Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Virology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
REPLICATION

Kaposi’s Sarcoma-Associated Herpesvirus Encodes a bZIP Protein with Homology to BZLF1 of Epstein-Barr Virus

Su-Fang Lin, Dan R. Robinson, George Miller, Hsing-Jien Kung
Su-Fang Lin
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44016, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dan R. Robinson
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44016, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
George Miller
Departments of Molecular Biophysics and Biochemistry,
Pediatrics, and
Epidemiology and Public Health, School of Medicine, Yale University, New Haven, Connecticut 06520
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hsing-Jien Kung
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44016, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.73.3.1909-1917.1999
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a recently discovered human gamma herpesvirus strongly implicated in AIDS-related neoplasms. We report here the identification in the KSHV genome of a gene for a protein designated K-bZIP and belonging to the basic-leucine zipper (bZIP) family of transcription factors. K-bZIP shows significant homology to BZLF1, which plays a key role in the replication and reactivation of Epstein-Barr virus. K-bZIP is a homodimerizing protein of 237 amino acids with a prototypic bZIP domain at the C terminus. The N-terminal portion of K-bZIP is derived from the K8 open reading frame which, through in-frame splicing, adjoins the ZIP domain. This structure was revealed by rapid analysis of cDNA ends, followed by cloning of the entire cDNA. A 1.35-kb transcript encoding K-bZIP was detected in BCBL-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate. The synthesis of this transcript was blocked by the protein synthesis inhibitor cycloheximide but not by the viral DNA synthesis inhibitor phosphonoacetate, a result which classifies it as an early lytic gene. RNase protection analysis further mapped the major transcription start site for the 1.35-kb K-bZIP mRNA and identified two other splice variants which encode proteins with the N-terminal portion of K-bZIP but lacking the C-terminal ZIP domain. Full-length K-bZIP forms dimers with itself, and the C terminus encompassing the ZIP domain is required for this process. Our studies set the stage for understanding the role of K-bZIP in the replication and reactivation of the KSHV genome.

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV), the eighth type of human herpesvirus, was discovered in 1994 from a skin lesion of a Kaposi’s sarcoma patient (10). Phylogenic analysis of nucleic acid sequences placed KSHV in the lymphotropic gamma Herpesviridae family, showing significant homologies with herpesvirus saimiri and Epstein-Barr virus (EBV) (36). While HVS and EBV are considered oncogenic agents in primates (19, 32), definitive evidence for the tumorigenic potential of KSHV is lacking. However, a number of viral gene products, such as ORF K1, ORF K12 (kaposin), ORF K9 (vIRF), and ORF 72 (v-cyclin D), were shown to have mitogenic and transforming properties when overexpressed in certain cell types (11, 22, 29, 37). KSHV is also armed with several cellular homologues with immunomodulatory functions, including vIL6, vMIPs, and vGPCR (2, 6, 27, 35, 40). These gene products are likely to be involved in the progression of KS, a disease originating from uncontrolled paracrine signalings of vascular endothelium and spindle cells (15).

Although the presence of KSHV DNA has been repeatedly demonstrated in KS lesions, KS cell lines established in vitro usually do not harbor viral genomes (1, 18). However, various KSHV-infected human B-cell lines derived from primary effusion lymphomas are available for molecular studies (7, 8, 41). Complete sequences of the viral genomes from one such line and one KS biopsy specimen have been independently determined (38, 42). In the primary effusion lymphoma lines, most of the viral genes are not expressed, suggesting that the resident virus is predominantly in a latent state (33, 41, 43). The addition of phorbol esters or sodium butyrate to the culture medium activates the expression of viral lytic genes and results in the release of virus particles (28, 33). The identities of the KSHV target genes directly responding to stimulation by phorbol esters or sodium butyrate are not clear, nor is the gene expression cascade leading to the lytic phase. Nonetheless, for many other gamma herpesviruses, the viral immediate-early gene(s) responsible for the activation of lytic genes has been determined (13, 14, 39, 47-49). Among the notable examples is the BZLF1 (also known as ZEBRA, Zta, or EB1) product of EBV which, when overexpressed, can reactivate latent EBV, enabling it to enter the lytic cycle (14, 16, 30, 31). BZLF1 is also involved in the replication of EBV DNA in the lytic stage (17).

The genomic organizations of KSHV and EBV are similar in certain regions. By positional analogies (i.e., downstream of the BRRF2-BRRF1-BRLF1 complex), KSHV ORF K8 appears to be a homolog of BZLF1. Indeed, the N-terminal domain of ORF K8 shows some similarity to that of BZLF1. However, the leucine zipper (ZIP) motif, which is crucial to the function of BZLF1, is conspicuously missing from ORF K8. In addition, there is no canonical poly(A) signal within 1 kb downstream from ORF K8, and a potential splice donor site (44) can be identified immediately before the terminator UAG codon (nucleotide 75567). We therefore hypothesized that splicing may be involved in the generation of functional ORF K8. In this regard, it is noteworthy that the BZLF1 transcript also undergoes two splicing events, and the C-terminal domains are linked together (31).

Here, we report the successful cloning, by rapid analysis of cDNA ends (RACE) and reverse transcription (RT)-PCR, of multiply spliced transcripts encoding ORF K8 and the discovery of a prototypic ZIP domain encoded by one of the exons. Expression of these transcripts is absent in latent BCBL-1 cells but can be induced by phorbol esters. This induction is sensitive to cycloheximide but not to phosphonoacetic acid (PAA), a result which classifies these transcripts as early genes. The most abundant transcript yields a protein, designated K-bZIP, of 237 amino acids with a basic-ZIP (bZIP) motif. Functional analysis shows that K-bZIP forms homodimers. We have also mapped the transcriptional start site of the K-bZIP gene, which reveals the putative promoter sequence. Our studies provide a framework for studying the role of this protein in KSHV replication and the latency phase/lytic phase switch.

