Inhibition of Retrovirus Replication
by Human Schlafen 11
Murine Schlafen (Slfn) Family
In search for IRF3-regulated genes, we found multiple members of the Schlafen (Slfn) gene family…
Putative AAA domain
Slfn1
Slfn2
Slfn3
Slfn4
Slfn5
Slfn8
Slfn9
Slfn10
Slfn14
Putative RNA helicase domain
Human Slfn Family
Putative AAA domain
Slfn12
Slfn12L
Slfn5
Slfn11
Slfn13
Slfn14
Putative RNA helicase domain
Discovery 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
In the next 12 years…
•
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)
AAA-domain
“ATPases Associated with various cellular Activities”
• 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
Snider et al. Genome Biology 2008 9:216
Slfn expression profiles
mRNA Levels of Slfns in Human Fibroblasts (HFF) upon Stimulation:
 Ct(TBP-TargetGene)
SLFN5
SLFN11
SLFN12
SLFN13
SLFN12L
SLFN14
ISG54
Unstimulated
7.59±0.02
6.22±0.06
6.98±0.05
2.95±0.15
U.D.
U.D.
5.84±0.06
IFNb
9.76±0.12
7.83±0.10
8.20±0.09
3.67±0.05
2.20±0.09
U.D.
11.67±0.02
pdAdT
9.61±0.04
7.44±0.03
7.37±0.25
2.67±0.26
-0.22±0.34
U.D.
12.89±0.03
pIC
10.42±0.05
5.97±0.10
8.57±0.02
0.67±0.46
-2.88±0.02
U.D.
13.45±0.08
mRNA Levels of Slfns in Cell lines:
 Ct(TBP-TargetGene)
SLFN5
SLFN11
SLFN12
SLFN13
SLFN12L
SLFN14
HEK293
2.40±0.40
6.65±0.04
2.01±0.08
-2.04±0.08
U.D.
-8.20±0.33
HEK293T
-0.40±0.02
U.D.
U.D.
-12.1±0.65
U.D.
-7.70±0.30
Hela
5.01±0.03
U.D.
5.00±0.02
-11.32±0.98
U.D.
U.D.
CEM
6.69±0.46
4.58±0.06
-6.70±0.74
-2.597±0.14
U.D.
-8.72±0.38
Jurkat
6.84±0.07
5.73±0.06
-1.82±0.10
-4.72±0.12
-3.60±0.49
-4.23±0.51
huSlfn11 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 HIV
MSCV
HIV
HIV Infection and Replication Cycle
Do Slfns act as effectors in the innate immune response?
huSlfn11 restricts production of MSCV or HIV
MSCV
HIV
Truncation mutants of huSlfn11
Divergent AAA domain
RNA helicase
Slfn5 (891 a.a.)
Slfn11 (901 a.a.)
Slfn11N (1-579 a.a.)
Slfn11C (533-901 a.a.)
huSlfn11N restricts production of MSCV or HIV
MSCV
HIV
huSlfn11 does not restrict production of AAV or HSV
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 huSlfn11
Intracellular vRNA levels are not affected by huSlfn11
huSlfn11 specifically inhibits viral protein synthesis
huSlfn11 specifically inhibits viral protein synthesis
huSlfn11 appears to inhibit viral protein translation by interference with -1 frameshifting
CEM cells and HIVLAI
THANKS...
David Lab
Salk Institute
Manqing Li
Matt Weitzmann
Sebastian Landry
Hilary Sandig
Elaine Kao
Kirsten Vroom
Irene Pedersen
Dennis Otero
Hiroshi Moro
Yoshinari Tanabe
Takeaki Nishibori
John Young
UCSD-CFAR
John Guatelli
Davey Smith