Transcript Slide 1

Finding Host Proteins
Required for HIV Replication
Abe Brass
Partners AIDS Research Center and GI Unit
Mass. General Hospital
Harvard Medical School
How do we find what HIV
needs to replicate?
Rationale
• Employ new methods in mammalian
genetics to find host factors that HIV
depends upon (HDFs).
• HDFs provide targets for anti-retroviral
therapy and chemical prophylaxis.
Rationale
• HIV may be hard pressed to evade
HDF-directed therapies.
• Comprehensive information about the
lifecycle of the virus will benefit the HIV
research community.
Overview
•
•
•
•
•
RNAi Mechanism
RNAi Tools
Finding HIV-dependency factors
Three HIV siRNA Screens
TNPO3
RNAi Mechanism
RNAi Tools
Genetic Screens
• Deplete protein expression with shRNAs
or siRNAs.
• Test how depletion impacts phenotype
with simple in vitro functional assay.
• Unbiased whole genome screens bring
new targets into the “pipeline”.
Genetic Screens
• The way a genetic screen is designed can
profoundly influence which genes are
uncovered
• Different screen platforms yield different
results (i.e. libraries, viruses, cell lines,
transfection conditions and efficiencies,
readouts)
• Some weak hits may be the most
important unlike small molecule screens
(knockdown efficiency unknown).
Caveats
• False positives (OTEs). Present, but
minimized through bioinformatic
functional clustering, expression studies
and reagent redundancy.
• False negatives. Why didn’t this host
factor score? Saturation is the goal, but
hard to obtain by generating
hypomorphs with the current siRNA
technology. Optimized validated
reagents will help.
shRNA Libraries
•Whole genome, 3 shRNAs/gene
•Packaged into Retroviral Pools, Stable knockdown
•Focused Libraries: Kinases, Ubiquitin pathway
•shRNAs have unique barcodes
•Formats : MSCV, lentivirus, Inducible.
shRNA Libraries
mir30-shRNA
Retrovirus
LTR
mir30-5’
mir30-3’
barcode
LTR
Phenotype
Integration
mir30-shRNA
Processing of shRNA
Target mRNAsingle copy
knockdown
shRNA Libraries
Control
Experimental
PCR recovery of and color label of
barcode
Competitive hybridization
to barcode microarray
shRNA dropped out
following selection
shRNA enriched
following selection
Barcode: unique 60 nt sequences that allow the
abundance of any particular retroviral shRNA in a
complex population to be followed using microarray
hybridization.
siRNA Libraries
• Arrayed format-”one gene per well”
• High throughput whole genome screens
done with liquid handling robotics.
• Transiently transfect siRNAs, RISC
active for 6 days post transfection.
• Image based=scanning microscope.
• Reporter gene=plate reader.
Finding HIV-dependency factors
Luciferase
Percent infected
120
90
Part one
60
30
0
CD4
Tat
Beta-gal (RLU)
400000
300000
200000
100000
0
Part two
siRNA Library
• Dharmacon: SMARTpool library, 4
siRNAs per pool, whole human genome
(21,121 genes).
• Initial screen done with pools.
• Validation round done with the four
individual siRNAs.
Screen Results
• 16 genes scored with
• Out of 21,121
4/4 individual siRNAs
genes, 386 scored 2
SD below control
• 44 genes 3/4
• 99 genes 2/4
• 1.8% hit rate
• 115 genes 1/4
• 273 of 386 siRNA
pools were confirmed
• Each of the four
by at least 1 siRNA
individual siRNA
(71%)
were retested
• Reagent Redundancy
tries to minimize
OTEs, but some of the
¼ are “knowns”.