MATERIALS AND METHODS

Cell culture.BCBL-1 cells (41) were grown at 37°C in RPMI 1640 supplemented with 10% fetal bovine serum in the presence of 5% CO2. Virus replication was induced by the treatment of log-phase cells with TPA (12- O -tetradecanoylphorbol-13-acetate) (20 ng/ml) for various times. COS-1 cells (24) were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum in the presence of 5% CO2. When transient expression was specified, BCBL-1 cells were electroporated with the desired plasmids by a standard protocol (3), while COS-1 cells were transfected with plasmids by use of Lipofectamine as described by the manufacturer (Gibco BRL, Gaithersburg, Md.). Forty-eight hours after transfection, cellular protein lysates or RNA was prepared and stored at −70°C until further use.

RACE.The RACE assays were carried out essentially by the procedures reported by Frohman et al. (21) with the following modifications. Specifically, poly(A)+ RNA was purified by use of oligo(dT)-cellulose (type 7; Pharmacia, Piscataway, N.J.) spin columns. Poly(A)+ RNA (2.5 μg) then was primed with oligonucleotide SS-dT (CGTAGGTTACCGTATCGGATAGCGGCCGCATTTTTTTTTTTTTTTTTT) for 3′ RACE or dT20 for 5′ RACE, and RT was carried out with avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim, Biochemicals, Indianapolis, Ind.) at 42°C for 1 h under the conditions specified by the manufacturer. The resulting first-strand cDNA was used directly as a DNA template in 3′ RACE. The DNA template for 5′ RACE was prepared by further treatment of the first-strand cDNA with 60 U of terminal deoxynucleotidyltransferase (Gibco BRL) in the presence of 0.2 mM dATP for 15 min at 37°C. Second-strand synthesis was then primed with primer SS-dT, and the mixture was incubated with 120 U of T4 DNA polymerase, 24 U of Escherichia coli DNA ligase, and 5 U of RNase H in a buffer containing 0.2 mM deoxynucleoside triphosphates, 100 mM KCl, 10 mM ammonium sulfate, 5 mM MgCl2, 0.15 mM β-NAD, 20 mM Tris-HCl (pH 7.5), and 50 μg of bovine serum albumin per ml for 4 h at 15°C. The primers used for 5′ RACE were SS (CGTAGGTTACCGTATCGGATAG) and K8-AS (TTTTCCCCACCGTCAGTATTGTCC) (or K8-AS-N [CTTTCTCAGAATTGTCCGTTCCCG]). The primers used for 3′ RACE were SS and K8-S (AGAGGAACGCTTATGCACTAAGGC). Full-length cDNAs of K-bZIP were synthesized with K8FL-S1 (TTCCGAGACTGAAGTGTTCGCAAG) and K8FL-AS1 (GACAAGTCCCAGCAATAAACCCAC) or with K8FL-S2 (TGCCAAATGCCCAGAATGAAGGAC) and K8FL-AS1. PCR conditions for denaturation, annealing, and polymerization were 93°C for 50 s, 56°C for 1 min, and 68°C for 3 min for 5′ RACE and 93°C for 50 s, 62°C for 1 min, and 68°C for 3 min for 3′ RACE, respectively. High-fidelity Taq polymerase (Boehringer) was used in all the RACE assays, and 30 cycles of amplification were used for each reaction. PCR products were subsequently cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.). The DNA sequence of each insert was determined on both strands by the dideoxynucleotide chain termination method.

Plasmids.Plasmid C1 is a pCR2.1-TOPO-based clone which contains the full-length cDNA of the K-bZIP coding region generated by RT-PCR. Inserts of C1 were transferred to pcDNA3.1 (Invitrogen) derivatives to introduce either the hemagglutinin (HA) tag (MGYPYDVPDYASGP) or the T7 tag (MASMTGGQQMGGP) to the N terminus. The resulting plasmids were denoted pHA-KBZIP and pT7-KBZIP, respectively. pBS-P2 was obtained by cloning a PCR fragment spanning nucleotides 74745 to 74947 of viral DNA (42) into pBluescript KS(+) (Stratagene, La Jolla, Calif.) with XbaI andSacII as cloning sites. pBS-IVS was constructed by cloning a PCR fragment spanning nucleotides 75376 to 75621 of viral DNA into pBluescript KS(+) with BamHI and SacI as cloning sites. All the plasmids were sequenced on both strands to confirm that no mutations were introduced during the cloning process.

RNase protection assay. XhoI-linearized pBS-P2 or pBS-IVS was used as a template for the synthesis of [α-32P]UTP-labeled antisense RNA probes by use of a MAXIscript kit (Ambion, Austin, Tex.). Fifty nanograms of probe (specific activity, 2 × 106 cpm/μg) was hybridized to 15 μg of total RNA from BCBL-1 cells treated with TPA for 20 h. RNase protection reactions were carried out by use of a HybSpeed RPA kit (Ambion). Protected RNA fragments were resolved on a 6% polyacrylamide–8 M urea denaturing gel. A sequencing reaction containing ddA, ddC, and ddG of a DNA fragment with a known sequence (chicken CSK gene) was run in parallel to the sample, providing size markers.

Northern blot analysis.Cells were lysed in TRIZOL reagent (Gibco BRL), and total cellular RNA was isolated as specified by the manufacturer. Twenty micrograms of total RNA from each sample was separated by electrophoresis through a 6% formaldehyde–1% agarose gel and blotted overnight onto a nylon membrane (Nytran; Schleicher & Schuell) by standard procedures (3). DNA probes used in Fig.4 were either PCR amplified or obtained by digestion with restriction enzymes from genomic subclones of KSHV DNA. Detailed genomic locations of each probe were as follows: K8, nucleotides 74850 to 75104; and K8.1, nucleotides 75905 to 76207.

