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PubMed2003

The latency-associated nuclear antigen homolog of herpesvirus saimiri inhibits lytic virus replication.

Herpesvirus saimiri (HVS), a T-lymphotropic tumor virus of neotropical primates, and the Kaposi's sarcoma-associated human herpesvirus 8 (KSHV) belong to the gamma-(2)-herpesvirus (Rhadinovirus) subfamily and share numerous features of genome structure and organization. The KSHV latency-associated nuclear antigen (LANA) protein appears to be relevant for viral persistence, latency, and transformation. It binds to DNA, colocalizes with viral episomal DNA, and presumably mediates efficient persistence of viral genomes. LANA further represses the transcriptional and proapoptotic activities of the p53 tumor suppressor protein. Here we report on the ORF73 gene of HVS strain C488, which is the positional and structural homolog of KSHV LANA. The ORF73 gene in OMK cells can encode a 62-kDa protein that localizes to the nucleus in a pattern similar to that of LANA. We show that the ORF73 gene product can regulate viral gene expression by acting as a transcriptional modulator of latent and lytic viral promoters. To define the HVS ORF73 function in the background of a replication-competent virus, we constructed a viral mutant that expresses ORF73 under the transcriptional control of a mifepristone (RU-486)-inducible promoter. The HVS ORF73 gene product efficiently suppresses lytic viral replication in permissive cells, indicating that it defines a critical control point between viral persistence and lytic replication.

Animals↗
PubMed2001

Transcription activation of polyadenylated nuclear rna by rta in human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus.

Human herpesvirus 8 (HHV-8) (also known as Kaposi's sarcoma-associated herpesvirus) encodes a novel noncoding polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) during the early phase of lytic replication. PAN RNA is the most abundant transcript of HHV-8, comprising 80% of total poly(A)-selected transcripts in HHV-8-infected cells during lytic replication. We directly measured the abundance of PAN RNA by visualizing 1.1- to 1.2- kb PAN RNA in an ethidium bromide-stained gel from poly(A)-selected RNA. We further pursued the mechanisms by which PAN RNA expression is induced to such high levels. rta, an immediate-early gene of HHV-8, is a transactivator that is sufficient and necessary to activate lytic gene expression in latently infected cells. Ectopic expression of Rta was previously shown to induce PAN RNA expression from the endogenous viral genome and activate the PAN promoter in a reporter system. Here, we have identified the Rta-responsive element (RRE) in the PAN promoter. Deletion analysis revealed that the RRE is present in a region between nucleotides -69 and -38 of the PAN promoter. A promoter construct containing the 69 nucleotides upstream of the transcription start site of the PAN promoter was activated by Rta in the absence or presence of the HHV-8 genome. Rta activated the PAN promoter up to 7,000-fold in 293T cells and 2,000-fold in B cells. Electrophoretic mobility shift assays demonstrated that Rta formed a highly stable complex with the RRE of the PAN promoter. Our study suggests that Rta can induce PAN RNA expression by direct binding of Rta to the RRE of the PAN promoter. This study has highlighted an important mechanism controlling PAN RNA expression and also provides a model system for investigating how Rta transactivates gene expression during lytic replication.

Animals↗
PubMed2006

Intracellular Tat of human immunodeficiency virus type 1 activates lytic cycle replication of Kaposi's sarcoma-associated herpesvirus: role of JAK/STAT signaling.

Human immunodeficiency virus type 1 (HIV-1) infection significantly increases the risk of Kaposi's sarcoma (KS) occurrence in individuals infected with Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV infection appears to be necessary but not sufficient for KS development without other cofactors. However, factors that facilitate KSHV to cause KS have not been well defined. Previously, we determined that human herpesvirus 6 was one of the cofactors that activated lytic cycle replication of KSHV. Here, we demonstrate that the Tat protein of HIV-1 is a potentially important factor in the pathogenesis of KS, as determined by production of lytic phase mRNA transcripts and viral proteins in BCBL-1 cells. Mechanistic studies showed ectopic expression of Tat induced the production of human interleukin-6 (huIL-6) and its receptor (huIL-6Ra) and activated STAT3 signaling. Neutralization of huIL-6 or huIL-6R or inhibition of STAT3 signaling enhanced the replication. In addition, IL-4/STAT6 signaling also partially contributed to Tat-induced KSHV replication. These findings suggest that Tat may participate in KS pathogenesis by inducing KSHV replication and increasing KSHV viral load. These data also suggest that JAK/STAT signaling may be of therapeutic value in AIDS-related KS patients.

