Role of Defective Oct-2 and OCA-B Expression in Immunoglobulin Production and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation in Primary Effusion Lymphoma

ABSTRACT Primary effusion lymphoma (PEL) is a distinct type of B-cell non-Hodgkin lymphoma characterized by the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8). Despite having a genotype and gene expression signature of highly differentiated B cells, PEL does not usually express surface or cytoplasmic immunoglobulin (Ig). We show the lack of Oct-2 and OCA-B transcription factors to be responsible, at least in part, for this defect in Ig production. Like Ig genes, ORF50, the key regulator of the switch from latency to lytic reactivation, contains an octamer motif within its promoter. We therefore examined the impact of Oct-2 and OCA-B on ORF50 activation. The binding of Oct-1 to the ORF50 promoter has been shown to significantly enhance ORF50 transactivation. We found that Oct-2, on the other hand, inhibited ORF50 expression and consequently lytic reactivation by competing with Oct-1 for the octamer motif in the ORF50 promoter. Our data suggest that Oct-2 downregulation in infected cells would be favorable to KSHV in allowing for efficient viral reactivation.

Download Full-text

Suppression of Tetradecanoyl Phorbol Acetate-Induced Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus by K1 Signal Transduction

Journal of Virology ◽

10.1128/jvi.76.23.12185-12199.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 12185-12199 ◽

Cited By ~ 28

Author(s):

Bok-Soo Lee ◽

Mini Paulose-Murphy ◽

Young-Hwa Chung ◽

Michelle Connlole ◽

Steven Zeichner ◽

...

Keyword(s):

Signal Transduction ◽

Kaposi's Sarcoma ◽

Ectopic Expression ◽

Kaposi’S Sarcoma ◽

Viral Reactivation ◽

Dependent Manner ◽

Tetradecanoyl Phorbol Acetate ◽

Lytic Reactivation ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region and elicits cellular signal transduction through this motif. To investigate the role of K1 signal transduction in KSHV replication, we expressed full-length K1 and CD8-K1 chimeras in BCBL1 cells. Unlike its strong signaling activity in uninfected B lymphocytes, K1 did not induce intracellular calcium mobilization or NF-AT activation at detectable levels in KSHV-infected BCBL1 cells. Instead, K1 signaling dramatically suppressed KSHV lytic reactivation induced by tetradecanoyl phorbol acetate (TPA) stimulation, but not by ORF50 ectopic expression. Mutational analysis showed that the cytoplasmic ITAM sequence of K1 was required for this suppression. Viral microarray and immunoblot analyses demonstrated that K1 signaling suppressed the TPA-mediated increase in the expression of a large subset of viral lytic genes in KSHV-infected BCBL1 cells. Furthermore, electrophoretic mobility shift assays demonstrated that TPA-induced activation of AP-1, NF-κB, and Oct-1 activities was severely diminished in BCBL1 cells expressing the K1 cytoplasmic domain. The reduced activities of these transcription factors may confer the observed reduction in viral lytic gene expression. These results demonstrate that K1-mediated signal transduction in KSHV-infected cells is profoundly different from that in KSHV-negative cells. Furthermore, K1 signal transduction efficiently suppresses TPA-mediated viral reactivation in an ITAM-dependent manner, and this suppression may contribute to the establishment and/or maintenance of KSHV latency in vivo.

Download Full-text

The Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor Has Broad Signaling Effects in Primary Effusion Lymphoma Cells

Journal of Virology ◽

10.1128/jvi.77.1.57-67.2003 ◽

2003 ◽

Vol 77 (1) ◽

pp. 57-67 ◽

Cited By ~ 89

Author(s):

Mark Cannon ◽

Nicola J. Philpott ◽

Ethel Cesarman

Keyword(s):

G Protein ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Cell Cycle Control ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

