FIG. 8.

UNG2 shRNA-treated PEL cells showed reduced numbers of episomal copies and produced fewer virions after reactivation. (A) Detection of UNG2 in shRNA-treated BCBL-1 and BC-3 cells showed a significant knockdown of UNG2 levels because of UNG2 shRNA-treated cells but not with control luciferase shRNA. β-Actin blot assays show equal protein loading. (B) UNG2 shRNA-treated BCBL-1 and BC-3 cells showed 40 to 70% decreases in viral genome copy numbers during latency. Viral copies were calculated by real-time qPCR with the K1 gene as the target for amplification. (C) Relative numbers of virions produced after TPA and sodium butyrate treatment of BCBL-1 and BC-3 cells selected with UNG2 and Luc shRNAs. Virions were quantified by isolating DNA from purified virions and quantification by K1 gene amplification in a real-time qPCR. (D) Immunostaining of LANA in HEK293 cells infected with virions obtained from UNG2 and Luc shRNA-treated cells. Equal numbers of virions from UNG2 and Luc shRNA-selected cells were used for infection of HEK293 cells. The average number of LANA-positive cells per optical field was determined, and the results are presented as a bar graph. On the basis of LANA staining, virions obtained from UNG2 shRNA-treated PEL cells showed three- to fourfold reduced infectivity.