FIG 2.

The HPV18 central URR element, region 4, is unnecessary for replicon maintenance. (A) Diagram of replicons containing the central URR and origin with deletions in region 4 (green). Control replicons include the CpGneo (vector), the CpGneo vectors with the minimum HPV18 origin (ori), region 4 and the origin (Reg4+ori), and the central URR and origin (central URR+ori). (B) Keratinocyte colonies arising from continuous G418 selection were stained with methylene blue approximately 14 days posttransfection. (C) Cellular DNA collected from five passages of cotransfected cells were digested and analyzed by Southern blotting and viral DNA detected by hybridization with a radioactively labeled CpGneo vector probe. The top panel represents DNA samples cut with an enzyme that does not cut the replicon, permitting visualization of extrachromosomal, supercoiled replicon DNA. The bottom panel shows DNA cut with BstXI which separates the replicon into two fragments. Lanes containing a size marker (M) contain two fragments generated from digesting the CpGneo vector with BstXI. The monomeric, supercoiled form (s/c) of the replicon is indicated. (D) HPV18 genome (left) and replicon (right) copy numbers were measured by qPCR. The data shown are representative (panels B and C) or an average of two biological replicates (panel D).