The RING-CH Ligase K5 Antagonizes Restriction of KSHV and HIV-1 Particle Release by Mediating Ubiquitin-Dependent Endosomal Degradation of Tetherin
Figure 2
RNAi-depletion of K5 during lytic replication suppresses KSHV particle release.
(A) Three shRNA hairpins were designed to target K5 and co-expressed with K5-HA in 293T cells. The efficiency of reduction of K5 expression was assessed 48 hours later by western blot of total cell lysates detecting the HA tag. Stripped blots were re-probed for alpha tubulin to demonstrate equal loading. (B & C) r219-HeLa cells were transduced by lentiviral vectors encoding K5 shRNAs at an MOI of 5, reseeded 72 hours later and transfected with RTA expression plasmid the following day. Supernatants were harvested 48 hours post-RTA transfection and KSHV titer expressed as infectious unit/ml (B) and DNase-I resistant genome copies/ml (C) were determined as in Figure 1. At the time of harvest (48 hours post RTA transfection), ORF37 and GAPDH mRNA were measured in the cell lysates by Taqman Q-RT-PCR to assess effect of the hairpins on KSHV reactivation and ORF37 expression (D). At the time of harvest, cells were also kept to assess the effect of the hairpins on genome replication. Cellular DNA was extracted and KSHV episomes measured by QPCR for ORF37 (E). Results are expressed as KSHV genomes per nanogram of total cellular DNA. Results are the mean of 2 independent experiments and errors are standard error of the mean.
doi: https://doi.org/10.1371/journal.ppat.1000843.g002