Finding Host ProteinsRequired for HIV Replication

Abe Brass

Partners AIDS Research Center and GI Unit Mass. General HospitalHarvard 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.

mir30-5’ mir30-3’ barcode LTR

mir30-shRNARetrovirus

Integration mir30-

shRNA

Phenotype

Processing of shRNA Target

mRNA-single copy knockdown

LTR

shRNA Libraries

shRNA Libraries

Barcode: unique 60 nt sequences that allow the abundance of any particular retroviral shRNA in a complex population to be followed using microarray hybridization.

Control Experimental

PCR recovery of and color label of barcode

Competitive hybridization

to barcode microarray

shRNA dropped out following selection shRNA enriched

following selection

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

CD4

Tat

Luciferase

Part two

Part one

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• Out of 21,121

genes, 386 scored 2 SD below control

• 1.8% hit rate

• Each of the four individual siRNA were retested

• 16 genes scored with 4/4 individual siRNAs

• 44 genes 3/4• 99 genes 2/4• 115 genes 1/4• 273 of 386 siRNA pools

were confirmed by at least 1 siRNA (71%)

• Reagent Redundancy tries to minimize OTEs, but some of the ¼ are “knowns”.

Known Host Factors Found in the ScreenA4GALT (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 ScreenADAM10 (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

Cells KD Virus Infection Readout Hits

Zhou et al

HeLa-Bgal 24 h HXB2 48 hr; 96 hrβ-gal

reporter activation

207

Konig et al

293T 48 h

NL 4.3 luc

vector, VSV-G

24 hr Luc reporter 295

Brass et al

TZM-bl,HeLa 72 h HIV-1-

IIIB48 hr; 48 hr in

new cells

p24 ; reporter

activation 280

Comparison of Three HIV siRNA Screens

Stephen Goff Cell 135 2008

1813

9 207

280

295

Brass et al

Zhou et alKonig et al

RelAMed6Med7

783total

HDFs Found by Two Independent ScreensADRBK1 DDX3X Med7

AKT1 DMXL1 MID1IP1ANAPC2 IDH1 (1) Mre11A

ANKRD30A (1) Jak1 (1) Nup153Cav2 MAP4 Nup155CD4 Med14 Rab28 (1)

CHST1 Med19 RanBP2CTDP1 (1) Med28 RelA

CXCR4 Med4 RNF26 (1)CyclinT1 (1) Med6 TCEB3

WNK1 TRIM55 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

Virion RTC PIC ProvirusPIC

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