Immunoprecipitation and Western immunoblot analysis.Immunoprecipitation was performed as described previously (25). Briefly, cells were rinsed in ice-cold phosphate-buffered saline and lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg of pepstatin A per ml, 0.2 U of aprotinin per ml, 0.5 μg of leupeptin per ml). Two hundred micrograms of cell lysate was cleared by centrifugation and mixed with 1 to 2 μg of monoclonal antibodies against HA (BAbCO, Richmond, Calif.) or T7 (Novagen, Madison, Wis.) peptides for 2 h at 4°C with gentle rotation. The immunocomplex was then captured by the addition of a protein A-protein G-Sepharose mixture (Zymed, South San Francisco, Calif.) and rocking for an additional 2 h. Beads were washed three times in RIPA buffer and then boiled for 10 min in 60 μl of 2× SDS sample buffer (125 mM Tris-Cl [pH 6.8], 4% SDS, 2 mM EDTA, 20% glycerol, 0.6% bromphenol blue). Protein samples from total cell lysates or immunoprecipitations were resolved by SDS–10% polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Biotechnology System, Boston, Mass.) by use of a semidry apparatus (Pharmacia). After being blocked in TBST (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 1% Tween 20)–5% bovine serum albumin for 1 h at room temperature, the filters were incubated with primary antibodies (1:1,500 dilution) for 2 h at room temperature. The filters were subsequently washed with TBST three times for 10 min each time. The filters were then incubated with horseradish peroxidase-conjugated goat anti-mouse antibodies (1:2,000 dilution) for 1 h, washed three times with TBST, and developed with enhanced chemiluminescence (ECL) reagents (Amersham, Arlington Heights, Ill.).

DNA and protein sequence analyses.DNA sequences of RACE clones were compiled, aligned, and analyzed with MacVector (version 6.0) software. Deduced amino acid secondary structures were analyzed by use of Chou-Fasman, Kyte-Doolittle, or Robson-Garnier programs. A comprehensive collection of bZIP transcription factor families was obtained from a database of protein families, Pfam (45). Alignment of consensus sequences among different bZIP proteins was performed by ClustalW analysis.

Nucleotide sequence accession number.The cDNA sequence of K-bZIP described in this study has been deposited in GenBank under accession no. AF072866 .

RESULTS

Major transcripts originating from the ORF K8 gene are spliced.Previously, we noted that ORF K8 shows homology with EBV BZLF1 but lacks a canonical ZIP domain. We hypothesized that the ORF K8 gene encodes only part of the protein, with the remaining portion being connected by in-frame splicing(s). To investigate this possibility, the RACE approach was used to obtain cDNAs containing both ends of the transcript encompassing the ORF K8 gene. The use of antisense primer K8-AS in the 5′ RACE reaction generated a PCR product with an estimated size of 150 bp (Fig. 1B), while a nested primer, K8-AS-N, produced a smaller band of about 75 bp. The size difference of these two PCR products correlated well with the difference in the map locations of these two primers (compare Fig. 1A and Fig. 1B). Both bands were cloned, and five transformants each were randomly selected for sequencing.

Fig. 1.
  • Open in new tab
  • Download powerpoint
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.

Compilation of these sequences revealed a major transcriptional start site located 5 bp upstream of the first ATG of the ORF K8 gene (nucleotide 74850). The cDNA sequences in this region amplified by 5′ RACE were identical to the genomic sequence, indicating that no splicing was involved. However, a more complicated pattern was seen in the 3′ RACE reaction with sense primer K8-S (Fig. 1C). Three major bands of 1.4, 0.8, and 0.75 kb were seen, with the 0.75-kb band being the most intense. The presence of multiple bands suggested that splicing may have occurred in this region.

To confirm this notion, sequences from 15 individual 3′ RACE cDNAs were determined and compared to the genomic sequence. The results are summarized in Fig. 2A. Alignment of the cDNA sequences with the genomic sequence revealed the presence of three introns (IVS1, IVS2, and IVS3). Based on the presence or absence of these introns, three types of alternatively spliced transcripts could be discerned. Type I has all three intervening sequences (IVSs) spliced out, type II retains IVS2, and type III preserves both IVS1 and IVS2. All three transcripts used a canonical polyadenylation signal (AATAAA) located at nucleotide 76714. These results provide conclusive evidence that ORF K8 transcripts undergo splicing and that such splicing events result in the attachment of a potential ZIP domain to the ORF K8 protein (see below).

Fig. 2.
  • Open in new tab
  • Download powerpoint
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.

Molecular cloning of K-bZIP.To ensure that the splicing schemes derived from the 5′ and 3′ RACE products were authentic, full-length cDNAs of the transcripts were synthesized by RT-PCR with primers derived, respectively, from the 5′- and 3′-terminal sequences (e.g., K8FL-S1 and K8FL-AS1). As shown in Fig. 1D, lane 1, a major band(s) of about 1.2 kb was amplified with primers K8FL-S1 and K8FL-AS1. When a nested primer, K8FL-S2, was used, a 1-kb PCR fragment was amplified (Fig. 1D, lane 2), as expected. These sizes are consistent with those of the spliced products (the unspliced transcripts would be 2 and 1.8 kb, respectively).

