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PubMed2005

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 alter mitochondrial morphology during lytic replication.

Epstein-Barr virus (EBV) is a human DNA virus that is responsible for the syndrome infectious mononucleosis, and is associated with several forms of cancer. During both lytic and latent viral infection, viral proteins manipulate the host's cellular components to aid in viral replication and maintenance. Here, it is demonstrated that induction of EBV lytic replication results in a dramatic reorganization of mitochondria accompanied by a significant alteration of mitochondrial membrane potential and a rapid and transient increase in the microtubular cytoskeleton. Moreover, we show that expression of the EBV immediate-early genes BZLF1 and BRLF1 contributes to the mitochondrial alteration but not the increase in the microtubule cytoskeleton, suggesting that the mechanism for the observed cytoplasmic restructuring involves a number of coordinated viral and host proteins.

Cell Line↗
PubMed1999

Demethylation of the Epstein-barr virus origin of lytic replication and of the immediate early gene BZLF1 is DNA replication independent. Brief report.

Epstein-Barr virus (EBV) episomal DNA is extensively methylated in Burkitt lymphoma derived cell lines. In this study we examined whether lytic viral cycle reactivation is dependent on demethylation of critical viral genes. Viral replication was induced in the Burkitt's lymphoma cell line Daudi by the combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium-butyrate. Two regions necessary for EBV replication, the BZLF1 immediate early region and the origin of lytic cycle replication (ori Lyt) were demethylated during the early phase of the lytic virus cycle. Demethylation was observed while production of new (unmethylated) viral DNA was blocked by phosphonoformic acid (PFA). This suggests that demethylation, which may be instrumental for the onset of the lytic cycle, is an active process independent of viral DNA replication

Antigens, Viral↗
PubMed2004

Epstein-Barr virus lytic replication elicits ATM checkpoint signal transduction while providing an S-phase-like cellular environment.

When exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or arrest of cell-cycle progression, providing time for DNA repair. Induction of the Epstein-Barr virus (EBV) lytic program elicited a cellular DNA damage response, with activation of the ataxia telangiectasia-mutated (ATM) signal transduction pathway. Activation of the ATM-Rad3-related (ATR) replication checkpoint pathway, in contrast, was minimal. The DNA damage sensor Mre11-Rad50-Nbs1 (MRN) complex and phosphorylated ATM were recruited and retained in viral replication compartments, recognizing newly synthesized viral DNAs as abnormal DNA structures. Phosphorylated p53 also became concentrated in replication compartments and physically interacted with viral BZLF1 protein. Despite the activation of ATM checkpoint signaling, p53-downstream signaling was blocked, with rather high S-phase CDK activity associated with progression of lytic infection. Therefore, although host cells activate ATM checkpoint signaling with response to the lytic viral DNA synthesis, the virus can skillfully evade this host checkpoint security system and actively promote an S-phase-like environment advantageous for viral lytic replication.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1983

Electron microscopy study of early lytic replication forms of bacteriophage P1 DNA.

P1 replication intermediates were isolated from the intracellular DNA of lytically infected cells and analyzed by electron microscopy. At early times in infection replication intermediates were mainly of two types, circular theta- and sigma-shaped molecules plus a small proportion of linear bubble-shaped molecules. At later times in infection sigma molecules were the predominant replicating form. In contrast, sigma molecules were rarely found in recombination deficient, recA, infected cells. These observations show that early P1 DNA synthesis occurs, in part, by a circular mode of replication and imply that transition to a later, probably rolling circle, phase of replication is controlled by the bacterial general recombination system.

Coliphages↗
PubMed2000

Inflammatory cytokines and the reactivation of Kaposi's sarcoma-associated herpesvirus lytic replication.

Kaposi's sarcoma (KS) is a complex proliferative lesion long suspected of being dependent on exogenous paracrine signaling molecules to stimulate its proliferative, angiogenic, and inflammatory components. In particular, both clinical and experimental observations have pointed to a potential role for inflammatory cytokines as permissive factors for KS development, but KS pathogenesis is also critically dependent on infection by an exogenous herpesvirus, the KS-associated herpesvirus (KSHV). To examine the possible links between inflammatory cytokines and KSHV replication, we tested for the ability of such cytokines to induce lytic viral reactivation in the latently infected BCBL-1 cell line. Interferon-gamma consistently activated KSHV replication, whereas tumor necrosis factor, interleukin-1, interleukin-2, interleukin-6, granulocyte-macrophage colony stimulating factor, and basic fibroblast growth factor did not. Glucocorticoids also failed to induce lytic KSHV growth in these cells, but ionomycin, a calcium ionophore, induced replication and strongly augmented the known inductive effects of phorbol esters. Interferon-alpha had a dose-dependent inhibitory effect on KSHV induction by ionomycin. The identification of interferon-gamma as an activator and interferon-alpha as an inhibitor of KSHV induction in vitro correlates well with in vivo observations and demonstrates for the first time that inflammatory cytokines can directly modulate KSHV replication.

