Article Figures & Data
Figures
- FIG. 1.
Northern analysis of immediate-early gene expression. Total RNA from infected cells was prepared at the indicated times after infection and electrophoresed through 1.0% agarose gels, transferred to nylon membranes, and hybridized with a radioactive double-stranded DNA probe containing either the Orf50 (A) or the Orf57 (B) gene sequence. To ensure equivalent loading, membranes were also hybridized with a GAPDH probe (C). Orf50 and Orf57 gene expression was analyzed at various times after infection in the absence of metabolic inhibitors and at 12 h p.i. in the presence or absence of cycloheximide (CHX) or at 36 h p.i. in the presence or absence of PAA. Arrows indicate sizes of the transcripts that would be predicted from the sequence of the specific RRV gene.
- FIG. 2.
Splicing pattern of the RRV Orf50, R8, and R8.1 transcripts. The genomic locations of the Orf50, R8, and R8.1 genes for RRV are shown as boxes. Nucleotide numbers for the RRV gene splice donor and splice acceptor sites are shown. Small arrows under genes, locations and numbers of primers used for 5′ and 3′ RACE. The exact locations of the primers are listed in Table 1. A weak putative TATA box is present at a position 31 bp upstream of the Orf50 gene transcription start site. The multiply spliced transcripts of all three genes are also shown as a schematic diagram. Long arrows above the boxes represent the direction of gene transcription.
- FIG. 3.
Northern analysis of early gene expression. Total RNA from infected cells was prepared at the indicated times after infection and electrophoresed through 1.0% agarose gels, transferred to nylon membranes, and hybridized with a radioactive double-stranded DNA probe containing either the RRV R1 (A) or the RRV vIL-6 (B) gene sequence (B). To ensure equivalent loading, membranes were also hybridized with a GAPDH probe (C). R1 and vIL-6 gene expression was analyzed at various times after infection in the absence of metabolic inhibitors and at 12 h p.i. in the presence or absence of cycloheximide (CHX) or at 40 h p.i. in the presence or absence of PAA. Arrows indicate sizes of transcripts that would be predicted from the sequences of the specific RRV genes.
- FIG. 4.
Northern analysis of LANA, vFLIP, and vCyclin gene expression. Total RNA from infected cells was prepared at the indicated times after infection and electrophoresed through 1.0% agarose gels, transferred to nylon membranes, and probed as described in Materials and Methods. To ensure equivalent loading, membranes were stripped and rehybridized with a GAPDH probe (data not shown). (A) Orf73 gene expression was analyzed at various times after infection in the absence of metabolic inhibitors. The membrane was hybridized with a radioactive sense-specific riboprobe. (B) An identical membrane was hybridized with an antisense-specific riboprobe. (C) vFLIP gene expression was analyzed at various times after infection in the absence of metabolic inhibitors and at 12 and 36 h p.i. in the presence of cycloheximide (CHX) or PAA, respectively. vCyclin gene expression was analyzed at various times after infection in the absence of metabolic inhibitors and at 12 and 36 h p.i. in the presence of cycloheximide or PAA, respectively. Arrows indicate sizes of the transcripts that would be predicted from the sequences of the specific RRV genes.
- FIG. 5.
Northern analysis of late gene expression. Total RNA from infected cells was prepared at the indicated times after infection and electrophoresed through 1.0% agarose gels, transferred to nylon membranes, and hybridized with a radioactive double-stranded DNA probe containing either the RRV Orf62 gene sequence (A) or the RRV SCIP gene sequence (B). To ensure equivalent loading, membranes were also hybridized with a GAPDH probe (C). Orf62 and SCIP gene expression was analyzed at various times after infection in the absence of metabolic inhibitors and at 12 and 40 h p.i. in the presence or absence of cycloheximide (CHX) or PAA, respectively. Arrows indicate sizes of the transcripts that would be predicted from the sequences of the specific RRV genes.
- FIG. 6.
