Fig 1.
Presence of ORF75 RNA in nearly all LANA-expressing cells in KS skin tissue.
A) Skin KS lesion from an HIV+ patient with KS and KSHV-MCD, dual-stained with ORF75 RNAscope (yellow) followed by LANA IHC (purple) and counterstained with DAPI (blue). Dual RNAscope-IHC image shown. The yellow box and dashed white box regions are shown in higher magnification in B) and C) respectively. B) Higher magnification of yellow box (Fig 1A). Negative staining for RNAscope is with RNAscope 2.5 LS negative control probe dapB gene. Negative staining for IHC has no primary antibody. The top panel represents dual RNAscope-IHC image showing negative staining (left) and ORF75 RNA/LANA protein staining (right). The bottom two panels represent same region with individual ORF75 RNAscope and LANA IHC staining, respectively. C) The KS-lesion region in the white box is enlarged to show colocalization of ORF75 RNA and LANA protein; the dashed green box is then enlarged further to highlight individual cells.
Fig 2.
ORF75 RNA is constitutively expressed in latently infected endothelial cells.
A) Schematic of the establishment of the TIME.219 KSHV-infected immortalized endothelial cell line by infection of TIME cells with recombinant KSHV.219 virus. Blue and red arrows indicate the period of infection without and with puromycin selection, respectively. Sample collection days during the initial period of selection are shown. Representative images of de novo infection at day 6 and after stable selection (TIME.219) are shown. B) qPCR analysis of representative KSHV latent and lytic genes in the latently infected TIME.219 cell line and the BCBL-1 (PEL) cell line. N=3 biological replicates of the established cell lines TIME.219 (obtained after day 30) and BCBL-1. qPCR analysis was done with 40 cycles and a GAPDH internal reference control. Expression was normalized to corresponding LANA expression of TIME.219 and BCBL-1, respectively. Error bar indicate ± standard deviations of 3 separate experiments. Black arrows highlight the ORF75 gene. C) qPCR analysis of representative latent and lytic KSHV genes at 1, 3, and 6 days post infection (DPI) after de novo infection of TIME cells with rKSHV.219 virus. Connected lines indicate gene expression trend of the same gene throughout the course of experiment.
Fig 3.
ORF75 promoter has a very high basal transcription activity.
A) (Top) Schematic of the ORF75 promoter region with various putative transcription factor binding elements. (Bottom) Table showing the genomic location, orientation and sequence of various promoter elements with respect to ORF75’s start codon (ATG). B) Schematic showing a map of the ORF75 protein coding transcript and the ORF75 promoter-luciferase plasmids. Note that the gene map is shown in the conventional orientation, but the plasmids are shown in the opposite orientation. C) Promoter luciferase assay of ORF75 full-length (p75) construct in unstimulated HEK293T cells. pGL3 is the empty vector control. Assayed at 4 days post transfection. D) Promoter luciferase assay of ORF75, ORF74, ORF72, LANA, ORF57 and RTA promoter activity in uninfected HEK293T cells. Assayed at 72h post transfection. E) Promoter luciferase assay in HEK293T cells of various ORF75 promoter mutation constructs. Schematics of the mutation constructs are shown on the left and the corresponding basal transcription activity on the right. Assayed at 72h post transfection. Numbers on the top of each bar in Fig 3C, 3D and 3E indicates average fold change normalized to pGL3 vector control. Shown are the means ± standard deviations of at least 3 separate experiments. P-values (*p ≤ 0.05, ns not significant) are calculated using two-sided paired t-test. Relative luciferase unit indicated is normalized to Beta-galactosidase that was used as transfection control. GL: genomic location as per NC_009333 KSHV reference genome.
Fig 4.
ORF75 promoter’s high basal transcription activity is regulated by Sp1 DNA elements.