The entire sequence of type I cDNA superimposed on the genome sequence is shown in Fig. 2B. The protein coding sequences are contained within the first three exons. There are two basic regions, one in exon I and the other in exon II. A prototypic ZIP domain can be found in exon III. The basic region of exon II and the ZIP domain of exon III form a typical bZIP domain (discussed below). We have tentatively assigned the first methionine of the open reading frame as the putative initiation codon and, hence, amino acid 1. Amino acid 4 is also a potential initiator methionine, within a better Kozak context. There are two in-frame termination codons (Fig. 2A), one located in IVS2 (nucleotide 75567) and the other located within exon III (nucleotide 75789). Type I cDNA (Fig. 2A) encodes a protein of 237 amino acids. Because the transcript is fully spliced, it skips the termination codon in IVS2 and contains an intact bZIP domain. The type I cDNA product is therefore denoted K-bZIP. Type II cDNA retains IVS2, and the protein, 189 amino acids long, terminates within IVS2. Type III cDNA contains both IVS1 and IVS2 and encodes a protein of 239 amino acids, as was previously predicted from the genomic sequence of the ORF K8 gene (42). Both type II and type III cDNAs are predicted to encode proteins that carry the basic regions but not the ZIP domain.

bZIP domain of K-bZIP.Perhaps the most interesting structural feature of K-bZIP is the presence of a heptad repeat of 4 leucines and 1 isoleucine, which constitutes the ZIP domain involved in dimerization. The ZIP domain is preceded by an arginine- and lysine-rich region which presumably functions to bind DNA. Compared to other known cellular bZIP proteins, such as Jun and Fos, K-bZIP shows similarity in the ZIP domain and, to a lesser extent, in the arginine- and lysine-rich region (Fig. 2C) (23, 26). The space between the basic region and the ZIP domain is invariable among the bZIP proteins, and K-bZIP is no exception. Figure 2D shows the alignment of the bZIP domain of K-bZIP with those of two other herpesvirus bZIP proteins, EBV BZLF1 and Marek’s disease virus Meq. The overall similarity between K-bZIP and BZLF1 is about 37%.

Mapping of the transcription start site of K-bZIP by an RNase protection assay.The 5′ RACE results suggested a major transcription start site for all the transcripts. To precisely map the 5′ start site, plasmid pBS-P2 was constructed by the insertion of a DNA fragment extending from −105 to +98 (with the A of the first ATG set as 1) into a pBluescript vector. [α-32P]UTP-labeled antisense RNA (relative to the K-bZIP gene) was transcribed from pBS-P2 in vitro and used as a probe in an RNase protection assay (Fig.3A, lane 4). When the probe was incubated with RNAs from TPA-treated BCBL-1 cells and monitored by RNase digestion, protection of a distinct 103-nucleotide fragment was observed. An additional 203-nucleotide fragment, presumably derived from the readthrough transcript, was also detected. This protection was specific, since RNAs from uninduced BCBL-1 cells or with unrelated sequences (yeast RNA) did not give rise to such a signal (Fig. 3A, lane 3, and data not shown). As the schematic drawings in Fig. 3A show, the transcription start site of K-bZIP is located at nucleotide −5, a position which agrees well with the 5′ RACE data. In addition, a canonical TATA box found 29 nucleotides upstream of the transcription start site (Fig. 2B) could serve as the promoter for the K-bZIP transcripts.

Fig. 3.
  • Open in new tab
  • Download powerpoint
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.

Splice variants of K-bZIP.RNase protection assays were also performed to verify the splicing pattern of K-bZIP as well as to quantify the relative amounts of the three types of transcripts. As illustrated in the schematic drawings in Fig. 3B, the RNA probe used in these assays encompasses the 3′ half of IVS1, all of exon II, and the 5′ half of IVS2. Therefore, a type III or unspliced message which retains both IVS1 and IVS2 would protect a fragment of 246 nucleotides, type II transcripts would protect a 150-nucleotide fragment, and type I transcripts would protect a 93-nucleotide fragment. When total RNAs from TPA-induced BCBL-1 cells were used, all three predicted fragments were detected, while the intensities of the bands were distinct (Fig. 3B). Band intensity was further quantified by storage phosphor screen analysis and corrected against the length of the protected fragment. The molar ratio of type I, II, and III transcripts was determined to be 16:4:1. These results confirm the coexistence of differentially spliced variants of K-bZIP in chemically induced BCBL-1 cells and the notion that type I transcripts represent the predominant species.

Expression kinetics of K-bZIP.To determine the stage of the viral life cycle in which KSHV K-bZIP was expressed, BCBL-1 cells were treated with TPA in combination with a protein synthesis inhibitor (cycloheximide) or a herpesvirus DNA polymerase inhibitor (PAA). Total RNAs from each sample were probed with a K-bZIP-specific DNA fragment. As shown in Fig. 4A, without treatment, there was little expression of this gene; the expression of the 1.35-kb K-bZIP transcript(s) began as early as 6 h, peaked at 24 h, and was maintained to 72 h after TPA treatment. The expression of the K-bZIP transcript(s) was blocked by cycloheximide but not by PAA, indicating that it is an early gene. As a control, a duplicate blot was hybridized with an ORF K8.1-specific probe (Fig.4B). The ORF K8.1 gene resides in the IVS3 region and shares sequences with exon IV of the ORF K8 gene. It encodes two immunogenic glycoproteins via differential splicing (9). Despite the close proximity of the ORF K8 and ORF K8.1 genes, the expression kinetics of these two genes were quite different. The synthesis of ORF K8.1 began much later, at 24 h, and peaked at 48 h after TPA treatment. Furthermore, the expression of the ORF K8.1 transcript was sensitive to PAA treatment. Thus, the ORF K8.1 gene is classified as a late gene. These results suggest that within this 2-kb gene complex reside two independently regulated promoters. In a Northern blot analysis with the K-bZIP probe, we noted two transcripts of about 4 kb. We determined these to be readthrough transcripts initiated at the promoter for ORF 50, the homologue of EBV BRLF1 (31) (data not shown). Interestingly, one of these transcripts was not detected by the ORF K8.1 probe, indicating that this transcript lacks all or a portion of IVS3.

Fig. 4.
  • Open in new tab
  • Download powerpoint
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).