Animals↗
PubMed2004

Requirement of a 12-base-pair TATT-containing sequence and viral lytic DNA replication in activation of the Kaposi's sarcoma-associated herpesvirus K8.1 late promoter.

Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 late promoter consists of a minimal 24-bp sequence, with a TATA-like, 12-bp promoter core, AATATTAAAGGG, and is active on a reporter only in butyrate-induced KSHV-infected cells. The activity of the K8.1 promoter can be enhanced (>15-fold) by the KSHV left-end lytic origin of DNA replication (oriLyt-L) sequence while providing inefficient replication of plasmid DNA and is inhibited by viral DNA replication inhibitors, suggesting that activation of the K8.1 promoter on the reporter is involved in KSHV lytic DNA replication largely by trans.

Base Sequence↗
PubMed2006

Asynchronous progression through the lytic cascade and variations in intracellular viral loads revealed by high-throughput single-cell analysis of Kaposi's sarcoma-associated herpesvirus infection.

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus-8) is frequently tumorigenic in immunocompromised patients. The average intracellular viral copy number within infected cells, however, varies markedly by tumor type. Since the KSHV-encoded latency-associated nuclear antigen (LANA) tethers viral episomes to host heterochromatin and displays a punctate pattern by fluorescence microscopy, we investigated whether accurate quantification of individual LANA dots is predictive of intracellular viral genome load. Using a novel technology that integrates single-cell imaging with flow cytometry, we found that both the number and the summed immunofluorescence of individual LANA dots are directly proportional to the amount of intracellular viral DNA. Moreover, combining viral (immediate early lytic replication and transcription activator [RTA] and late lytic K8.1) and cellular (syndecan-1) staining with image-based flow cytometry, we were also able to rapidly and simultaneously distinguish among cells supporting latent, immediate early lytic, early lytic, late lytic, and a potential fourth "delayed late" category of lytic replication. Applying image-based flow cytometry to KSHV culture models, we found that de novo infection results in highly varied levels of intracellular viral load and that lytic induction of latently infected cells likewise leads to a heterogeneous population at various stages of reactivation. These findings additionally underscore the potential advantages of studying KSHV biology with high-throughput analysis of individual cells.

Antigens, Viral↗
PubMed1985

Lytic cytomegalovirus replication and the hormones of human pregnancy.

The frequency of human cytomegalovirus (CMV) excretion during pregnancy denoting active infection has been demonstrated to increase as gestation advances and at term involves a significant percentage of women. This increase enhances the risk of congenital infection of the fetus. Thus it appears that some factor(s) unique to the condition of pregnancy favors susceptibility to maternal CMV infection. We designed our studies to investigate the possible association of the continuously rising levels of selected hormones and this increased susceptibility. Progesterone, 17 beta-estradiol, and cortisol were added to tissue culture media in final concentrations to match those occurring in term pregnancy serum. Two strains of human foreskin cells, one neonatal and the other fetal, were treated with either single or paired combinations of hormone-containing media. Lytic CMV replication in neonatal foreskin cells was enhanced by a maximum of 5.7-fold when these cells were treated with cortisol. Such enhancement did not occur in the fetal cells. No synergistic effects were seen when cortisol was used in combination with other hormones nor when neonatal foreskin cells were replicated for at least three generations in either single or paired combinations of hormones prior to use. Differential hormonal enhancement of CMV replication in vitro suggests a possible mechanism for the increased incidence of CMV infection observed during human pregnancy.