A). (Left) schematic of truncation mutants, deletion mutants, and binding site mutants of the ORF75 promoter used to make luciferase constructs. (Right) promoter luciferase activity of these constructs in 293T cells. Numbers on the top of each bar indicates average fold change relative to empty vector pGL3 control. B) Schematic showing the location and sequence of the proximal Sp1 (pSp1) element and ARE elements in the ORF75 promoter and the mutants. C) Sequences of the pSp1 consensus element of ORF75 promoter probe and the mutant probes used in EMSA. The promoter probe is a 50 nt long dsDNA labelled with 5’ IR dye CW700. The mutated pSp1 element is in purple and underlined. The terminal ATG is the start codon of ORF75 protein. D) EMSA showing binding of Sp1, Sp3, and Sp4 proteins with the double-stranded DNA (dsDNA) IR-probe of ORF75 promoter in a 8% native PAGE gel. Equal concentration of HEK293T lysates over-expressing the various Sp proteins were used in the assay. E) Same as D), but with competitive specific and Sp1 element mutated probes. Shown are the means ± standard deviations of at least 3 separate experiments. P-values (*p ≤ 0.05, **p ≤ 0.01, ns not significant) are calculated using two-sided paired t-test. Representative gel shift assay blots shown. All gel shift assay observations were reproduced in at least 3 separate experiments. Beta-galactosidase was used for transfection normalization. See S7 Fig for full blots.
Fig 5.
ORF75 promoter’s Sp1 DNA elements are regulated by Sp transcription factors.
A) Promoter luciferase assay of ORF75 promoter constructs along with over-expression of various Sp proteins in HepG2 cells. Sp proteins were expressed from a CMV-driven pN3 vector. ORF75 full-length (p75) and truncated (p75-T2) promoters are shown. Numbers on the top of each bar represents average fold change normalized to untreated pGL3 for each group of expression plasmid set as 1. The panel below the histogram represents the representative western blot for the over-expression proteins. B) ORF75 promoter (p75-T2) luciferase activity in the presence of Sp1-specific inhibitor Mithramycin A in 293T cells. 48h post transfection. C) Supershift assay. Equal amounts of HEK293T lysates (30ug) were preincubated with 1, 1.5 and 2µg of anti-Sp1 (21962-1-AP, Proteintech) or anti-Sp3 (26584-1-AP, Proteintech) before incubation with ORF75 promoter probe (p75 pSp1 probe) followed by gel shift assay. 6% native TBE gel. D) Same as in C) but with HEK293T nuclear lysates and 3µg each of different Sp1 antibodies. Sp1 antibody (AP) (21962-1-AP, Proteintech), Sp1 antibody (1C6) (sc-420, Santa Cruz), Sp1 antibody (E3) (sc-17824, Santa Cruz). Shown are the means ± standard deviations of 3 separate experiments. P-values (**p ≤ 0.01, ****p ≤ 0.0001, ns not significant) are calculated using two-sided paired t-test. Beta-galactosidase was used for transfection normalization. See S7 Fig for full blots.
Fig 6.
Sp1 protein is the key transcription factor regulating ORF75 promoter’s basal activity.