K-bZIP forms homodimers in vivo.One characteristic of bZIP proteins is their ability to form homo- or heterodimers. As the first step in assessing the function of K-bZIP, we studied homodimer formation by K-bZIP. K-bZIP was differentially tagged with either the HA or the T7 epitope sequence at the N terminus. These two constructs were coexpressed in COS-1 cells. If homodimers were formed, coprecipitation of HA-K-bZIP and T7-K-bZIP would be expected. In the experiment shown in Fig. 5A, the cell extracts were first immunoprecipitated with T7 antibody, followed by Western blotting with HA antibody. HA antibody failed to detect K-bZIP in the T7 antibody immunoprecipitates of vector-transfected control cells, HA-K-bZIP-transfected cells, or T7-K-bZIP-transfected cells, attesting to the specificity of the antibody. An intense band corresponding to HA-K-bZIP was, however, readily detected in the T7 antibody immunoprecipitates of HA-K-bZIP- and T7-K-bZIP-cotransfected cells, suggesting that these two types of molecules form a complex. This conclusion was further reinforced by a reciprocal experiment in which HA antibody was used to immunoprecipitate the complex, followed by Western blotting to detect T7-K-bZIP (Fig. 5B). The above data provide strong evidence that K-bZIP forms homodimers.

Fig. 5.
  • Open in new tab
  • Download powerpoint
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.

While previous studies (e.g., 23) strongly implicated the leucine heptad repeats or the ZIP domain in dimer formation, we wished to confirm that this is the case for K-bZIP. We noted that the type II transcript encodes a protein, designated K-bZIPΔLZ, whose truncation point coincides with the start of the ZIP domain. K-bZIPΔLZ was tagged with the T7 epitope at the N terminus and coexpressed with HA-K-bZIP in COS-1 cells, followed by immunoprecipitation and Western blot analysis. In contrast to the results obtained with full-length K-bZIP (Fig. 5), K-bZIPΔLZ (Fig.6A, lane 5) failed to immunoprecipitate K-bZIP and vice versa (Fig. 6B, lane 3), suggesting the involvement of the ZIP domain in dimer formation.

Fig. 6.
  • Open in new tab
  • Download powerpoint
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.

DISCUSSION

In this report, we describe the identification and cloning of the gene for a novel KSHV protein with a prototypical bZIP structure, designated K-bZIP. The K-bZIP mRNA is generated through multiple in-frame splicings and thus was not recognized by direct examination of open reading frames in the genomic sequences. K-bZIP shows significant similarity to EBV BZLF1 (4, 31) with respect to genomic location, splicing pattern, and overall homology.

BZLF1 has been extensively characterized and is known to form homodimers in vivo and to recognize a group of sequences (BZLF1-responsive elements [ZREs]) that are related to the AP-1 consensus sequence and that are found in a number of EBV early promoters (16). The BZLF1 gene is also one of the master genes that, through binding to ZRE sequences, invokes the viral lytic cycle (12, 30, 31, 34). In addition, BZLF1 directly binds to the replication origin of the lytic cycle (OriL) and is essential for the synthesis of the concatemeric form of viral DNA for viral packaging (17).

We show here that K-bZIP also forms homodimers and, judging from its bZIP structure, likely will bind AP-1 or AP-1-like sequences. Experiments are in progress to determine the DNA binding specificity of K-bZIP dimers. While there are similarities between K-bZIP and BZLF1, there are also important differences. We note that the ZIP repeats of K-bZIP are much more extensive, including four leucines and one isoleucine, compared to those of BZLF1, which have only one leucine and one methionine. On the other hand, BZLF1 has a basic region with an arginine and lysine content much higher than that of K-bZIP. In addition, the DNA contact residues conserved in Jun, Fos, and BZLF1 are not well conserved in K-bZIP (Fig. 2) (23, 26, 46). K-bZIP homodimers therefore may recognize a DNA sequence motif distinct from conventional AP-1 or ZRE sequences.

Another interesting finding is the existence of differentially spliced variants of K-bZIP. Neither type II nor type III variants encode the ZIP domain, but they retain an intact N-terminal sequence, including the basic region. In the present study, it was found that the type I K-bZIP message is 4 times more abundant than the type II transcript and at least 15 times more abundant than the type III transcript. Whether proteins encoded by the type II and type III messages serve as modulators of the type I-encoded protein or themselves have independent functions remains to be ascertained. What we do know, based on the experiment shown in Fig. 6, is that type II proteins and, by the same token, type III proteins do not form dimers.

The induction kinetics, as well as the sensitivity toward inhibitors, suggest that the K-bZIP gene belongs to the class of early lytic genes. These results also parallel a report showing that the expression of BZLF1 was severely attenuated in the absence of de novo protein synthesis (20). In contrast, the ORF K8.1 gene, which is located within the third intron of K-bZIP and which shares the 3′ portion of exon IV, is regulated as a late gene. Thus, there is a complex regulatory mechanism of genes within this small transcriptional unit. In addition, a doublet (4.4- and 3.8-kb transcripts in Fig. 4A) and a single band (4.4-kb transcript in Fig. 4B) with a slower migration mobility were detected by K-bZIP- and ORF K8.1-specific probes, respectively. cDNA cloning of these transcripts demonstrated that they originate from Rta/ORF 50, the homologue of the EBV BRLF1 immediate-early protein (31) (data not shown). Indeed, similar bicistronic transcripts for both EBV BRLF1 and BZLF1 were identified and shown to be capable of translating both open reading frames in COS-1 cells (31). Furthermore, in a more synchronous induction system, the expression of monocistronic BZLF1 dramatically decreases at 8 h postinduction, while the bicistronic transcripts for both BRLF1 and BZLF1 are still detectable until 48 h postinduction (20). If a similar mechanism operates in KSHV, the translation of K-bZIP protein from the bicistronic or polycistronic RNAs, despite its low level, may bear significance. The lack of the 3.8-kb transcript in the Northern analysis when ORF K8.1 was used as a probe indicates that the ORF K8.1 gene is present only in the 4.4-kb message. The ORF K8.1 gene is located in the K-bZIP IVS3 (594 bp), an intron which is efficiently spliced in K-bZIP transcripts (RACE results and Fig. 3B). Since IVS3, where the ORF K8.1 coding sequence begins, is efficiently spliced out of K-bZIP transcripts, it is likely that the 3.8-kb transcript is derived from the 4.4-kb transcript by the removal of K-bZIP IVS3.

