Post on 01-Apr-2015
Inhibition of Retrovirus Replication by Human Schlafen 11
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
Human Slfn FamilyHuman Slfn Family
Slfn5
Slfn11
Slfn13
Slfn12
Slfn12L
Putative AAA domain
Slfn14Putative RNA helicase domain
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
• 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…
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”
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:
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?
Retroviral Replication Cycle
huSlfn11 does not alter Infection by MSCV or HIVhuSlfn11 does not alter Infection by MSCV or HIV
MSCV
HIV
HIV Infection and Replication Cycle
Do Slfns act as effectors in the innate immune response?
huSlfn11 restricts production of MSCV or HIVhuSlfn11 restricts production of MSCV or HIV
MSCV
HIV
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
huSlfn11N restricts production of MSCV or HIVhuSlfn11N restricts production of MSCV or HIV
MSCV
HIV
huSlfn11 does not restrict production of AAV or HSVhuSlfn11 does not restrict production of AAV or HSV
Mechanism?Mechanism?
Extracellular p24 and vRNA levels correlate with viral titers by infection assay =>
Fewer viral particles are produced
Virus budding & release are not affected by huSlfn11Virus budding & release are not affected by huSlfn11
Intracellular vRNA levels are not affected by huSlfn11Intracellular vRNA levels are not affected by huSlfn11
huSlfn11 specifically inhibits viral protein synthesishuSlfn11 specifically inhibits viral protein synthesis
huSlfn11 specifically inhibits viral protein synthesishuSlfn11 specifically inhibits viral protein synthesis
huSlfn11 appears to inhibit viral protein translation by interference with -1 frameshifting
CEM cells and HIVLAI
David LabManqing LiHilary SandigElaine KaoKirsten VroomIrene PedersenDennis OteroHiroshi Moro Yoshinari TanabeTakeaki Nishibori
Salk InstituteMatt WeitzmannSebastian Landry
John Young
THANKS...THANKS...
UCSD-CFARJohn GuatelliDavey Smith