Cells, Cultured↗
PubMed2004

Murine gammaherpesvirus 68 open reading frame 31 is required for viral replication.

Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta- and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein. The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MHV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.

Animals↗
PubMed2006

Modulation of Kaposi's sarcoma-associated herpesvirus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen-activated protein kinase pathways during primary infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma, a dominant AIDS-related tumor of endothelial cells, and several other lymphoproliferative malignancies. While activation of the phosphatidylinositol 3-kinase-protein kinase C-MEK-ERK pathway is essential for KSHV infection, we have recently shown that KSHV also activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways during primary infection (J. Xie, H. Y. Pan, S. Yoo, and S.-J. Gao, J. Virol. 79:15027-15037, 2005). Here, we found that activation of both JNK and p38 pathways was also essential for KSHV infection. Inhibitors of all three MAPK pathways reduced KSHV infectivity in both human umbilical vein endothelial cells (HUVEC) and 293 cells. These inhibitory effects were dose dependent and occurred at the virus entry stage of infection. Consistently, inhibition of all three MAPK pathways with dominant-negative constructs reduced KSHV infectivity whereas activation of the ERK pathway but not the JNK and p38 pathways enhanced KSHV infectivity. Importantly, inhibition of all three MAPK pathways also reduced the yield of infectious virions during KSHV productive infection of HUVEC. While the reduction of infectious virions was in part due to the reduced infectivity, it was also the result of direct modulation of KSHV lytic replication by the MAPK pathways. Accordingly, KSHV upregulated the expression of RTA (Orf50), a master transactivator of KSHV lytic replication, and activated its promoter during primary infection. Furthermore, KSHV activation of RTA promoter during primary infection was modulated by all three MAPK pathways, predominantly through their downstream target AP-1. Together, these results indicate that, by modulating multiple MAPK pathways, KSHV manipulates the host cells to facilitate its entry into the cells and postentry productive lytic replication during primary infection.

Cells, Cultured↗
PubMed1999

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo.

B-Lymphocytes↗
PubMed2006

Short, discontinuous exposure to butyrate effectively sensitizes latently EBV-infected lymphoma cells to nucleoside analogue antiviral agents.

Antiviral drugs alone have been unsuccessful in the treatment of Epstein-Barr virus (EBV)-associated malignancies because the virus maintains a latent state of replication in these tumors. In recent years, novel therapeutic approaches are being investigated wherein lytic replication of the virus is induced prior to the use of cytotoxic antiviral drugs. The choice of suitable agents to induce lytic replication has been a critical step in this novel approach. We have previously demonstrated that butyrate derivatives induce a lytic pattern of EBV gene expression in patient-derived EBV-positive lymphoblastoid cell lines and, together with nucleoside analogue ganciclovir, effectively reduce or eliminate tumor growth in humans. Butyrate has drawbacks as a therapeutic agent, however, as constant intravenous infusion is required to achieve detectable plasma levels of this drug. In this study, we investigated whether discontinuous exposure to butyrate is capable of initiating lytic phase gene expression and thymidine kinase induction, and sensitizing EBV-positive lymphoma cells to ganciclovir-mediated cell growth arrest and apoptosis. We demonstrate that multiple daily 6-h exposures of the EBV-positive Burkitt's lymphoma cell line P3HR1 to butyrate induced sustained expression of the EBV lytic phase protein BMRF. Viral thymidine kinase was also induced by intermittent exposure, although to a lower level than with continuous exposure treatment. However, discontinuous exposure to butyrate in combination with ganciclovir induced a similar level of tumor cell growth inhibition as did continuous treatment, as measured by serial enumeration of viable cells, MTT cell proliferation assays and measurement of cellular DNA content. We further demonstrated that those cells which survived initial exposure to butyrate plus ganciclovir remained susceptible to further cycles of combination treatment. These findings suggests that continuous infusion of butyrate may not be necessary for maintaining viral thymidine kinase gene expression and sensitization to antiviral agents in EBV-associated tumors, and that therapeutic regimens which employ more convenient, discontinuous exposure to butyrate may also be effective clinically.