In summary, we have identified in the KSHV genome a gene for a bZIP protein and its splicing variants which lack the ZIP domain. We have also mapped the start and poly(A) sites of the transcriptional unit. The transcription of K-bZIP follows the kinetics of an early lytic gene. Functional analysis has revealed that K-bZIP is capable of forming a complex with itself. The transactivation or repression ability and the target genes of K-bZIP remain to be characterized.

ACKNOWLEDGMENTS

We thank K. Everiss for critical reading of the manuscript.

This work was supported by grants from the USDA (93-37024-9340), the NCI (CA46613), and the Council for Tobacco Research (4034).

FOOTNOTES

    • Received 4 September 1998.
    • Accepted 12 November 1998.
  • Copyright © 1999 American Society for Microbiology

REFERENCES

  1. 1.↵
    1. Aluigi M. G.,
    2. Albini A.,
    3. Carlone S.,
    4. Repetto L.,
    5. De Marchi R.,
    6. Icardi A.,
    7. Moro M.,
    8. Noonan D.,
    9. Benelli R.
    KSHV sequences in biopsies and cultured spindle cells of epidemic, iatrogenic and Mediterranean forms of Kaposi’s sarcoma.Res. Virol. 147 1996 267 275
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    1. Arvanitakis L.,
    2. Geras-Raaka E.,
    3. Varma A.,
    4. Gershengorn M. C.,
    5. Cesarman E.
    Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation.Nature 385 1997 347 350
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    1. Ausubel F. M.,
    2. Brent R.,
    3. Kingston R. E.,
    4. Moore D. D.,
    5. Seidman J. G.,
    6. Smith J. A.,
    7. Struhl K.
    Current protocols in molecular biology. 1995 John Wiley & Sons, Inc. New York, N.Y
  4. 4.↵
    1. Baer R.,
    2. Bankier A. T.,
    3. Biggin M. D.,
    4. Deininger P. L.,
    5. Farrell P. J.,
    6. Gibson T. J.,
    7. Hatfull G.,
    8. Hudson G. S.,
    9. Satchwell S. C.,
    10. Seguin C.,
    11. et al.
    DNA sequence and expression of the B95-8 Epstein-Barr virus genome.Nature 310 1984 207 211
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Bartkiewicz M.,
    2. Gold H.,
    3. Altman S.
    Identification and characterization of an RNA molecule that copurifies with RNase P activity from HeLa cells.Genes Dev. 3 1989 488 499
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Boshoff C.,
    2. Endo Y.,
    3. Collins P. D.,
    4. Takeuchi Y.,
    5. Reeves J. D.,
    6. Schweickart V. L.,
    7. Siani M. A.,
    8. Sasaki T.,
    9. Williams T. J.,
    10. Gray P. W.,
    11. Moore P. S.,
    12. Chang Y.,
    13. Weiss R. A.
    Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines.Science 278 1997 290 294
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Cesarman E.,
    2. Chang Y.,
    3. Moore P. S.,
    4. Said J. W.,
    5. Knowles D. M.
    Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.N. Engl. J. Med. 332 1995 1186 1191
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    1. Cesarman E.,
    2. Moore P. S.,
    3. Rao P. H.,
    4. Inghirami G.,
    5. Knowles D. M.,
    6. Chang Y.
    In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi’s sarcoma-associated herpesvirus-like (KSHV) DNA sequences.Blood 86 1995 2708 2714
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Chandran B.,
    2. Smith M. S.,
    3. Koelle D. M.,
    4. Corey L.,
    5. Horvat R.,
    6. Goldstein E.
    Reactivities of human sera with human herpesvirus-8-infected BCBL-1 cells and identification of HHV-8-specific proteins and glycoproteins and the encoding cDNAs.Virology 243 1998 208 217
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.↵
    1. Chang Y.,
    2. Cesarman E.,
    3. Pessin M. S.,
    4. Lee F.,
    5. Culpepper J.,
    6. Knowles D. M.,
    7. Moore P. S.
    Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma.Science 266 1994 1865 1869
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Chang Y.,
    2. Moore P. S.,
    3. Talbot S. J.,
    4. Boshoff C. H.,
    5. Zarkowska T.,
    6. Godden K.,
    7. Paterson H.,
    8. Weiss R. A.,
    9. Mittnacht S.
    Cyclin encoded by KS herpesvirus.Nature 382 1996 410 (Letter.)
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Chatila T.,
    2. Ho N.,
    3. Liu P.,
    4. Liu S.,
    5. Mosialos G.,
    6. Kieff E.,
    7. Speck S. H.
    The Epstein-Barr virus-induced Ca2+/calmodulin-dependent kinase type IV/Gr promotes a Ca2+-dependent switch from latency to viral replication.J. Virol. 71 1997 6560 6567
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Chevallier-Greco A.,
    2. Manet E.,
    3. Chavrier P.,
    4. Mosnier C.,
    5. Daillie J.,
    6. Sergeant A.
    Both Epstein-Barr virus (EBV)-encoded trans-acting factors, EB1 and EB2, are required to activate transcription from an EBV early promoter.EMBO J. 5 1986 3243 3249
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    1. Countryman J.,
    2. Miller G.
    Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment of heterogeneous viral DNA.Proc. Natl. Acad. Sci. USA 82 1985 4085 4089
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Ensoli B.,
    2. Barillari G.,
    3. Gallo R. C.
    Cytokines and growth factors in the pathogenesis of AIDS-associated Kaposi’s sarcoma.Immunol. Rev. 127 1992 147 155
    OpenUrlCrossRefPubMedWeb of Science
  16. 16.↵
    1. Farrell P. J.,