A) Promoter luciferase assay of ORF75 promoter in Schneider Drosophila line 2 (SL2) with or without exogenous human Sp1 expression. Sp1 protein was expressed from pPAC vector driven by ACTIN 5C promoter. pPAC0 is the empty vector. Numbers on the top of each bar represents average fold change normalized to pGL3 with pPAC0 set as 1. Assayed at 72h post nucleofection. The western blot panel below shows the expression of exogenous Sp1 protein in SL2 cells. Data are presented as mean ± SD of three individually nucleofected samples. B) Same as in A), except with ORF75 p75-T4 truncated and Sp1 element-deleted promoter constructs. Numbers on the top of each bar represents average fold change normalized to p75 with pPAC0 set as 1. C) Promoter luciferase assay of various mutant ORF75 promoters in Schneider Drosophila line 2 (SL2) with exogenous human Sp1 expression. Presence of various elements are shown in the promoter schematic as different shapes. Absence of a shape in the promoter schematic indicates corresponding element mutation. Data shown are presented as mean ± SD of three individually nucleofected samples. Assayed at 72h post nucleofection. Numbers on the top of each bar represents average fold change normalized to pGL3 set as 1. P-values (***p ≤ 0.001, ****p ≤ 0.0001, ns not significant) are calculated using two-sided paired t-test. D) qPCR expression analysis of Sp1 mRNA in shControl 293T and two shSp1 293T stable Sp1 knockdown cell lines. Sp1 mRNA expression is normalised to shControl. Internal reference gene GAPDH. E) Promoter luciferase assay of full length ORF75 promoter (p75) and truncated promoter (p75-T4) in shControl and shSp1 293T knockdown cell lines. Assayed at 3 DPI. Shown are the means ± standard deviations of 3 separate experiments. Numbers on the top of each bar represents average fold change normalized to shControl values for p75 and p75-T2 set as 1. P-values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns not significant) are calculated using two-sided unpaired t-test. Beta-galactosidase was used for transfection normalization. See S7 Fig for full blots.
Fig 7.
ORF75 promoter exhibits relatively higher activity in TIVE cells than BJAB cells.
A) Promoter luciferase assay of ORF75 full length promoter in TIVE (endothelial cell line) and BJAB (B-cell line) cells. Results shown are the fold change over the respective pGL3 empty vector for each cell type. Data shown are ± standard deviations of 3 separate experiments. P-values (**p ≤ 0.01) are calculated using two-sided paired t-test B) Western blot analysis of Sp1, Sp3 and Sp4 protein levels from different cell lines using whole cell lysates. Blue and black arrows indicates full-length Sp1 and alternate SP3 forms, respectively. C) EMSA and WB analysis. Nuclear lysates of various uninfected cell lines were analysed for their Sp1 binding activity to the proximal Sp1 element of ORF75 promoter through EMSA. D) Same as in C) but with nuclear lysates of KSHV infected and uninfected cell lines. The lower three panels represent parallel WB of the nuclear lysates. *In the LANA WB, the asterisk indicates a non-specific band. E) Same as A), except four different ORF75 promoters were used for the promoter luciferase assay in BJAB and TIVE cells. Assayed at 72h post transfection. Numbers on the top of each bar indicates average fold upregulation relative to empty vector pGL3 for each cell type set as 1. Shown are the means ± standard deviations of 3 separate experiments. P-values (**p ≤ 0.01, ****p ≤ 0.0001, ns not significant) are calculated using two-sided unpaired t-test. All blots and gel shift assays were at least replicated in three separate experiments. Blots stripped and reprobed, see S7 Fig for more information.
Fig 8.
Degradation of full-length Sp1 protein and accumulation of its variants during KSHV lytic induction.
A) WB of whole cell lysate (RIPA buffer) of early passage cell lines showing alternate forms of Sp1 protein in KSHV infected B-cell lines (4-12% BIS-TRIS gel, MES buffer). Sp1, Sp4 and ACTIN are same blot stripped and reprobed. Sp3 blot was run parallel. B) WB of BCBL-1 cells at different time points of lytic reactivation blotted with anti-Sp1 antibody. ORF45 and vIL6 were used here as control for lytic reactivation (10% BIS-TRIS gel, MES buffer). Sp1, ORF45 and ACTIN are same blot stripped and reprobed. vIL6 blot was run separately with the same samples. C) EMSA and western blot analysis. 10 µg of whole cell lysates (M-PER, Invitrogen) of BCBL-1 cells at different time points of lytic reactivation were analysed for their Sp1 protein binding activity to the proximal Sp1 element of ORF75 promoter through EMSA in a 8% TBE gel. The bottom three panel represent parallel WBs showing accumulation pattern of full-length Sp1 and 45 kDa Sp1 protein isoform during NaB induced lytic induction (10% BIS-TRIS gel, MES buffer). Black and red triangle indicates full-length Sp1 and 45 kDa Sp1 form binding to the proximal Sp1 DNA element of the ORF75 promoter, respectively. Sp1 and ACTIN are same blot stripped and reprobed. D) WB of uninfected TIME and infected TIME.219 cell line at different time points of lytic reactivation using TPA. LANA was used as control for infected TIME.219 cells and vIL6 was used as a control for lytic reactivation (8% Bolt BIS-TRIS gel, MES buffer). Black and red arrow indicates full-length and alternate Sp1 forms in WBs, respectively. SP1, vIL6, and ACTIN were on one blot, which was stripped and reprobed, while SP3 and LANA were on a parallel blot, which was also stripped and reprobed. Shown are representative blots, all observations were reproduced in at least 3 separate experiments. See S8 and S9 Figs for full blots.