Antiviral Agents↗
PubMed2006

gamma-2 Herpes virus post-transcriptional gene regulation.

gamma-2 herpes viruses, which include Kaposi's sarcoma-associated herpes virus, are an important subfamily of herpes virus because of their oncogenic potential. Herpes virus saimiri (HVS) is the prototype gamma-2 herpes virus and is a useful model to study the basic mechanisms of lytic replication in this subfamily. Like all herpes viruses, HVS has two distinct life cycles, latent persistence and lytic replication. Analysis of herpes virus genomes has demonstrated that, in contrast to cellular genes, most virus genes that are expressed lytically do not have introns. Herpes viruses replicate in the nucleus of the host cell, and therefore require that the viral intron-lacking mRNAs are exported from the nucleus to allow virus mRNA translation. This review focuses upon the role of HVS ORF 57, a post-transcriptional regulatory protein, which is conserved in all herpes viruses. HVS ORF 57 is a multifunctional protein involved in both trans-activation and trans-repression of target mRNAs. The major role of the ORF 57 protein in mediating viral mRNA export is considered, and the ORF 57-host cell interactions that are required for this function are discussed.

Gene Expression Regulation, Viral↗
PubMed2001

Cell-type-specific regulation of the two foamy virus promoters.

The foamy virus (FV) genome contains two promoters, the canonical long terminal repeat (LTR) promoter, containing three consensus AP-1 binding sites, and an internal promoter (IP) within the env gene. We investigated the regulation of the two promoters in lytic and persistent infections and found that in the presence of a constitutive source of the viral transactivator protein Tas, transactivation of the LTR promoter and that of the IP differ. In lytic infections, both the LTR promoter and the IP are efficiently transactivated by Tas, while in persistent infections, the IP is efficiently transactivated by Tas, but the LTR promoter is not. Analysis of proteins expressed from the LTR promoter and the IP during infection indicated that IP transcription is more robust than that of the LTR promoter in persistently infected cells, while the opposite is true for lytically infected cells. Coculture experiments also showed that LTR promoter transcription is greatest in cells which support lytic replication. Replacement of much of the LTR promoter with the IP leads to increased viral replication in persistent but not lytic infections. We also found that the induction of persistently infected cells with phorbol 12-myristate 13-acetate (PMA) greatly enhanced viral replication and transcription from the SFVcpz(hu) (new name for human FV) LTR promoter. However, mutation of three consensus AP-1 binding sites in the FV LTR promoter did not affect viral replication in lytically or persistently infected cells, nor did the same mutations affect LTR promoter transactivation by Tas in PMA-treated cells. Our data indicate that differential regulation of transcription is important in the outcome of FV infection but is unlikely to depend on AP-1.

Animals↗
PubMed1991

Multicomponent origin of cytomegalovirus lytic-phase DNA replication.

Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dbp. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A+T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.

Base Sequence↗
PubMed2005

A redox-sensitive cysteine in Zta is required for Epstein-Barr virus lytic cycle DNA replication.

Epstein-Barr virus (EBV) reactivation from latency is known to be sensitive to redox regulation. The immediate-early protein Zta is a member of the basic-leucine zipper (bZIP) family of DNA binding proteins that stimulates viral and cellular transcription and nucleates a replication complex at the viral lytic origin. Zta shares with several members of the bZIP family a conserved cysteine residue (C189) that confers redox regulation of DNA binding. In this work, we show that replacement of C189 with serine (C189S) eliminated lytic cycle DNA replication function of Zta. The mechanistic basis for this replication defect was investigated. We show that C189S was not significantly altered for DNA binding activity in vitro or in vivo. We also show that C189S was not defective for transcription activation of EBV early gene promoters. C189S was deficient for transcription activation of several viral late genes that depend on lytic replication and therefore was consistent with a primary defect of C189S in activating lytic replication. C189S was not defective in binding methylated DNA binding sites and was capable of activating Rta from endogenous latent viral genomes, in contrast to the previously characterized S186A mutation. C189S was slightly impaired for its ability to form a stable complex with Rta, although this did not prevent Rta recruitment to OriLyt. C189S did provide some resistance to oxidation and nitrosylation, which potently inhibit Zta DNA binding activity in vitro. Interestingly, this redox sensitivity was not strictly dependent on C189S but involved additional cysteine residues in Zta. These results provide evidence that the conserved cysteine in the bZIP domain of Zta plays a primary role in EBV lytic cycle DNA replication.