    2. Rowe D. T.,
    3. Rooney C. M.,
    4. Kouzarides T.
    Epstein-Barr virus BZLF1 trans-activator specifically binds to a consensus AP-1 site and is related to c-fos.EMBO J. 8 1989 127 132
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    1. Fixman E. D.,
    2. Hayward G. S.,
    3. Hayward S. D.
    Replication of Epstein-Barr virus oriLyt: lack of a dedicated virus-encoded origin-binding protein and dependence on Zta in cotransfection assays.J. Virol. 69 1995 2998 3006
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Flamand L.,
    2. Zeman R. A.,
    3. Bryant J. L.,
    4. Lunardi-Iskandar Y.,
    5. Gallo R. C.
    Absence of human herpesvirus 8 DNA sequences in neoplastic Kaposi’s sarcoma cell lines.J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 13 1996 194 197
    OpenUrlPubMedWeb of Science
  19. 19.↵
    1. Fleckenstein B.,
    2. Desrosiers R. C.
    Herpesvirus saimiri and herpesvirus ateles The herpesviruses Roizman B. 1 1982 253 332 Plenum Publishing Corp. New York, N.Y
    OpenUrl
  20. 20.↵
    1. Flemington E. K.,
    2. Goldfeld A. E.,
    3. Speck S. H.
    Efficient transcription of the Epstein-Barr virus immediate-early BZLF1 and BRLF1 genes requires protein synthesis.J. Virol. 65 1991 7073 7077
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Frohman M. A.,
    2. Dush M. K.,
    3. Martin G. R.
    Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.Proc. Natl. Acad. Sci. USA 85 1988 8998 9002
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Gao S. J.,
    2. Boshoff C.,
    3. Jayachandra S.,
    4. Weiss R. A.,
    5. Chang Y.,
    6. Moore P. S.
    KSHV ORF K9 (vIRF) is an oncogene which inhibits the interferon signaling pathway.Oncogene 15 1997 1979 1985
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    1. Glover J. N.,
    2. Harrison S. C.
    Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA.Nature 373 1995 257 261
    OpenUrlCrossRefPubMedWeb of Science
  24. 24.↵
    1. Gluzman Y.
    SV40-transformed simian cells support the replication of early SV40 mutants.Cell 23 1981 175 182
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    1. Grasso A. W.,
    2. Wen D.,
    3. Miller C. M.,
    4. Rhim J. S.,
    5. Pretlow T. G.,
    6. Kung H. J.
    ErbB kinases and NDF signaling in human prostate cancer cells.Oncogene 15 1997 2705 2716
    OpenUrlCrossRefPubMedWeb of Science
  26. 26.↵
    1. Kerppola T.,
    2. Curran T.
    Transcription. Zen and the art of Fos and Jun.Nature 373 1995 199 200
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Kledal T. N.,
    2. Rosenkilde M. M.,
    3. Coulin F.,
    4. Simmons G.,
    5. Johnsen A. H.,
    6. Alouani S.,
    7. Power C. A.,
    8. Luttichau H. R.,
    9. Gerstoft J.,
    10. Clapham P. R.,
    11. Clark-Lewis I.,
    12. Wells T. N. C.,
    13. Schwartz T. W.
    A broad-spectrum chemokine antagonist encoded by Kaposi’s sarcoma-associated herpesvirus.Science 277 1997 1656 1659
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Lagunoff M.,
    2. Ganem D.
    The structure and coding organization of the genomic termini of Kaposi’s sarcoma-associated herpesvirus.Virology 236 1997 147 154
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    1. Lee H.,
    2. Veazey R.,
    3. Williams K.,
    4. Li M.,
    5. Guo J.,
    6. Neipel F.,
    7. Fleckenstein B.,
    8. Lackner A.,
    9. Desrosiers R. C.,
    10. Jung J. U.
    Deregulation of cell growth by the K1 gene of Kaposi’s sarcoma-associated herpesvirus.Nat. Med. 4 1998 435 440
    OpenUrlCrossRefPubMedWeb of Science
  30. 30.↵
    1. Lieberman P. M.,
    2. Hardwick J. M.,
    3. Sample J.,
    4. Hayward G. S.,
    5. Hayward S. D.
    The Zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in target promoter and enhancer regions.J. Virol. 64 1990 1143 1155
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Manet E.,
    2. Gruffat H.,
    3. Trescol-Biemont M. C.,
    4. Moreno N.,
    5. Chambard P.,
    6. Giot J. F.,
    7. Sergeant A.
    Epstein-Barr virus bicistronic mRNAs generated by facultative splicing code for two transcriptional trans-activators.EMBO J. 8 1989 1819 1826
    OpenUrlPubMedWeb of Science
  32. 32.↵
    1. Miller G.
    Epstein-Barr virus: biology, pathogenesis, and medical aspects Fields virology Fields B. N., Knipe D. M. 2 1990 1921 1958 Raven Press, Ltd. New York, N.Y
    OpenUrl
  33. 33.↵
    1. Miller G.,
    2. Heston L.,
    3. Grogan E.,
    4. Gradoville L.,
    5. Rigsby M.,
    6. Sun R.,
    7. Shedd D.,
    8. Kushnaryov V. M.,
    9. Grossberg S.,
    10. Chang Y.
    Selective switch between latency and lytic replication of Kaposi’s sarcoma herpesvirus and Epstein-Barr virus in dually infected body cavity lymphoma cells.J. Virol. 71 1997 314 324
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Miller G.,
    2. Rabson M.,
    3. Heston L.
    Epstein-Barr virus with heterogeneous DNA disrupts latency.J. Virol. 50 1984 174 182
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Moore P. S.,
    2. Boshoff C.,
    3. Weiss R. A.,
    4. Chang Y.
    Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV.Science 274 1996 1739 1744
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Moore P. S.,
    2. Gao S. J.,
    3. Dominguez G.,
    4. Cesarman E.,
    5. Lungu O.,
    6. Knowles D. M.,
