Fig 2.

Hypoxia increases the production of LANA RNA and LANA protein levels. (A) Diagram of the mRNA transcripts produced from LTc and LTi as previously reported (10, 31, 33, 38, 44, 45). An unspliced transcript of 5.8 kb encoding ORFs 71 to 73 is produced from the LTc start site. Alternative splicing (as shown by dotted lines) generates mRNA transcripts of 5.4 kb and 1.7 kb for ORFs 71 to 73 and ORFs 71 to 72, respectively. A 5.5-kb transcript produced from the LTi start site also encodes ORFs 71 to 73 (31). (B and C) Northern blot analysis showing increased ORF73 (LANA) mRNA in BC-3 and JSC-1 cells exposed to hypoxia (1% O₂). Cells were cultured in normoxia (N) or hypoxia (H) or in the presence of 20 ng/ml TPA (T). After 22 h, RNA was extracted and 5 μg of RNA was probed using probes specific for ORF73 (B) or K12 (C). LANA mRNAs are upregulated in hypoxia compared to normoxia but not by TPA treatment. K12 is upregulated substantially by exposure to TPA but only minimally or not at all by hypoxia. (D) Western blot showing protein levels of LANA in BC-3 and JSC-1 cells cultured in 1% O₂ (H) for up to 72 h in hypoxia compared to those cultured in normoxia (N). After culture in hypoxia or normoxia, cells were lysed and 20 μg of protein was run on two gels. One gel (upper) was probed with antibody specific for LANA, HIF-1α, and actin, and the other (lower) was probed with antibody to HIF-2α and actin. The gels were stripped between successive probes. HIF-1α and HIF-2α levels were stabilized up to 48 h in hypoxia and then decreased in both BC-3 and JSC-1 cells. ImageJ analysis, shown in the right panel, showed LANA levels increased approximately 2- to 3-fold in BC-3 and 2.3-fold in JSC-1 cells. (E) vIRF3 levels in BC-3 and JSC-1 cells exposed to hypoxia for 24 h and through 72 h. Its levels remained essentially unchanged in hypoxia.