. 4. mir 29a enchances HIV 1 Mrna interaction with RISC and P body. 5. What happen after HIV 1 mrna have been transported into P body? 30

31 P body depletion increases viral protein synthesis without affecting its mrna level 31

32 The proposed model of HIV-1 mrna be regulated by P body 32

33 Framework 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. 5. What happen after HIV 1 mrna have been transported into P body? mir 29a facilitates suppression of HIV 1 translation by enhancing the association of viral mrna with P body 33

34 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 mrna interaction with RISC and P body. 5. mir 29a facilitates suppression of HIV 1 translation by enhancing the association of viral mrna with P body 34

35 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. 35

36 36

37 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. 37

38 HIV strains Divided by env gene similarity. 38

39 M O 39

40 Thanks for your attention 40

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