G Protein Coupled Receptor ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

G Protein Coupled

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV-8]) is a gamma-2-herpesvirus responsible for Kaposi's sarcoma as well as primary effusion lymphoma (PEL). KSHV is a lymphotropic virus that has pirated many mammalian genes involved in inflammation, cell cycle control, and angiogenesis. Among these is the early lytic viral G protein-coupled receptor (vGPCR), a homologue of the human interleukin-8 (IL-8) receptor. When expressed, vGPCR is constitutively active and can signal via mitogen- and stress-activated kinases. In certain models it activates the transcriptional potential of NF-κB and activator protein 1 (AP-1) and induces vascular endothelial growth factor (VEGF) production. Despite its importance to the pathogenesis of all KSHV-mediated disease, little is known about vGPCR activity in hematopoietic cells. To study the signaling potential and downstream effects of vGPCR in such cells, we have developed PEL cell lines that express vGPCR under the control of an inducible promoter. The sequences required for tetracycline-mediated induction were cloned into a plasmid containing adeno-associated virus type 2 elements to enhance integration efficiency. This novel plasmid permitted studies of vGPCR activity in naturally infected KSHV-positive lymphocytes. We show that vGPCR activates ERK-2 and p38 in PEL cells. In addition, it increases the transcription of reporter genes under the control of AP-1, NF-κB, CREB, and NFAT, a Ca2+-dependent transcription factor important to KSHV lytic gene expression. vGPCR also increases the transcription of KSHV open reading frames 50 and 57, thereby displaying broad potential to affect viral transcription patterns. Finally, vGPCR signaling results in increased PEL cell elaboration of KSHV vIL-6 and VEGF, two growth factors involved in KSHV-mediated disease pathogenesis.

Download Full-text

Angiogenesis and Hematopoiesis Induced by Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Interleukin-6

Blood ◽

10.1182/blood.v93.12.4034.412k38_4034_4043 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4034-4043 ◽

Cited By ~ 18

Author(s):

Yoshiyasu Aoki ◽

Elaine S. Jaffe ◽

Yuan Chang ◽

Karen Jones ◽

Julie Teruya-Feldstein ◽

...

Keyword(s):

Lymph Nodes ◽

Interleukin 6 ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Biological Activities ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nih3t3 Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]) is a herpesvirus linked to the development of Kaposi’s sarcoma (KS), primary effusion lymphoma, and a proportion of Castleman’s disease. KSHV encodes viral interleukin-6 (vIL-6), which is structurally homologous to human and murine IL-6. The biological activities of vIL-6 are largely unknown. To gain insight into the biology of vIL-6, we expressed vIL-6 in murine fibroblasts NIH3T3 cells and inoculated stable vIL-6–producing clones into athymic mice. vIL-6 was detected selectively in the blood of mice injected with vIL-6–expressing clones. Compared with controls, vIL-6–positive mice displayed increased hematopoiesis in the myeloid, erythroid, and megakaryocytic lineages; plasmacytosis in spleen and lymph nodes; hepatosplenomegaly; and polyclonal hypergammaglobulinemia. vIL-6–expressing NIH3T3 cells gave rise to tumors more rapidly than did control cells, and vIL-6–positive tumors were more vascularized than controls. Vascular endothelial growth factor (VEGF) was detected at higher levels in the culture supernatant of vIL-6–expressing cells compared with controls, and immunohistochemical staining detected VEGF in spleen, lymph nodes, and tumor tissues from mice bearing vIL-6–producing tumors but not control tumors. Thus, vIL-6 is a multifunctional cytokine that promotes hematopoiesis, plasmacytosis, and angiogenesis. Through these functions, vIL-6 may play an important role in the pathogenesis of certain KSHV-associated disorders.

Download Full-text

Kaposi's Sarcoma-Associated Herpesvirus LANA2 Is a B-Cell-Specific Latent Viral Protein That Inhibits p53

Journal of Virology ◽

10.1128/jvi.75.1.429-438.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 429-438 ◽

Cited By ~ 213

Author(s):

Carmen Rivas ◽

Ai-En Thlick ◽

Carlo Parravicini ◽

Patrick S. Moore ◽

Yuan Chang

Keyword(s):

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Blot Hybridization ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