Cytokines↗
PubMed2006

Marek's disease virus: lytic replication, oncogenesis and control.

Marek's disease (MD) is caused by a ubiquitous, lymphotropic alphaherpesvirus, MD virus (MDV). MD has been a major concern in the poultry industry owing to the emergence of increasingly virulent strains over the last few decades that were isolated in the face of comprehensive vaccination. The disease is characterized by a variety of clinical signs; among them are neurological symptoms, chronic wasting and, most notably, the development of multiple lymphomas that manifest as solid tumors in the viscera and musculature. Much work has been devoted to study MD-induced oncogenesis and the genes involved in this process. Among the many genes encoded by MDV, a number have been shown recently to affect the development of tumors in chickens, one protein directly causing transformation of cells (Meq) and another being involved in maintaining transformed cells (vTR). Other MDV gene products modulate and are involved in early lytic in vivo replication, thereby increasing the chance of transformation occurring. In this review, we will summarize specific genes encoded by MDV that are involved in the initiation and/or maintenance of transformation and will focus mostly on current vaccination and control strategies against MD, particularly how modern molecular biological methods may be used to improve strategies to combat the disease in the future.

Animals↗
PubMed2004

Forced lytic replication impairs host colonization by a latency-deficient mutant of murine gammaherpesvirus-68.

A regulated switch between latent and lytic gene expression is common to all known herpesviruses. However, the effects on host colonization of altering this switch are largely unknown. We deregulated the transcription of the gene encoding the major lytic transactivator of murine gammaherpesvirus-68, ORF50, by inserting a new and powerful promoter element in its 5' untranslated region. In vitro, the mutant virus (M50) transcribed ORF50 at a high level and showed more rapid lytic spread in permissive fibroblast cultures, but in vivo, the M50 virus showed a severe deficit in latency establishment, with no sign of the infectious mononucleosis-like illness normally associated with wild-type infection. Although a low level of M50 viral DNA was detectable by PCR in spleens, replication-competent virus could not be recovered beyond 10 days post-infection. The M50 virus was also attenuated in immunocompromised mice. Thus a gammaherpesvirus unable to shut off lytic cycle gene expression showed severely restricted host colonization.

Animals↗
PubMed2004

Short duration of elevated vIRF-1 expression during lytic replication of human herpesvirus 8 limits its ability to block antiviral responses induced by alpha interferon in BCBL-1 cells.

Human herpesvirus 8 (HHV-8) encodes multiple proteins that disrupt the host antiviral response, including viral interferon (IFN) regulatory factor 1 (vIRF-1). The product of the vIRF-1 gene blocks responses to IFN when overexpressed by transfection, but the functional consequence of vIRF-1 that is expressed during infection with HHV-8 is not known. These studies demonstrate that BCBL-1 cells that were latently infected with HHV-8 expressed low levels of vIRF-1 that were associated with PML bodies, whereas much higher levels of vIRF-1 were transiently expressed during the lytic phase of HHV-8 replication. The low levels of vIRF-1 that were associated with PML bodies were insufficient to block alpha IFN (IFN-alpha)-induced alterations in gene expression, whereas cells that expressed high levels of vIRF-1 were resistant to some changes induced by IFN-alpha, including the expression of the double-stranded-RNA-activated protein kinase. High levels of vIRF-1 were expressed for only a short period during the lytic cascade, so many cells with HHV-8 in the lytic phase responded to IFN-alpha with increased expression of antiviral genes and enhanced apoptosis. Furthermore, the production of infectious virus was severely compromised when IFN-alpha was present early during the lytic cascade. These studies indicate that the transient expression of high levels of vIRF-1 is inadequate to subvert many of the antiviral effects of IFN-alpha so that IFN-alpha can effectively induce apoptosis and block production of infectious virus when present early in the lytic cascade of HHV-8.

B-Lymphocytes↗
PubMed1997

Selective switch between latency and lytic replication of Kaposi's sarcoma herpesvirus and Epstein-Barr virus in dually infected body cavity lymphoma cells.

