Inhibition of Retrovirus Replication by Human Schlafen 11.

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Inhibition of Retrovirus Replication by Human Schlafen 11

Transcript of Inhibition of Retrovirus Replication by Human Schlafen 11.

Page 1: Inhibition of Retrovirus Replication by Human Schlafen 11.

Inhibition of Retrovirus Replication by Human Schlafen 11

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Murine Schlafen (Slfn) FamilyMurine Schlafen (Slfn) Family

Slfn5

Slfn8

Slfn9

Putative AAA domain

Slfn14

Slfn3

Slfn4

Slfn10

Slfn1

Slfn2

In search for IRF3-regulated genes, we found multiple members of the Schlafen (Slfn) In search for IRF3-regulated genes, we found multiple members of the Schlafen (Slfn) gene family…gene family…

Putative RNA helicase domain

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Human Slfn FamilyHuman Slfn Family

Slfn5

Slfn11

Slfn13

Slfn12

Slfn12L

Putative AAA domain

Slfn14Putative RNA helicase domain

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Discovery of SlfnsDiscovery of Slfns

• The Schlafen (Slfn) family of genes are differentially regulated during thymocyte maturation and are preferentially expressed in the lymphoid tissues.

• Ectopic expression of the prototype member Slfn1 early in the T lineage alters cell growth and development.

• Expression of Slfn1 in fibroblasts and thymoma cells either retards or ablates cell growth.

• Slfn1 knockout mice had no significant phenotype

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• Transgenic T-cell specific expression of muSlfn8 in mice resulted in impaired T cell development (Geserick et al., Int Immunol. 2004)

• Mutation of muSlfn2 causes loss of immune quiescence in T cells and monocytes(Berger et al., Nature Immun. 2010)

• muSlfn1 causes a cell cycle arrest by inhibiting induction of cyclin D1 (Brady et al., JBC 2005)

• Role of Schlafen 2 (SLFN2) in the generation of interferon alpha-induced growth inhibitory responses (Katsoulidis et al., JBC 2009)

• Lack of reproducible growth inhibition by muSlfn1 and muSlfn2 in vitro (Zhao et al., Blood Cells Mol Dis. 2008)

In the next 12 years…In the next 12 years…

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AAA-domain

Snider et al. Genome Biology 2008 9:216

• membrane fusion• protein translocation• ER-associated degradation• proteolysis• peroxisome biogenesis• mitosis, meiosis• apoptosis• microtubule dynamics• motors• DNA replication• DNA recombination and repair• transcriptional regulation• homo/hetero-multimer formation

“ATPases Associated with various cellular Activities”

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mRNA Levels of Slfns in Human Fibroblasts (HFF) upon Stimulation:

ΔCt(TBP-TargetGene) Unstimulated IFNb pdAdT pICSLFN5 7.59±0.02 9.76±0.12 9.61±0.04 10.42±0.05SLFN11 6.22±0.06 7.83±0.10 7.44±0.03 5.97±0.10SLFN12 6.98±0.05 8.20±0.09 7.37±0.25 8.57±0.02SLFN13 2.95±0.15 3.67±0.05 2.67±0.26 0.67±0.46SLFN12L U.D. 2.20±0.09 -0.22±0.34 -2.88±0.02SLFN14 U.D. U.D. U.D. U.D.ISG54 5.84±0.06 11.67±0.02 12.89±0.03 13.45±0.08

Slfn expression profilesSlfn expression profiles

ΔCt(TBP-TargetGene) HEK293 HEK293T Hela CEM JurkatSLFN5 2.40±0.40 -0.40±0.02 5.01±0.03 6.69±0.46 6.84±0.07SLFN11 6.65±0.04 U.D. U.D. 4.58±0.06 5.73±0.06SLFN12 2.01±0.08 U.D. 5.00±0.02 -6.70±0.74 -1.82±0.10SLFN13 -2.04±0.08 -12.1±0.65 -11.32±0.98 -2.597±0.14 -4.72±0.12SLFN12L U.D. U.D. U.D. U.D. -3.60±0.49SLFN14 -8.20±0.33 -7.70±0.30 U.D. -8.72±0.38 -4.23±0.51

mRNA Levels of Slfns in Cell lines:

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huSlfn11 knock-down HEK293 cell lineshuSlfn11 knock-down HEK293 cell lines

Slfns are induced by Interferons and TLR ligands

Putative helicase domain (similarity to RIG-I ??)

=>

Do they function as pathogen sensors or activators of innate immune responses?

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Retroviral Replication Cycle

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huSlfn11 does not alter Infection by MSCV or HIVhuSlfn11 does not alter Infection by MSCV or HIV

MSCV

HIV

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HIV Infection and Replication Cycle

Do Slfns act as effectors in the innate immune response?

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huSlfn11 restricts production of MSCV or HIVhuSlfn11 restricts production of MSCV or HIV

MSCV

HIV

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Slfn5 (891 a.a.)

RNA helicase Divergent AAA domain

Slfn11 (901 a.a.)

Slfn11N (1-579 a.a.)

Slfn11C (533-901 a.a.)

Truncation mutants of huSlfn11Truncation mutants of huSlfn11

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huSlfn11N restricts production of MSCV or HIVhuSlfn11N restricts production of MSCV or HIV

MSCV

HIV

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huSlfn11 does not restrict production of AAV or HSVhuSlfn11 does not restrict production of AAV or HSV

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Mechanism?Mechanism?

Extracellular p24 and vRNA levels correlate with viral titers by infection assay =>

Fewer viral particles are produced

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Virus budding & release are not affected by huSlfn11Virus budding & release are not affected by huSlfn11

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Intracellular vRNA levels are not affected by huSlfn11Intracellular vRNA levels are not affected by huSlfn11

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huSlfn11 specifically inhibits viral protein synthesishuSlfn11 specifically inhibits viral protein synthesis

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huSlfn11 specifically inhibits viral protein synthesishuSlfn11 specifically inhibits viral protein synthesis

huSlfn11 appears to inhibit viral protein translation by interference with -1 frameshifting

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CEM cells and HIVLAI

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David LabManqing LiHilary SandigElaine KaoKirsten VroomIrene PedersenDennis OteroHiroshi Moro Yoshinari TanabeTakeaki Nishibori

Salk InstituteMatt WeitzmannSebastian Landry

John Young

THANKS...THANKS...

UCSD-CFARJohn GuatelliDavey Smith

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