An RNA Interference Strategy for Identifying Host Factors Required for HIV Infection

1
An RNA Interference Strategy for Identifying Host
Factors Required for HIV Infection

• Abe Brass
• Ragon Institute of MGH, MIT and Harvard U. and GI
  Unit MGH
•  

2
How do we find what HIV needs to replicate?
3
(No Transcript)
4
(No Transcript)
5
Genetic Screens

• Deplete protein expression with siRNAs
• Test how depletion impacts phenotype with simple
  in vitro functional assay
• New information about the viral lifecycle which
  may benefit research and treatment

6
(No Transcript)
7
Part one
Luciferase
CD4
Part two
Tat
8
Screen Results

• 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.

• Out of 21,121 genes, 386 scored 2 SD below
  control, 50 or less
• 1.8 hit rate
• Each of the four individual siRNA were retested

9
(No Transcript)
10
(No Transcript)
11
Three HIV siRNA Screens
12
783 total
Zhou et al
Konig et al
207
295
9
RelA Med6 Med7
18
13
280
Brass et al
13
(No Transcript)
14
REACTOME event HIV life cycle (99 genes)
3 screens together 32 unique genes (p 1e-11)
15
REACTOME HIV events
16
Comparison of Screens

• Different platforms yield different results
• Technology caveats false positives (OTEs) and
  false negatives (reagents not validated)
• Known factors and pathways recovered
• Suggests that many of the unique hits are
• relevant and screens are complementary
• Still a lot of work to be done

17
TNPO3
18
SR
19
HIV-1
20
Non-targeting
DMSO
Aphidicolin
21
siRNAs against TNPO3
TNPO3 siRNAs HeLa-T4 cells HIV-IIIB virus p24
staining
LNA2
Verite 1
Verite 2
Verite 3
LNA3
TNPO3
1
3041
OTP1
OTP2
OTP3
OTP4
LNA1
22
OTP
LNA
Verite
CXCR4
CXCR4
NT
NT
kDa
1 2 3
1 2 3 4
1 2 3
110
TNPO3
80
Ran
30
23
Provirus
PIC
24
(No Transcript)
25
(No Transcript)
26
10 Input
10 Input
10 Input
Salt
FLAG-IN
FLAG-IN
IP HA Western FLAG
HA-TNPO3
HA-LEDGF
HA-LEDGF
HA-Rad52
IP HA Western HA
PIC
IN
TNPO3
27
NT
TNPO3
GFP
28
(No Transcript)
29
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 HMS
• Felipe Diaz-Griffero, DFCI, HMS
• Harvard CFAR
• Bill and Melinda Gates Foundation