The BC-1 cell line, derived from a body cavity-based, B-cell lymphoma, is dually infected with Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In these studies, the relationships between these two gammaherpesviruses and BC-1 cells were characterized and compared. Single-cell cloning experiments suggested that all BC-1 cells contain both genomes. In more than 98% of cells, both viruses were latent. The two viruses could be differentially induced into their lytic cycles by chemicals. EBV was activated into DNA replication and late-gene expression by the phorbol ester tetradecanoyl phorbol acetate (TPA). KSHV was induced into DNA replication and late-gene expression by n-butyrate. Amplification of both EBV and KSHV DNAs was inhibited by phosphonoacetic acid. Induction of the KSHV lytic cycle by n-butyrate was accompanied by the disappearance of host-cell beta-actin mRNA. Induction of EBV by TPA was not accompanied by such an effect on host-cell gene expression. Induction of the KSHV lytic cycle by n-butyrate was associated with the expression of several novel polypeptides. Recognition of one of these, p40, served as the basis of development of an assay for antibodies to KSHV in the sera of infected patients. BC-1 cells released infectious EBV; however, there was no evidence for the release of encapsidated KSHV genomes by BC-1 cells, even though n-butyrate-treated cells contained numerous intranuclear nucleocapsids. The differential inducibility of these two herpesviruses in the same cell line points to the importance of viral factors in the switch from latency to lytic cycle.

Animals↗
PubMed2004

Evidence for both lytic replication and tightly regulated human herpesvirus 8 latency in circulating mononuclear cells, with virus loads frequently below common thresholds of detection.

To address whether human herpesvirus 8 (HHV-8) DNA in peripheral blood mononuclear cells (PBMCs) might be the product of latent or lytic infection and to shed light on sporadic detection of HHV-8 DNA in individuals seropositive for the virus, we studied the frequency of infected cells, total virus load, and virus load per infected cell in PBMCs from men coinfected with HHV-8 and human immunodeficiency virus (HIV), some of whom had Kaposi's sarcoma. The low frequencies of infected cells detected (fewer than one per million cells in some individuals) suggest that the prevalence of the virus in circulating leukocytes was underestimated in previous studies that employed more conventional sampling methods (single, small-volume specimens). Mean virus loads ranged from 3 to 330 copies per infected PBMC; these numbers can represent much higher loads in individual lytically infected cells (>10(3) genomes/cell) in mixtures that consist predominantly of latently (relatively few genomes) infected cells. The presence in some subjects of high HHV-8 mean genome copy numbers per infected cell, together with viral DNA being found in plasma only from subjects with positive PBMCs, supports earlier suggestions that the virus can actively replicate in PBMCs. In some individuals, mean virus loads were less than 10 genomes per infected cell, suggesting a tightly controlled purely latent state. HHV-8 genome copy numbers are substantially higher in latently infected cells derived from primary effusion lymphomas; thus, it appears that HHV-8 is able to adopt more than one latency program, perhaps analogous to the several types of Epstein-Barr virus latency.

DNA, Viral↗
PubMed1998

Four tRNA-like sequences and a serpin homologue encoded by murine gammaherpesvirus 68 are dispensable for lytic replication in vitro and latency in vivo.

Experimental infection of inbred strains of laboratory mice with murine herpesvirus 68 (MHV-68), a natural pathogen of wild rodents, results in acute productive infection of the lung followed by a latent infection of B lymphocytes. We have previously shown that MHV-68 encodes an open reading frame with similarity to poxvirus serpins, designated ORF1, and eight novel tRNA-like sequences. The latter are processed into mature, uncharged tRNAs and are abundantly expressed during both lytic and latent infection. In this study it is demonstrated that deletion of four of the tRNA-like sequences and ORF1 from the virus genome does not affect the ability of MHV-68 to replicate in vitro or to establish, and reactivate from, latency in vivo.

Animals↗
PubMed1996

Pathogenesis of chronic Epstein-Barr virus infection: detection of a virus strain with a high rate of lytic replication.