P53 Overexpression ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is associated with three proliferative diseases ranging from viral cytokine-induced hyperplasia to monoclonal neoplasia: multicentric Castleman's disease (CD), Kaposi's sarcoma (KS), and primary effusion lymphoma (PEL). Here we report a new latency-associated 1,704-bp KSHV spliced gene belonging to a cluster of KSHV sequences having homology to the interferon regulatory factor (IRF) family of transcription factors. ORFK10.5 encodes a protein, latency-associated nuclear antigen 2 (LANA2), which is expressed in KSHV-infected hematopoietic tissues, including PEL and CD but not KS lesions. LANA2 is abundantly expressed in the nuclei of cultured KSHV-infected B cells. Transcription of K10.5 in PEL cell cultures is not inhibited by DNA polymerase inhibitors nor significantly induced by phorbol ester treatment. Unlike LANA1, LANA2 does not elicit a serologic response from patients with KS, PEL, or CD as measured by Western blot hybridization. Both KSHV vIRF1 (ORFK9) and LANA2 (ORFK10.5) appear to have arisen through gene duplication of a captured cellular IRF gene. LANA2 is a potent inhibitor of p53-induced transcription in reporter assays. LANA2 antagonizes apoptosis due to p53 overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicin treatment of wild-type p53 U2OS cells. While LANA2 specifically interacts with amino acids 290 to 393 of p53 in glutathione S-transferase pull-down assays, we were unable to demonstrate LANA2-p53 interaction in vivo by immunoprecipitation. These findings show that KSHV has tissue-specific latent gene expression programs and identify a new latent protein which may contribute to KSHV tumorigenesis in hematopoietic tissues via p53 inhibition.

Download Full-text

Kaposi's Sarcoma-Associated Herpesvirus Latent and Lytic Gene Expression as Revealed by DNA Arrays

Journal of Virology ◽

10.1128/jvi.75.2.891-902.2001 ◽

2001 ◽

Vol 75 (2) ◽

pp. 891-902 ◽

Cited By ~ 271

Author(s):

Richard G. Jenner ◽

M. Mar Albà ◽

Chris Boshoff ◽

Paul Kellam

Keyword(s):

Gene Expression ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Dna Arrays ◽

Nylon Membrane ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is associated with three human tumors, Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. KSHV encodes a number of homologs of cellular proteins involved in the cell cycle, signal transduction, and modulation of the host immune response. Of the virus complement of over 85 open reading frames (ORFs), the expression of only a minority has been characterized individually. We have constructed a nylon membrane-based DNA array which allows the expression of almost every ORF of KSHV to be measured simultaneously. A PEL-derived cell line, BC-3, was used to study the expression of KSHV during latency and after the induction of lytic replication. Cluster analysis, which arranges genes according to their expression profile, revealed a correlation between expression and assigned gene function that is consistent with the known stages of the herpesvirus life cycle. Furthermore, latent and lytic genes thought to be functionally related cluster into groups. The correlation between gene expression and function also infers possible roles for KSHV genes yet to be characterized.

Download Full-text

Expression and Subcellular Localization of the Kaposi's Sarcoma-Associated Herpesvirus K15P Protein during Latency and Lytic Reactivation in Primary Effusion Lymphoma Cells

Journal of Virology ◽

10.1128/jvi.01370-17 ◽

2017 ◽

Vol 91 (21) ◽

Cited By ~ 2

Author(s):

Caitlin G. Smith ◽

Himanshu Kharkwal ◽

Duncan W. Wilson

Keyword(s):

Molecular Weight ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Kaposi’S Sarcoma ◽

Full Length ◽

Transcription Activator ◽

Lytic Reactivation ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Different Molecular Weight

ABSTRACT The K15P membrane protein of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with multiple cellular signaling pathways and is thought to play key roles in KSHV-associated endothelial cell angiogenesis, regulation of B-cell receptor (BCR) signaling, and the survival, activation, and proliferation of BCR-negative primary effusion lymphoma (PEL) cells. Although full-length K15P is ∼45 kDa, numerous lower-molecular-weight forms of the protein exist as a result of differential splicing and poorly characterized posttranslational processing. K15P has been reported to localize to numerous subcellular organelles in heterologous expression studies, but there are limited data concerning the sorting of K15P in KSHV-infected cells. The relationships between the various molecular weight forms of K15P, their subcellular distribution, and how these may differ in latent and lytic KSHV infections are poorly understood. Here we report that a cDNA encoding a full-length, ∼45-kDa K15P reporter protein is expressed as an ∼23- to 24-kDa species that colocalizes with the trans-Golgi network (TGN) marker TGN46 in KSHV-infected PEL cells. Following lytic reactivation by sodium butyrate, the levels of the ∼23- to 24-kDa protein diminish, and the full-length, ∼45-kDa K15P protein accumulates. This is accompanied by apparent fragmentation of the TGN and redistribution of K15P to a dispersed peripheral location. Similar results were seen when lytic reactivation was stimulated by the KSHV protein replication and transcription activator (RTA) and during spontaneous reactivation. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the latent and lytic phases. IMPORTANCE The K15P protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to play key roles in disease, including KSHV-associated angiogenesis and the survival and growth of primary effusion lymphoma (PEL) cells. The protein exists in multiple molecular weight forms, and its intracellular trafficking is poorly understood. Here we demonstrate that the molecular weight form of a reporter K15P molecule and its intracellular distribution change when KSHV switches from its latent (quiescent) phase to the lytic, infectious state. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the viral latent and lytic stages.

