Jurkat Cells

1
1. Select for latent infection
2. Map integration sites
3. siRNA library in individual clones to ID human
regions which affect activation
HIV-1
Massively Parallel Pyro- sequencing
Jurkat Cells HAART
HAART kills active HIV populations, selecting for
latent infections
or - Prostratin and valproic acid
4. Larger Scale low throughput verification
5. Check for survival due to integration site bias
Pool
Massively Parallel Pyro- sequencing
siRNAs affecting latent HIV activation
Bioinformatics
Figure 2. Technique overview
2
3. Select for latently infected populations by
treatment with HAART
1. Infect Jurkat cells with HIV
2. Verify Infection and HAART sensitivity
HIV-1 GFP
Infected Cells HAART
Jurkat Cells
Infected Cells

• Verify GFP expression (ie active
• infection) in 90 of clones by FACS
• Test a sample pool for sensitivity to
• various drug cocktails

5. Repeat infection until most cells are latently
infected
4. Verify Latent Integration
Latently Infected
Actively Infected
Uninfected
HIV
Infected Cells HAART
actin
Allow cells to grow and then qPCR a sample with
primers against viral genome to estimate percent
latently infected versus uninfected cells
Figure 1. Selection of latently infected cells
3
Table 1. siRNA screens to identify RNAs involved
in latent HIV activation
Experiment Infection HAART Prostratin/ Viproic Acid Assay to ID siRNAs involved in activation
Control A - - N/A (baseline for comparisons)
Experiment A - More Death, More GFP
Control B - N/A (baseline for comparisons)
Experiment B Less Death, Less GFP