In rare cases Epstein-Barr virus (EBV) leads to chronic active infection (CAEBV) which is characterized by persistant symptoms of infectious mononucleosis. Previously we described a case of persisting polyclonal B-cell lymphocytosis (PPBL) that was associated with CAEBV. Using reverse transcription and polymerase chain reaction we showed that in late passages of a spontaneous cell line, SM, latent EB viral genes such as EBNA1, EBNA2, EBNA3A/3B/ 3C, LMP1 and LMP2A were active. The master gene of the lytic cycle, BZLF1, was silent. This indicated that there was no general defect in immortalization and establishing latency by this CAEBV isolate SM. We obtained virus from the standard immortalizing strain B95-8 and the CAEBV strain SM from latently infected LCL quantified the number of virus particles by competitive PCR and demonstrated that the impaired capacity to immortalize umbilical cord blood lymphocytes was a virus strain-specific property, and was not due to an incapability to infect purified CD19+ B lymphocytes. Transcription of latency- and immortalization-associated genes such as EBNA1, EBNA2 and LMP2A was reduced, in contrast to a strongly enhanced activity of the master gene of the lytic cycle, BZLF1. A scenario for an antagonistic regulation of lytic and latent cycle genes is presented and a role for the pathogenesis of CAEBV is discussed.

B-Lymphocytes↗
PubMed1989

Analysis of viral proteins in human cytomegalovirus-infected cells during impaired lytic replication of herpes simplex virus.

Herpes simplex virus (HSV) latency can be established in vitro following arrest of virus replication and survival of infected cells in culture. Human cytomegalovirus (HCMV) has been shown to interact with HSV, resulting in reactivation of latent HSV. In addition, impaired replication of superinfecting HSV occurs in HCMV-infected human cells. HCMV-infected human embryonic lung cells inhibit production of infectious HSV despite replication of HSV DNA at levels comparable to those in control cultures infected only with HSV. Using radioimmunoprecipitation techniques, we found that the synthesis of HSV type 1 proteins of the alpha, beta/gamma, and gamma kinetic classes was impaired during the restricted replication of HSV in HCMV-infected HEL cells. However, synthesis of the HSV beta protein ICP-8 and HCMV alpha and beta proteins was not significantly affected in superinfected cell cultures.

Cells, Cultured↗
PubMed2004

Activation of bovine herpesvirus 4 lytic replication in a non-permissive cell line by overexpression of BoHV-4 immediate early (IE) 2 gene.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association, it establishes persistent infections in its natural host, the bovine, and in an experimental host, the rabbit. BoHV-4 immediate early 2 (IE2) RNA is the less abundant, spliced, 1.8 kb RNA. The predicted amino acid sequence, of the IE2 protein, reveals that it could encode a 61 kDa protein with amino acid sequence homology to the Epstein-Barr virus (EBV) transactivator R protein and its homologues including, herpesvirus saimiri (HVS), equine herpesvirus 2 (EHV-2), murine herpesvirus 68 and Kaposi's sarcoma-associated herpesvirus (KSHV). We examined recently the interaction of BoHV-4 with a human rhabdomyosarcoma cell line, RD-4, and found that although some infectious viruses can be produced, no cytopathic effect (CPE) was observed [J. Gen. Virol. 81 (2000) 1807]. Because IE2 could play a critical role in BoHV-4 productive infection and its overexpression in RD-4 cells could switch the non-permissive RD-4 status to a permissive one. RD-4 cells expressing stably BoHV-4 IE2 gene were generated. BoHV-4 IE2 induced an increased production of infectious viral particles sufficient to obtain an apparent cytopathic effect. It is concluded that BoHV-4 IE2 is a key factor in determining the outcome of BoHV-4 infection.

Animals↗
PubMed2006

The KSHV viral interleukin-6 is not essential for latency or lytic replication in BJAB cells.

Kaposi's Sarcoma-associated herpesvirus encodes a homolog of the human cellular interleukin-6 that may play a formative role in many KSHV-related diseases. While the viral IL-6 can signal similarly to its human counterpart little is known about the role of vIL-6 during KSHV infection. Using homologous recombination and selection in eukaryotic cells, a KSHV isolate was purified that does not express vIL-6 as was a control recombinant that left vIL-6 intact. The two viruses establish and maintain latency to similar levels in BJAB B-cells, reactivate to similar levels in B-cells and Monkey kidney cells and have very similar KSHV gene expression patterns. BJAB cells expressing KSHV survive better than the parental BJAB cells in low serum and the vIL-6 deletion does not abrogate this growth advantage. Thus vIL-6 is not essential for establishment, maintenance, or reactivation from latency in cell culture and is not involved in the survival of infected BJAB B-cells in low serum.

Animals↗
PubMed2004

Effective inhibition of Rta expression and lytic replication of Kaposi's sarcoma-associated herpesvirus by human RNase P.

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2'-O-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of approximately 150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of <10% in the Rta expression and viral growth was found in cells that were either not treated with an EGS or that were treated with a disabled EGS containing mutations that preclude recognition by RNase P. Our study provides direct evidence that EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors.

Antiviral Agents↗