    7. Garber R.,
    8. Pellett P. E.,
    9. McGeoch D. J.,
    10. Chang Y.
    Primary characterization of a herpesvirus agent associated with Kaposi’s sarcoma.J. Virol. 70 1996 549 558
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    1. Muralidhar S.,
    2. Pumfrey A. M.,
    3. Hassani M.,
    4. Sadaie M. R.,
    5. Azumi N.,
    6. Kishishita M.,
    7. Brady J. N.,
    8. Doniger J.,
    9. Medveczky P.,
    10. Rosenthal L. J.
    Identification of kaposin (open reading frame K12) as a human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus) transforming gene.J. Virol. 72 1998 4980 4988
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    1. Neipel F.,
    2. Albrecht J. C.,
    3. Fleckenstein B.
    Cell-homologous genes in the Kaposi’s sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity? J. Virol. 71 1997 4187 4192
    OpenUrlFREE Full Text
  39. 39.↵
    1. Nicholas J.,
    2. Coles L. S.,
    3. Newman C.,
    4. Honess R. W.
    Regulation of the herpesvirus saimiri (HVS) delayed-early 110-kilodalton promoter by HVS immediate-early gene products and a homolog of the Epstein-Barr virus R trans activator.J. Virol. 65 1991 2457 2466
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    1. Nicholas J.,
    2. Ruvolo V. R.,
    3. Burns W. H.,
    4. Sandford G.,
    5. Wan X.,
    6. Ciufo D.,
    7. Hendrickson S. B.,
    8. Guo H. G.,
    9. Hayward G. S.,
    10. Reitz M. S.
    Kaposi’s sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6.Nat. Med. 3 1997 287 292
    OpenUrlCrossRefPubMedWeb of Science
  41. 41.↵
    1. Renne R.,
    2. Zhong W.,
    3. Herndier B.,
    4. McGrath M.,
    5. Abbey N.,
    6. Kedes D.,
    7. Ganem D.
    Lytic growth of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) in culture.Nat. Med. 2 1996 342 346
    OpenUrlCrossRefPubMedWeb of Science
  42. 42.↵
    1. Russo J. J.,
    2. Bohenzky R. A.,
    3. Chien M. C.,
    4. Chen J.,
    5. Yan M.,
    6. Maddalena D.,
    7. Parry J. P.,
    8. Peruzzi D.,
    9. Edelman I. S.,
    10. Chang Y.,
    11. Moore P. S.
    Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).Proc. Natl. Acad. Sci. USA 93 1996 14862 14867
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Sarid R.,
    2. Flore O.,
    3. Bohenzky R. A.,
    4. Chang Y.,
    5. Moore P. S.
    Transcription mapping of the Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) genome in a body cavity-based lymphoma cell line (BC-1).J. Virol. 72 1998 1005 1012
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Sharp P. A.
    Split genes and RNA splicing.Cell 77 1994 805 815
    OpenUrlCrossRefPubMedWeb of Science
  45. 45.↵
    1. Sonnhammer E. L.,
    2. Eddy S. R.,
    3. Durbin R.
    Pfam: a comprehensive database of protein domain families based on seed alignments.Proteins 28 1997 405 420
    OpenUrlCrossRefPubMedWeb of Science
  46. 46.↵
    1. Taylor N.,
    2. Flemington E.,
    3. Kolman J. L.,
    4. Baumann R. P.,
    5. Speck S. H.,
    6. Miller G.
    ZEBRA and a Fos-GCN4 chimeric protein differ in their DNA-binding specificities for sites in the Epstein-Barr virus BZLF1 promoter.J. Virol. 65 1991 4033 4041
    OpenUrlAbstract/FREE Full Text
  47. 47.↵
    1. van Santen V. L.
    Characterization of a bovine herpesvirus 4 immediate-early RNA encoding a homolog of the Epstein-Barr virus R transactivator.J. Virol. 67 1993 773 784
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    1. Whitehouse A.,
    2. Cooper M.,
    3. Meredith D. M.
    The immediate-early gene product encoded by open reading frame 57 of herpesvirus saimiri modulates gene expression at a posttranscriptional level.J. Virol. 72 1998 857 861
    OpenUrlAbstract/FREE Full Text
  49. 49.↵
    1. Zalani S.,
    2. Holley-Guthrie E.,
    3. Kenney S.
    Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.Proc. Natl. Acad. Sci. USA 93 1996 9194 9199
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Kaposi’s Sarcoma-Associated Herpesvirus Encodes a bZIP Protein with Homology to BZLF1 of Epstein-Barr Virus
Su-Fang Lin, Dan R. Robinson, George Miller, Hsing-Jien Kung
Journal of Virology Mar 1999, 73 (3) 1909-1917; DOI: 10.1128/JVI.73.3.1909-1917.1999

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Virology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Kaposi’s Sarcoma-Associated Herpesvirus Encodes a bZIP Protein with Homology to BZLF1 of Epstein-Barr Virus
(Your Name) has forwarded a page to you from Journal of Virology
(Your Name) thought you would be interested in this article in Journal of Virology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Kaposi’s Sarcoma-Associated Herpesvirus Encodes a bZIP Protein with Homology to BZLF1 of Epstein-Barr Virus
Su-Fang Lin, Dan R. Robinson, George Miller, Hsing-Jien Kung
Journal of Virology Mar 1999, 73 (3) 1909-1917; DOI: 10.1128/JVI.73.3.1909-1917.1999
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

DNA-binding proteins
Herpesvirus 8, Human
Trans-Activators
transcription factors
Viral Proteins

Related Articles

Cited By...

About

  • About JVI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jvirology

@ASMicrobiology

       

 

JVI in collaboration with

American Society for Virology

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0022-538X; Online ISSN: 1098-5514