Figure 1.

Cul3 impedes viral replication. (a) Cul3 domain organization with three cullin repeat domains (CR1-3) that enable interaction with a substrate receptor, a cullin homology domain (CH), which recruits an E2-enzyme and a neddylation site that regulates activity of the Cullin-RING E3 ligase complex. (b,c) HEK293T cells were reversely transfected with two independent siRNAs targeting Cul3 (CUL3_5 and CUL3_9) or a non-targeting siRNA (Scr 1776) as control. 48 h after transfection, cells were infected with a firefly luciferase-encoding HIV-1 NL4-3 (VSVg-pseudotyped). Cells were either harvested 24 h post infection to check for Cul3 protein expression by western blot (b), or harvested after 24, 48, or 72 h to measure viral gene expression by quantifying luciferase signal (c) (n = 2 +/− SD). (d) HEK293T.CD4.CCR5 cells were reverse-transfected with two independent siRNAs targeting Cul3 (CUL3_5 and CUL3_9) or a non-targeting siRNA (Scr 1776) as control. 48 h after transfection, cells were infected with wild type HIV-1 NL4-3. 48 h post infection, cells were harvested, viral mRNA was isolated, and expression of viral mRNA was quantified using qRT-PCR (n = 3 +/− SD). (e) HEK293T cells were co-transfected either with the indicated amounts Cul3 or a LacZ expressing vector as control and a Renilla luciferase control plasmid. 20 h post transfection, cells were infected with a firefly luciferase-encoding HIV-1 NL4-3 (VSVg). 48 h post infection, firefly luciferase activity was determined and normalized to the activity of the Renilla luciferase control plasmid (n = 3 +/− SD). * p < 0.05; ** p < 0.01.