Figure 1

HERC5 restricts a late stage of HIV-1 replication. A, Inhibition of HIV-1 particle production. 293T cells were co-transfected with pR9 and either empty plasmid or pHERC5. Twenty-four hours after transfection, infectious HIV-1 virions released into the supernatant were quantified using GHOST(3) indicator cells. B, Inhibition of HIV-1 replication. HOS-CD4/CXCR4 cells were co-transfected with plasmids encoding a replication-competent HIV-1 provirus (R9) and HERC5 or an empty vector control. HIV-1 virions released into the supernatant were pelleted 72 hours after transfection and analyzed by Western blotting using anti-p24CA antibody. p24CA levels were quantified densitometrically. C, Representative Western blot using anti-p24CA of HIV-1 particles released into the supernatant. Data shown are the average of at least two independent experiments performed in triplicate. D-G, 293T cells were co-transfected with pLKO.1/scrambled_shRNA or pLKO.1/HERC5_shRNA and pR9. Twenty-four hours after transfection, HIV-1 particles released into the supernatant, cellular RNA and cellular protein were isolated. HERC5, HERC3 and β-actin RNA were detected by reverse transcription polymerase chain reaction (RT-PCR) (D) and quantified densitometrically (E). HIV-1 particles released into the supernatant and intracellular Pr55Gag protein were detected by Western blotting using anti-p24CA (F) and quantified densitometrically (G). Data shown are representative of at least two independent experiments.