Putative NLS1 is dispensable for Rta function. (A) Schematics of WT Rta (top) and RtaΔNLS1 (bottom). Numbers refer to amino acid positions. ex, exon; +++, basic-amino acid rich; LR, leucine repeat; ST, serine-threonine amino acid rich; AD, transcriptional activation domain. (B) BL-41 cells were coelectroporated with the indicated amounts of plasmids expressing WT Rta (WT) or RtaΔNLS1 (ΔNLS1), the pGL3-nut-1 promoter/reporter (expressing luciferase), and pcDNA3.1-His-lacZ (expressing β-galactosidase) as an internal control. The level of activation was calculated by comparison to that of the reporter coelectroporated with empty expression vector. (C) BL-41 cells were electroporated similarly to the description for panel B, but the pGL3-TK promoter/reporter (expressing luciferase) was tested. (D) BCBL-1 cells were coelectroporated with the indicated plasmids and a plasmid expressing hepatitis delta virus large delta antigen and treated with tetradecanoyl phorbol acetate (TPA), as indicated. At 72 h postelectroporation, the percentage of transfected cells expressing large delta antigen and coexpressing ORF59 or K8.1 was calculated for each transfection. Levels of induction were calculated by comparison to that in cells transfected with the empty vector, which was normalized as onefold. Error bars show standard deviations for each transfection.

Putative NLS2 is required for Rta function. BL-41 cells were coelectroporated with plasmids expressing the indicated proteins and the levels of activation calculated as described in the legend to Fig. 1B. Reporter plasmids were pGL3-ORF57 (A) or pGL3-Nut-1 (B), both expressing luciferase. Black triangles indicate relative amounts of transfected expression vectors. Error bars show standard deviations.

NLS2, but not putative NLS1, is required for nuclear localization of Rta. HeLa cells were transfected with plasmids expressing the indicated proteins and analyzed by indirect immunofluorescence using Rta-specific antiserum. Nuclei were visualized by DAPI stain. Images of representative cells were captured digitally and converted to grayscale.

NLS2 is sufficient to completely relocalize eGFP to the cell nuclei. HeLa cells were transfected with plasmids expressing the indicated proteins and analyzed by DAPI staining of nuclei. GFP and DAPI images of representative cells were captured digitally and converted to grayscale. Nuclear/cytoplasmic localization ratios (Nucl:Cyto) are shown under the images; SD, standard deviations; NA, not applicable.

4-OHT-regulated nuclear localization of Rta. HeLa cells were transfected with plasmids expressing WT Rta-ER (A) or RtaΔNLS1,2-ER (B), and half of each set of transfected cells was treated with 4-OHT. Cells were analyzed and visualized as described in the legend to Fig. 3.

Nuclear localization of Rta is required for transactivation. BL-41 cells were coelectroporated with plasmids expressing the indicated proteins, and half of each set of transfected cells was treated with 4-OHT. The level of activation was calculated as described in the legend to Fig. 1B. Reporter plasmids were pGL3-ORF57 (A) or pGL3-Nut-1 (B), both expressing luciferase. Error bars show standard deviations. +, treated; −, not treated.

Nuclear relocalization of Rta reactivates the complete KSHV lytic cycle. (A) Vero-rKSHV.219 cells were transfected with the RtaΔNLS1,2-ER expression vector. Three hours following transfection, half of the cells were left untreated or treated with 4-OHT, as indicated. At 40 h following 4-OHT addition, total cellular RNA was purified and analyzed by Northern blotting; probes are indicated at left. Vec, vector. (B) Vero-rKSHV.219 cells were transfected with RtaΔNLS1,2-ER plasmid and treated with 4-OHT as described for panel A. At the indicated times following 4-OHT addition, total cellular RNA was purified and analyzed by Northern blotting; probes are indicated at left. The blot was quantitated by phosphorimager; each signal for Nut-1/PAN and ORF50 was normalized to the corresponding signal for 7SK. The level of induction was calculated by comparison to the signal for untreated cells at each time point. (C) Vero-rKSHV.219 cells were transfected and treated with 4-OHT as described for panel B. At 24 h after the addition of 4-OHT, cells were analyzed by indirect immunofluorescence using ORF50/Mta-specific primary antiserum and Alexa Fluor 350-conjugated secondary antibody. GFP and RFP fluorescence and immunofluorescent images of representative cells were captured digitally. (D) Vero-rKSHV.219 cells were transfected and treated with 4-OHT as described for panel B. At 72 h following 4-OHT addition, total cellular RNA was purified and analyzed by real-time qRT-PCR with primers that detect the indicated true L transcripts. (E) Vero-rKSHV.219 cells were transfected with the indicated plasmids, and indicated cells were treated with 4-OHT as described for panel A. At 48 h following 4-OHT addition, viral reactivation was quantitated by measuring the percentage of cells expressing the true L marker K8.1. Positive controls were transfection of WT Rta plasmid or treatment with sodium butyrate (Na Bu). Results are from two experiments performed in triplicate, in each of which 500 K8.1 positive cells were counted. (F) Vero-rKSHV.219 cells were transfected with the indicated plasmids, and the indicated cells were treated with 4-OHT as described for panel A. Seven days after the addition of 4-OHT, cell supernatants were transferred to naïve 293 cell monolayers. KSHV-infected 293 cells were detected by GFP expression, and nuclei were detected with DAPI. Infection was quantitated by counting 500 GFP-positive cells in triplicate experiments. Error bars show standard deviations. +, treated; −, not treated.

Nut-1/PAN is a direct transcriptional target of Rta in infected cells. (A, B, C, D) Strategies for testing the protein synthesis inhibitors hygromycin and neomycin in Vero-rKSHV.219 cells are given above the panels. In the experiments whose results are shown in panels B, C, and D, each half of the experimental strategy (untreated or treated with 4-OHT) was performed in duplicate; panels labeled GFP and RFP show images representative of the results of each experiment (converted to grayscale). (E) Total cellular RNAs corresponding to cells in the experiments diagrammed in panels A, B, C, and D were purified and analyzed by Northern blotting; probes are indicated at left. (F) Vero-rKSHV.219 cells were transfected with the reporter vectors pGL3-promoter (expressing firefly luciferase; Promega) and pcDNA3.1-His-lacZ (expressing β-galactosidase; Invitrogen). Cells were left untreated or treated with hygromycin as described forpanel C, and reporter expression was analyzed 40 h after the addition of hygromycin. Error bars show standard deviations. (G) Vero-rKSHV.219 cells were left untreated or treated with hygromycin as described for panel C, and endogenous tubulin expression was analyzed by Western blot 40 h after the addition of hygromycin. −, untreated; +, treated with 4-OHT; hygro, hygromycin; neo, neomycin; Luc, luciferase; βgal, β-galactosidase.