Known Host Factors Found in the Screen
A4GALT (2/4 siRNAs)
DDX3X (2)
PSME2 (1)
AKT1 (2)
ERCC3 (3)
PURA (2)
AP2M1 (1 )
FBXW11 (4)
Rab9p40 (3)
Arf1 (2)
GCN5L2 (1)
RANBP1 (1)
CD4 (2)
H3F3A (1)
RelA (4)
CD147 (3)
HRS (SP)
SIP1 (1)
CRTC2 (1 )
HTATSF1 (1)
ST3GAL5 (1)
CRTC3 (3 )
IKBG (2)
TFAP4 (3)
CTDP1 (1)
La Autoantigen (2)
TFE3 (2)
CXCR-4 (2)
FAPP1 (1)
VPRBP (1)
CyclinT1 (1)
NMT1 (3 )
ZNRD1 (2)
Biologic Processes
Enzymes Found in the Screen
ADAM10 (3/4 siRNAs)
WNK1 (3)
Jak1 (1)
DDX55 (3)
PSPHL (2)
USP26 (2)
DDX49 (2)
DPM1 (2)
OTUD3 (2)
ATPV0A1 (2)
OST48 (3)
LPL (2)
GAPVD1(2)
PRKX1 (1)
HUWE1 (2)
PIGH (1)
STT3A (2)
HERC3 (3)
PIGY (2)
RNF170 (3)
EXOD1(2)
ABGL5 (2)
FNTA(4)
HERC6 (2)
FLJ32569 (2)
ALKBH8 (2)
DDX33 (2)
ITPKA (2)
NMT1 (3)
SET7 (3)
MOS (2)
DIMT1L(4)
ARF1 (2)
PIP5K1C (3)
C14orf125 (3)
CENTG1 (3)
Three HIV siRNA Screens
Comparison of Three HIV siRNA Screens
Cells
Zhou
HeLa-Bgal
KD Virus Infection Readout Hits
24 h
HXB2
48 hr; 96 hr
β-gal
reporter
activation
48 h
NL 4.3
luc
vector,
VSV-G
24 hr
Luc reporter
295
72 h
HIV-1- 48 hr; 48 hr in
IIIB
new cells
p24 ;
reporter
activation
280
et al
Konig
293T
et al
Brass
et al
TZM-bl,
HeLa
Stephen Goff
Cell 135 2008
207
Konig et al
Zhou et al
295
207
9
13
18
280
Brass et al
783
total
RelA
Med6
Med7
HDFs Found by Two Independent Screens
ADRBK1
AKT1
ANAPC2
ANKRD30A (1)
Cav2
CD4
CHST1
CTDP1 (1)
CXCR4
CyclinT1 (1)
WNK1
DDX3X
DMXL1
IDH1 (1)
Jak1 (1)
MAP4
Med14
Med19
Med28
Med4
Med6
TRIM55
Med7
MID1IP1
Mre11A
Nup153
Nup155
Rab28 (1)
RanBP2
RelA
RNF26 (1)
TCEB3
TNPO3
Why so little overlap?
• Different screen platforms yield different
results.
• Technology caveats: False positives
(OTEs) and false negatives (reagents not
validated).
• High throughput methodologies
• Secondary filters of the primary data
TNPO3
SR
1.2
HIV LTR
Late RT
1.2
0.8
0.8
0.4
0.4
0
0
Virion
RTC
PIC
PIC
Provirus
1.4
1.2
2-LTR
1
0.8
0.6
0.4
0.2
0
Conclusions
• Functional enrichment, finding known factors,
and confirmation studies suggests the
majority of the genes found in the screens
impact HIV replication.
• Clearly conventional validation work is
required, but host factor discovery has been
accelerated (33+ functionally confirmed host
factors in 10 months).
• Two screens yielded TNPO3, which is very
likely the factor that permits HIV-1, HIV-2,
EIAV, and SIV access to their host’s
genomes.
Thank You
• Stephen J. Elledge, HHMI, BWH, HMS
• Judy Lieberman, Nan Yan, Derek Dykxhoorn,
IDI, Childrens Hospital
• Ramnik Xavier, Yair Benita, CCIB, MGH
• Caroline Shamu and staff, ICCB-L HMSFelipe
Diaz-Griffero, DFCI, HMS
• Bill and Melinda Gates Foundation
• Harvard CFAR