Article Figures & Data
Figures
- Fig. 1.
RACE analyses of KSHV ORF K8 transcripts. (A) Schematic drawings of the genomic locations of ORF K8 and RACE primers used in this study. The nucleotide coordinates in parentheses are from reference 42. Primer orientation is depicted by arrows. The sketch is not drawn completely to scale. AATAAA, polyadenylation signal; ORF, open reading frame; SA, splice acceptor; SD, splice donor; TRL: terminal repeat of left end; TRR, terminal repeat of right end. (B to D) Agarose gel (1%) electrophoresis of RACE products. (B) 5′ RACE with K8-AS-N or K8-AS as 3′ primers. (C) 3′ RACE with K8-S as a 5′ primer. (D) Full-length cDNAs of K-bZIP amplified by K8FL-S1 and K8FL-AS1 (lane 1) or by K8FL-S2 and K8FL-AS1 (lane 2). Resulting PCR fragments were about 0.8 kb shorter than the sizes expected from the genomic sequence (1.2 kb rather than 2 kb in lane 1 and 1.0 kb rather than 1.8 kb in lane 2), suggesting that splicing events occurred.
- Fig. 2.
Sequence analysis of K-bZIP. (A) Summary of three types of cDNAs obtained by RACE cloning. Exons are represented as open boxes with roman numerals. IVSs between exons are represented as wavy lines. Peptides corresponding to each transcript are shown as solid lines. ∗, translation stop codon; +++, basic region. aa, amino acids. (B) Fine structure of the cDNA sequence of K-bZIP. Nucleotide sequences derived from the longest cDNA are boxed and are shown in uppercase. Introns are shown in lowercase, and consensus splice donor and splice acceptor sites are shown in bold. The 237 amino acids of K-bZIP deduced from three exons are depicted beneath the nucleotide sequences. The heptad repeat leucines and isoleucine are circled. Genomic coordinates (42) of the sequences are given at the left. Features discussed in the text are underlined: tga at 74627, translation stop codon of Rta/ORF 50; tataa at 74816, putative TATA box of K-bZIP; atg at 75915, translation initiation codon of ORF K8.1; AATAAA at 76714, polyadenylation recognition sequence. (C) Comparison of bZIP domain of K-bZIP to those of representative human bZIP transcription factors. Accession numbers for each sequence are as follows: CEBA (CCAAT/enhancer binding protein alpha, P49715); JUN (transcription factor AP-1, P05412); FOS (p55c-fosproto-oncogene protein, P01100); CREB (cyclic AMP response element binding protein, P16220). HHV8, eighth type of human herpesvirus. (D) Comparison of bZIP domain of K-bZIP to those of herpesvirus homologues: MEQ oncoprotein encoded by Marek’s disease virus (MDV) EcoQ fragment, A44083); BZLF1 (BZLF1 transactivator protein encoded by EBV, P03206). Amino acid sequences were aligned with the ClustalW program. Conserved leucine residues are marked by dots. Dark-gray shading indicates identical residues; light-gray shading indicates similar residues.
- Fig. 3.
RNase protection assays. (A) Mapping of the transcription start site of the K-bZIP transcript. Fifty nanograms of in vitro-transcribed [α-32P]UTP-labeled RNAs derived from pBS-P2 (lane 4) was hybridized with 15 μg of RNAs from TPA-treated BCBL-1 cells (lane 1) or with an equal amount of yeast RNA (lane 3) at 68°C for 10 min. An RNase A-RNase T1 mixture was then added to digest the unhybridized RNAs. The RNA hybrids were subsequently resolved on a 6% polyacrylamide–8 M urea denaturing gel. A sequencing reaction containing ddA, ddC, and ddG fragments was run in parallel as a DNA ladder marker (lane 2). Schematic drawings of the hybridization between the antisense probe (solid line) and the expected transcript (wavy line) are depicted to the left of each protected band. For simplicity, nucleotide 74850 (A of the first ATG in the K-bZIP gene) is arbitrarily defined as 1. (B) Mapping of the splice variants. Reactions were performed under the same conditions as those described for panel A, except that 50 ng of in vitro-transcribed [α-32P]UTP-labeled RNAs derived from pBS-IVS was used as a probe. The numbers to the left of lanes 1 are in units of base pairs.
- Fig. 4.
Expression kinetics of K-bZIP in BCBL-1 cells. BCBL-1 cells were treated with TPA for different times as indicated. For the 12- and 48-h time points, duplicate cell cultures were prepared and additionally treated with cycloheximide (CH, 100 μg/ml) or PAA (100 μM), respectively. Total RNAs were extracted at the end of the treatments. Twenty micrograms of RNA from each sample was loaded in each lane and transferred to a nylon membrane after electrophoresis. (A) The filter was hybridized with a K-bZIP-specific probe (nucleotides 74850 to 75104). (B) The filter was hybridized with an ORF K8.1-specific probe (nucleotides 75905 to 76207). RNA loading was assayed by hybridizing the same filter with DNA encoding H1 RNA of human RNase P (5) (bottom panel).
- Fig. 5.
Dimerization of K-bZIP. Total cellular protein from COS-1 cells transfected with pcDNA3.1 (lanes 1, 5, 9, and 13), pHA-KBZIP (lanes 2, 6, 10, and 14), pT7-KBZIP (lanes 3, 7, 11, and 15), or both pHA-KBZIP and pT7-KBZIP (lanes 4, 8, 12, and 16) was recovered and quantitated, and equal amounts were used in each reaction. Immunoprecipitation (IP) assays were performed by incubation of protein lysates with monoclonal antibodies against the T7 tag (lanes 5 to 8) or the HA tag (lanes 13 to 16), and the immunocomplex was captured with a protein A-protein G-Sepharose mixture. Protein samples from total cell lysates or from immunoprecipitations were subjected to Western blot (WB) analysis and probed with antibodies against the HA tag (A) or the T7 tag (B). Filters were developed by the ECL method. Kd, kilodaltons; Ig-H and Ig-L, heavy and light chains of immunoglobulin, respectively.
- Fig. 6.
The C terminus of K-bZIP is required for dimer formation. COS-1 cells were transiently transfected with HA-K-bZIP and T7-K-bZIPΔLZ for 48 h. Aliquots of total protein extract (lane 1) were immunoprecipitated (IP) either with anti-HA antibody (lane 3) or with anti-T7 antibody (lane 5) before Western blot analysis. Lanes 2 and 4 are blanks. Western blots (WB) were probed with antibodies against the HA tag (A) or the T7 tag (B). Filters were developed by the ECL method. Kd, kilodaltons.