Download Full-text

Angiogenesis and Hematopoiesis Induced by Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Interleukin-6

Blood ◽

10.1182/blood.v93.12.4034 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4034-4043 ◽

Cited By ~ 298

Author(s):

Yoshiyasu Aoki ◽

Elaine S. Jaffe ◽

Yuan Chang ◽

Karen Jones ◽

Julie Teruya-Feldstein ◽

...

Keyword(s):

Lymph Nodes ◽

Interleukin 6 ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Biological Activities ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nih3t3 Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]) is a herpesvirus linked to the development of Kaposi’s sarcoma (KS), primary effusion lymphoma, and a proportion of Castleman’s disease. KSHV encodes viral interleukin-6 (vIL-6), which is structurally homologous to human and murine IL-6. The biological activities of vIL-6 are largely unknown. To gain insight into the biology of vIL-6, we expressed vIL-6 in murine fibroblasts NIH3T3 cells and inoculated stable vIL-6–producing clones into athymic mice. vIL-6 was detected selectively in the blood of mice injected with vIL-6–expressing clones. Compared with controls, vIL-6–positive mice displayed increased hematopoiesis in the myeloid, erythroid, and megakaryocytic lineages; plasmacytosis in spleen and lymph nodes; hepatosplenomegaly; and polyclonal hypergammaglobulinemia. vIL-6–expressing NIH3T3 cells gave rise to tumors more rapidly than did control cells, and vIL-6–positive tumors were more vascularized than controls. Vascular endothelial growth factor (VEGF) was detected at higher levels in the culture supernatant of vIL-6–expressing cells compared with controls, and immunohistochemical staining detected VEGF in spleen, lymph nodes, and tumor tissues from mice bearing vIL-6–producing tumors but not control tumors. Thus, vIL-6 is a multifunctional cytokine that promotes hematopoiesis, plasmacytosis, and angiogenesis. Through these functions, vIL-6 may play an important role in the pathogenesis of certain KSHV-associated disorders.

Download Full-text

Functional characterization of the M-type K15-encoded membrane protein of Kaposi's sarcoma-associated herpesvirus

Journal of General Virology ◽

10.1099/vir.0.82807-0 ◽

2007 ◽

Vol 88 (6) ◽

pp. 1698-1707 ◽

Cited By ~ 21

Author(s):

Linding Wang ◽

Melanie M. Brinkmann ◽

Marcel Pietrek ◽

Matthias Ottinger ◽

Oliver Dittrich-Breiholz ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Target Genes ◽

Primary Effusion Lymphoma ◽

Functional Characterization ◽

Sequence Divergence ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Binding Motif ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and the plasma-cell variant of multicentric Castleman's disease. Its alternatively spliced K15 gene encodes several membrane proteins with varying numbers of transmembrane domains. Two highly diverged alleles of the K15 gene, termed predominant (P) and minor (M), exist and share only 33 % amino acid identity with one another, but retain conserved putative src homology (SH) 2- and SH3-binding motifs. K15-M is thought to have entered the KSHV genome as the result of recombination with a related γ 2-herpesvirus. The more common K15-P allele has been shown to activate the mitogen-activated protein kinases Erk2 and JNK1 and the nuclear factor κB (NF-κB) pathway. To explore possible functional differences between K15-P and K15-M that might have influenced their spread in the KSHV population, here, the ability of the M form of K15 to activate these pathways was investigated. Similarly to K15-P, K15-M induces the activation of the Erk2 and JNK1 kinases, the NF-κB transcription factor and the expression of a similar range of cellular inflammatory genes, as assessed by gene-expression microarray studies and reporter assays. In epithelial cells, the activation of most K15-M target genes is impaired by mutagenesis of Y490 in its SH2-binding motif Y490EEV, although this motif appears less important in endothelial cells. Therefore, K15-M and K15-P can trigger similar intracellular signalling pathways, despite their extensive sequence divergence.