Expression of RRV Orf50, R8, and R8.1. (A to C) Homology between the KSHV Orf50 and RRV Orf50 proteins (A), K8 and R8 proteins (B), and K8.1 and R8.1 proteins (C). The alignments of the respective KSHV and RRV proteins were performed with the ClustalW alignment program. Identical amino acids are highlighted in black, and similar amino acids are highlighted in grey. Dashes, amino acids missing in one protein sequence. (D) The RRV Orf50, R8, and R8.1 mRNAs were transcribed and translated in vitro with [35S]methionine, and the proteins were subjected to SDS-PAGE, dried, and exposed to a phosphorimager. RRV Orf50 ran at a mobility of ∼64 kDa, and R8 and R8.1 ran at a mobility of ∼30 kDa. (E) pCDNA3-RRV Orf50, pCDNA3-R8, pCDNA3-R8.1, and a pEGFPN1 control vector were each transfected into Cos-1 cells, and cells were harvested 48 h posttransfection. Cell extracts were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with an anti-AU1 antibody. Lane 1, pEGFPN1 control-transfected cells; lane 2, RRV Orf50 protein; lane 3, RRV R8 protein; lane 4, RRV R8.1 protein. RRV Orf50 ran at a mobility of ∼64 kDa, and R8 and R8.1 ran at a mobility of ∼30 kDa.
- FIG. 7.
Cellular localization of RRV Orf50, R8, and R8.1 proteins. The Orf50, R8, and R8.1 cDNAs were fused in frame with the N terminus of GFP in the pEGFPN1 expression vector. 293 cells were transfected with either the EGFPN1 vector alone (A), the RRV Orf50-EGFPN1 fusion plasmid (B), the RRV R8-EGFPN1 fusion plasmid (C), or the RRV R8.1-EGFPN1 fusion plasmid (D). (E) Higher magnification (×40) of the RRV R8.1-EGFPN1 fusion plasmid expressed in rhesus fibroblasts. The EGFPN1 protein was expressed in both the nucleus and the cytoplasm, while the RRV Orf50-EGFPN1 and RRV R8-EGFPN1 proteins were targeted to the nucleus in contrast to the RRV R8.1-EGFPN1 protein, which was targeted to the periplasm and cytoplasm of the cell.
- FIG. 8.
Conservation of gene function between the human and rhesus Orf50 transcriptional transactivators. (A) RRV Orf50 is a transcriptional transactivator of the RRV Orf57, R8, and gB promoters. The promoters for five RRV genes, the Orf57, R8, gB, vIRF-1, and Orf50 genes, were cloned upstream of a luciferase reporter construct. The promoter plasmids were transfected with or without the pCDNA3-RRV Orf50 expression vector into 293 cells, and luciferase activity was assayed 48 h posttransfection. All assays were performed in triplicate and normalized to β-Gal to control for transfection efficiency. RRV Orf50 could transactivate the R8 promoter (R8p) greater than 500-fold and the Orf57 (Orf57p) and gB (gBp) promoters greater than 100-fold. It did not significantly activate its own promoter (Orf50p) or that of vIRF-1 (vIRF-1p). (B) KSHV Orf50 can transcriptionally transactivate RRV promoters. The RRV R8, Orf57, and gB promoter plasmids were transfected with or without the pCDNA3-RRV Orf50 expression vector into 293 cells, and luciferase activity was assayed 48 h posttransfection. All assays were performed in triplicate and normalized to β-Gal to control for transfection efficiency. These promoters were significantly transactivated by both the KSHV and RRV Orf50 proteins.
Tables
- TABLE 1.
Primers used for 5′ and 3′ RACE for mapping the RRV Orf50, R8, and R8.1 transcripts
Primer no. RACE RRV genomic location (nt) Sites mapped (nt) 1 5′ 66760 Orf50 transcription start site (68285), Orf50 exon 1 (65348-65368), Orf50 exon 2 (66312) 2 3′ 67941 R8 exon 1 (68341-68787), R8 exon 2 (68902-69024), R8 exon 3 (69146-69280), Orf50 transcription stop (68171) 3 5′ 68400 R8 exon 1 (68341), Orf50 exon 2 (68024) 4 3′ 68342 R8 exon 1 (68787), R8β exon 1 (69024), R8 exon 2 (68902-69024), R8 exon 3 (69146-69280) 5 5′ 68972 R8 exon 2 (68902), R8 exon 1 (68341-68787) 6 3′ 69200 R8 exon 3 (69280), R8.1 exon 1 (69423-69865), R8.1 exon 2 (69951-70366) 7 5′ 70359 R8.1 exon 1 (69423), R8 exon 3 (69146-69280), R8 exon 2 (69024) 8 3′ 69423 R8.1 exon 2 (70366)
