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Inhibition of Retrovirus Replication by Human Schlafen 11.
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Transcript of Inhibition of Retrovirus Replication by Human Schlafen 11.
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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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