FIG 9.
Contributions of vIRF-2 and its interaction with USP7 to productive replication. (A) Infectious titers of HHV-8 produced from lytically reactivated (doxycycline-treated) TRExBCBL1-RTA PEL cells in the absence and presence of vIRF-2 depletion. Duplicate cultures were transduced with lentiviral vector-delivered nonsilencing (NS) control shRNA or vIRF-2 mRNA-targeting shRNAs (sh1, sh2, or sh3), and the media from doxycycline-treated or untreated cultures were harvested 4 days after lytic induction with doxycycline (1 μg/ml for first 24 h) or mock treatment. These inocula were used to infect naive iSLK cells, and the percentages of infected cells were determined by LANA immunostaining (see Materials and Methods). P values (Student's t test, two tailed) for effects of shRNAs 1, 2, and 3 in lytically reactivated cells are 0.005, 0.011, and 0.010, respectively. (B) Samples of the same media were used to determine the yields of encapsidated (DNase I-resistant) HHV-8 genomic DNA by qPCR using primers directed to LANA open reading frame (ORF73) sequences (see Materials and Methods). P values (Student's t test, two tailed) for effects of shRNAs 1, 2, and 3 are 0.023, 0.003, and 0.021, respectively. (C) Diagrammatic representation of mutations introduced into BAC16 to generate the respective vIRF-2 knockout (TTG), vIRF-2.S247A-substituted (247), and revertant (R) viruses used in subsequent phenotypic analyses. (D) Restriction analyses (NheI + XhoI) of wild-type (WT), point-mutated (initiator codon ATG to TTG [TTG] and vIRF-2 S247-to-A247 codon mutation [247]), and wild-type-reverted (TTGR and 247R) BAC16 HHV-8 genomes, showing overall integrity of the genomes (indistinguishable from wild-type BAC16). The arrow indicates the position of the 4.3-kbp fragment containing the vIRF-2 gene. (E) LANA immunoblotting of extracts of iSLK cultures infected with the same infectious doses (determined by LANA staining, as outlined for panel A) of the different viruses and subsequently hygromycin selected to remove uninfected cells. These cultures were used, in duplicate, for lytic induction and assessments of infectious virus titers (F) and encapsidated viral DNA yields (G) following 9 days of doxycycline and sodium butyrate treatment (see Materials and Methods). P values (determined by two-tailed Student's t test) for TTG and 247 effects, relative to the WT, on lytic replication are 0.037 and 0.0001 for data in panel F and 0.049 and 0.018 for data in panel G. (H) Immunoblots (left) of extracts from lytically reactivated BAC16-infected iSLK cultures (harvested 3 days postinduction), showing lack of detectable full-length vIRF-2 expression (but possibly some low-level expression of truncated, alternatively initiated vIRF-2) by the TTG mutant and equivalent expression of vIRF-2 and vIRF-2.S247A by the respective wild-type/revertant and 247 viruses. USP7 and β-actin immunoblotting provided loading controls; p53 was probed to identify any modulation by vIRF-2 or its interaction with USP7. The blots on the right show (expected) vIRF-2 specificity of the TTG and S247A mutations, with unaffected expression of vIRF-1 and vIRF-3 in the respective extracts.