Fig 9.
ORF75 protein enhances expression of immediate early genes.
A) (Left) Promoter luciferase assay of the ORF75-T2 promoter along with co-expression of increasing amount of a plasmid expressing flag-tagged ORF75 (F-ORF75) protein. (Right) Parallel WB analysis of whole cell lysates from the promoter luciferase assay showing F-ORF75 expression. + and ++ indicate 1:2 and 1:4 ratios of ORF75 promoter to protein expression plasmid F-ORF75, respectively. B) Same as in A), except RTA was co-expressed alone or together with F-ORF75 along with the ORF75 promoter luciferase construct. C) Same as in A), except F-ORF75 was co-expressed along with 3 different length LANA promoter luciferase constructs (LANA I: 570 bp, LANA CI: 1.2 kb, LANA CP: 795 bp. D and E) F-ORF75 protein was co-expressed with two different lengths of RTA (ORF50) promoter luciferase fusion constructs. F) Same as D), except with an ORF57 promoter luciferase fusion construct. In B-F, 1:4 ratio of ORF75 promoter to protein expression plasmid (F-ORF75 or pcDNA3.1) was used. G) Schematic for the assay of KSHV gene expression in 293T-BAC16 cells transfected with F-ORF75, RTA, or a pCDNA3.1 control. Flag-ORF75 and RTA overexpression vectors or a pcDNA3.1 control vector were transfected into 293T cells latently infected with KSHV followed by qPCR analysis of endogenous KSHV genes (ORF75, LANA, ORF57 and vIL6) 48 hr post transfection. Fig 9G was created in BioRender. H) RT-qPCR of select genes in either empty or ORF75 and RTA expression vector transfected cells. Fold change normalized to non-transfected 293T-BAC16 cells. Endogenous ORF75 was detected using a primer pair spanning the 5’UTR and CDS. I) Same as H) except that RTA gene expression was analyzed in F-ORF75 transfected 293T-BAC16 cells. Other information: Numbers on the top of each bar in A through F indicates average fold upregulation relative to control set as 1. Error bar indicate ± standard deviations of 3 experiments. pcDNA3.1 plasmid was used as vector control. Promoter luciferase assay in 293T cells was performed at 72h. More information on the different promoters used here is provided in S5D Fig. See S9 Fig for full blots.
Fig 10.
Simplified summary schema showing the regulation of ORF75 promoter’s basal transcription activity. The activity is primarily regulated by the binding of the Sp1 protein to Sp1 elements. There is evidence that in all cell lines, the Sp1 complex interacts with the proximal Sp1 element, the Sp1-like elements, and the CCAAT boxes to activate this transcription. Also, a repressor, whose identity is unknown but acts via the distal region of the ORF75 promoter, variably inhibits basal transcription. This repressor’s effect determines the promoter’s activity across different cell lines, even in the presence of Sp1 activation. In B-cells, the distal region of the ORF75 promoter strongly represses its activity, leading to lower ORF75 transcription during latency. By contrast, endothelial and epithelial cells experience less repression and more activation of the ORF75 promoter due to the proximal Sp1 element. This results in higher ORF75 transcript levels, even though there is no lytic viral DNA replication. This figure was created in BioRender.