Figure 4. NPM depletion triggers induction of the full lytic replication cascade in PEL cells.

(A) BC-3 and BCBL-1 cells transduced with the non-target sh-Scr, sh1-NPM, or sh2-NPM were stained with antibodies against an early lytic marker ORF59 (anti-ORF59) and analyzed by indirect immunofluorescence. Images are representative of two independent experiments. Scale bar = 100 µm. (B) Quantification of the ORF59 positive BC-3 and BCBL-1 cells in panel A. Values are means of two independent experiments ± SD. (C) Total RNA from the BCBL-1 cells used in panel A were assayed for the abundance of LANA, ORF50, ORF57, and K8.1 transcripts by real-time quantitative PCR. The relative abundances of the latent and lytic transcripts in NPM depleted cells were compared to control sh-Scr infected cells. Transcripts for human beta-actin were used to normalize the results. Data is representative of two independent experiments ± SD. (D) Whole cell extracts of the BCBL-1 cells used in A were analyzed by immunoblotting with anti-K8.1, anti-NPM and anti-tubulin antibodies. (E) Supernatant from BCBL-1 cells transduced with sh1-NPM were collected and concentrated (conc. sup.), or used unconcentrated (non-conc. sup.) to infect naive SLK cells. Two days postinfection, establishment of latency was monitored by LANA expression in SLK cells by immunofluorescence microscopy. Images are representative of two independent experiments. Scale bar = 20 µm. Insets highlight representative cells at higher magnification. (F) Quantification of the number of LANA positive SLK cells two days post infection either with non-concentrted supernatant or concentrated supernatant derived from BCBL-1 cells transduced with control sh-Scr or sh1-NPM. Data is representative of two independent experiments ± SD.