Amino Acid Sequence↗
PubMed2005

Targeting human herpesvirus-8 for treatment of Kaposi's sarcoma and primary effusion lymphoma.

PURPOSE OF REVIEW: Human herpesvirus-8, also called the Kaposi's sarcoma herpesvirus, is present in all cases of Kaposi's sarcoma and primary effusion lymphoma and in some cases of multicentric Castleman's disease. This review discusses mechanisms by which human herpesvirus-8 contributes to tumorigenesis and how this knowledge can be used to target the virus for the treatment of these tumors. RECENT FINDINGS: Most primary effusion lymphomas and Kaposi's sarcoma tumor cells are latently infected with human herpesvirus-8 and hence resistant to antiherpesvirus drugs that are dependent on lytic replication. In contrast, many of the cells infected with human herpesvirus-8 in multicentric Castleman's disease support lytic replication, so that clinical improvement frequently occurs in response to treatment with antiherpesvirus drugs. The resistance of latently-infected tumor cells to antiherpesvirus drugs can be overcome by inducing human herpesvirus-8 to reenter the lytic cascade in the presence of antiherpesvirus drugs. This leads to apoptosis of virally infected cells without increasing production of infectious virus. Alternatively, the replication and maintenance of the human herpesvirus-8 episome during latency can be disrupted by glycyrrhizic acid or hydroxyurea so that the virus no longer contributes to tumorigenesis. Both the innate and acquired immune systems can also be augmented to help prevent or treat human herpesvirus-8-associated tumors. SUMMARY: Novel strategies targeting human herpesvirus-8, which is present in all cases of Kaposi's sarcoma and primary effusion lymphoma, provide opportunities for selectively killing tumor cells.

Animals↗
PubMed1999

Inhibition of Epstein-Barr virus replication by a benzimidazole L-riboside: novel antiviral mechanism of 5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole.

Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. D-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5, 6-dichloro-2-bromo-1-beta-D-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to the L-riboside 1263W94 [5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1. 1 microM, compared with 10 microM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.

Acyclovir↗
PubMed2002

Kinetics of expression of rhesus monkey rhadinovirus (RRV) and identification and characterization of a polycistronic transcript encoding the RRV Orf50/Rta, RRV R8, and R8.1 genes.

Rhesus monkey rhadinovirus (RRV) is a close relative of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8). RRV serves as an in vitro and an in vivo model for KSHV, and the mapping of its transcription program during lytic replication is significant since it represents de novo infection in the absence of stimulation with phorbol esters. Further, the RRV lytic system facilitates the making of recombinant viruses, and hence transcription profiling of the wild-type virus is important. Currently, the kinetics of lytic gene expression of RRV, the function of the RRV Orf50/Rta gene, and the presence of the RRV R8 and R8.1 genes are not known. This study details the transcription profile seen during RRV lytic replication and shows that RRV latency-associated nuclear antigen, viral FLIP (vFLIP), and vCyclin are transcribed during the RRV lytic phase. In addition, this study describes the identification of three new spliced products of the RRV Orf50, R8, and R8.1 genes, which are structural homologs of the KSHV Orf50, K8, and K8.1 genes, respectively. Characterization of the RRV Orf50 protein identifies it as a strong transcriptional transactivator capable of activating three early RRV promoters. Interestingly, the KSHV Orf50 transactivator can also activate these simian virus promoters, suggesting that there exists a conservation of gene function between the key transcription factors of KSHV and RRV.

Amino Acid Sequence↗
PubMed2006

Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus by morpholino oligomers.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.

Antigens, Viral↗