Nucleophosmin Phosphorylation by v-Cyclin-CDK6 Controls KSHV Latency
Figure 5
Phosphorylation of NPM correlates with KSHV latency.
(A) Whole-cell extracts of KSHV-positive (BC-3, BCBL-1, BC-1, JSC-1, IHH) and KSHV-negative (IHE) cell lines were subjected to SDS-PAGE followed by Western blotting and analyzed for pNPM Thr199, total NPM (NPM), v-cyclin and vIRF3. Tubulin was used as a loading control. (B) Cell extracts of BC-3, JSC-1 and IHE cells were immunoprecipitated using anti-LANA or rat IgG as a control. Immunocomplexes were resolved by SDS-PAGE and analyzed by Western blotting with anti-LANA and anti-NPM antibodies. Input (5%) is indicated on the right. (C) Total RNA from cells in panel A was assayed for the abundance of ORF50 and ORF57 transcripts by real-time quantitative PCR. Transcripts of human β-actin were used to normalize the values. Data is representative of three independent experiments ± SD. The relative abundances of the lytic transcripts were compared to those in BC-3 cells, which was set to one. (D) Quantification of the ORF59 positive cells used in panels A and C. Values are means of three independent experiments ± SD. (E) BCBL-1 or JSC-1 cells were acutely transduced with retroviruses expressing GFP (v-cyclin -) or v-cyclin (v-cyclin +). Whole-cell extracts were resolved by SDS-PAGE at 48 hours post-transduction and immunoblotted for pNPM Thr199, total NPM (NPM), and v-cyclin. Tubulin served as a loading control. (F) Total RNAs from cells in panel E were assayed for the abundance of ORF50, ORF57 and K8.1 transcripts by real-time quantitative PCR. Transcripts of human β-actin served as an endogenous control. The relative abundances of the lytic transcripts in v-cyclin over-expressing BCBL-1 and JSC-1 cells (v-cyclin +) were compared to those in the corresponding control cells (v-cyclin -), which were set as one. Data is representative of two independent experiments ± SD.