Download Full-text

Spectrum of Kaposi's Sarcoma-Associated Herpesvirus, or Human Herpesvirus 8, Diseases

Clinical Microbiology Reviews ◽

10.1128/cmr.15.3.439-464.2002 ◽

2002 ◽

Vol 15 (3) ◽

pp. 439-464 ◽

Cited By ~ 197

Author(s):

Dharam V. Ablashi ◽

Louise G. Chatlynne ◽

James E. Whitman, ◽

Ethel Cesarman

Keyword(s):

Dna Sequences ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

B Cell Lymphoma ◽

Human Herpesvirus 8 ◽

Herpesvirus 8 ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

SUMMARY Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), discovered in 1994, is a human rhadinovirus (gamma-2 herpesvirus). Unlike other human herpesviruses (herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, cytomegalovirus, HHV-6, and HHV-7), it is not widespread in the general population and has many unique proteins. HHV-8 is strongly associated with all subtypes of Kaposi's sarcoma (KS), multicentric Castleman's disease, and a rare form of B-cell lymphoma, primary effusion lymphoma. In addition, HHV-8 DNA sequences have been found in association with other diseases, but the role of the virus in these diseases is largely unconfirmed and remains controversial. The seroprevalence of HHV-8, based on detection of latent and lytic proteins, is 2 to 5% in healthy donors except in certain geographic areas where the virus is endemic, 80 to 95% in classic KS patients, and 40 to 50% in HIV-1 patients without KS. This virus can be transmitted both sexually and through body fluids (e.g., saliva and blood). HHV-8 is a transforming virus, as evidenced by its presence in human malignancies, by the in vitro transforming properties of several of its viral genes, and by its ability to transform some primary cells in culture. It is not, however, sufficient for transformation, and other cofactors such as immunosuppressive cytokines are involved in the development of HHV-8-associated malignancies. In this article, we review the biology, molecular virology, epidemiology, transmission, detection methods, pathogenesis, and antiviral therapy of this newly discovered human herpesvirus.

Download Full-text

Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Transcriptional Activator Rta Is an Oligomeric DNA-Binding Protein That Interacts with Tandem Arrays of Phased A/T-Trinucleotide Motifs

Journal of Virology ◽

10.1128/jvi.77.17.9399-9411.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9399-9411 ◽

Cited By ~ 32

Author(s):

Wei Liao ◽

Yong Tang ◽

Yu-liang Kuo ◽

Bao-Ying Liu ◽

Chi-Jie Xu ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Transcriptional Activator ◽

Kaposi’S Sarcoma ◽

Viral Reactivation ◽

Human Herpesvirus 8 ◽

Dependent Manner ◽

Herpesvirus 8 ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) encodes an immediate early transcriptional activator, Rta, which mediates viral reactivation from latency and lytic viral replication. Here we report the purification and characterizations of HHV-8 Rta and its interaction with Rta-responsive DNA elements. The Rta response element (RtaRE) in the promoter of the KSHV/HHV-8 K8 open reading frame was mapped to a 47-bp sequence (RtaRE1) and a 60-bp sequence (RtaRE2) upstream of the TATA motif. A comparison of the K8 RtaREs with other viral RtaREs revealed a pattern of multiple A/T triplets spaced with a periodicity of 10 or 20 bp. Substitutions of the in-phase A/T trinucleotides of the RtaRE1 with G/C bases greatly diminished Rta responsiveness and Rta binding. By contrast, base substitutions in an out-of-phase A/T-trinucleotide sequence had no effect. Importantly, multimers of (A/T)3N7 and N5(A/T)5N6(A/T)4 motifs supported a strong Rta response in a copy number-dependent manner. No specific sequence motifs in the spacer regions could be discerned. Potent Rta response, however, was obtained with phased A/T trinucleotides with 7-bp spacers of arbitrary sequences with high G/C content. Lengthening of the phased A/T motifs or lowering of the G/C content of the spacers resulted in a reduction in Rta response. Finally, Escherichia coli-derived Rta is an oligomer of 440 kDa in molecular size and binds RtaRE as an oligomer. These results support a model of Rta transactivation wherein the subunits of the Rta oligomer make multiple contacts with a tandem array of phased A/T triplets in the configuration of (A/T)3(G/C)7 repeats.

Download Full-text