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Finding Host Proteins Required for HIV Replication

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.

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Finding Host Proteins Required for HIV Replication

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  1. Finding Host ProteinsRequired for HIV Replication Abe Brass Partners AIDS Research Center and GI Unit Mass. General Hospital Harvard Medical School

  2. How do we find what HIV needs to replicate?

  3. 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.

  4. 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.

  5. Overview • RNAi Mechanism • RNAi Tools • Finding HIV-dependency factors • Three HIV siRNA Screens • TNPO3

  6. RNAi Mechanism

  7. RNAi Tools

  8. 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”.

  9. 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).

  10. 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.

  11. 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.

  12. Phenotype Processing of shRNA Target mRNA- single copy knockdown mir30-shRNA shRNA Libraries mir30-shRNA Retrovirus LTR mir30-5’ mir30-3’ barcode LTR Integration

  13. PCR recovery of and color label of barcode Competitive hybridization to barcode microarray shRNA dropped out following selection shRNA enriched following selection shRNA Libraries Control Experimental Barcode: unique 60 nt sequences that allow the abundance of any particular retroviral shRNA in a complex population to be followed using microarray hybridization.

  14. 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.

  15. Finding HIV-dependency factors

  16. Part one Luciferase CD4 Part two Tat

  17. 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.

  18. 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”.

  19. Biologic Processes

  20. Three HIV siRNA Screens

  21. Comparison of Three HIV siRNA Screens Stephen Goff Cell 135 2008

  22. 783 total Zhou et al Konig et al 295 207 9 RelA Med6 Med7 18 13 280 Brass et al

  23. 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

  24. TNPO3

  25. SR

  26. PIC Provirus Virion RTC PIC

  27. 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.

  28. 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

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