Artemisinin-Derived Dimer Diphenyl Phosphate Is an Irreversible Inhibitor of Human Cytomegalovirus Replication

ABSTRACTWe previously reported that among a series of artemisinin-derived monomers and dimers, dimer diphenyl phosphate (838) was the most potent inhibitor of human cytomegalovirus (CMV) replication. Our continued investigation of a prototypic artemisinin monomer (artesunate [AS]) and dimer (838) now reveals that both compounds have specific activity against CMV but do not inhibit lytic replication of human herpesvirus 1 or 2 or Epstein-Barr virus. AS and 838 inhibited CMV replication during the first 24 h of the virus replication cycle, earlier than the time of ganciclovir (GCV) activities and prior to DNA synthesis. Neither compound inhibited virus entry. Quantification of DNA replication and virus yield revealed a similar level of inhibition by GCV, but AS and 838 had a 10-fold-higher inhibition of virus yield than of DNA replication, suggesting that artemisinins could inhibit CMV through multiple steps: a predominant early inhibition and possibly an additional step following DNA replication. During the strong early CMV inhibition, the transcription of immediate-early genes was not significantly downregulated, and viral protein expression was reduced only after 48 h. AS and GCV were reversible CMV inhibitors, but the inhibition of CMV replication by 838 was irreversible. Combinations of GCV and 838 as well as GCV and AS were highly synergistic. Finally, treatment with 838, but not AS, prior to CMV infection demonstrated strong anti-CMV activity. These findings illustrate the unique activities of dimer 838, including early and irreversible CMV inhibition, possibly by tight binding to its target.

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Effect of phosphorylation on the transactivation activity of Epstein–Barr virus BMRF1, a major target of the viral BGLF4 kinase

Journal of General Virology ◽

10.1099/vir.0.83546-0 ◽

2008 ◽

Vol 89 (4) ◽

pp. 884-895 ◽

Cited By ~ 25

Author(s):

Pei-Wen Yang ◽

Shih-Shin Chang ◽

Ching-Hwa Tsai ◽

Yi-Hsin Chao ◽

Mei-Ru Chen

Keyword(s):

Dna Replication ◽

Nuclear Localization ◽

Epstein Barr Virus ◽

Human Herpesvirus ◽

Lytic Replication ◽

Barr Virus ◽

Transactivation Activity ◽

Synergistic Activation ◽

Functional Regulation ◽

Epstein Barr

Modification of human herpesvirus DNA polymerase processivity factors (PFs) by phosphorylation occurs frequently during viral lytic replication. However, functional regulation of the herpesvirus PFs through phosphorylation is not well understood. In addition to processivity, the PF BMRF1 of Epstein–Barr virus can function as a transactivator to activate the BHLF1 promoter within the lytic origin of replication (oriLyt), which is assumed to facilitate DNA replication through remodelling viral chromatin structure. BMRF1 is known to be phosphorylated by the viral BGLF4 kinase, but its impact on BMRF1 function is unclear. Seven candidate BGLF4 target sites were predicted within a proline-rich region between the DNA-processivity and nuclear-localization domains of BMRF1. We show that four of these residues, Ser-337, Thr-344, Ser-349 and Thr-355, are responsible for the BGLF4-induced hyperphosphorylation of BMRF1. In functional analyses, a phosphorylation-mimicking mutant of BMRF1 shows similar nuclear localization, as well as DNA-binding ability, to the wild type; however, it displays stronger synergistic activation of the BHLF1 promoter with Zta. Notably, BGLF4 downregulates BMRF1 transactivation and enhances the transactivation activity of Zta and the synergistic activation of BMRF1 and Zta on the BHLF1 promoter. Our findings suggest that BGLF4 may modulate the activation of the oriLyt BHLF1 promoter coordinately through multiple mechanisms to facilitate optimal oriLyt-dependent viral DNA replication.

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Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus

Journal of Virology ◽

10.1128/jvi.75.3.1378-1386.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1378-1386 ◽

Cited By ~ 134

Author(s):

Jeffrey Vieira ◽

Patricia O'Hearn ◽

Louise Kimball ◽

Bala Chandran ◽

Lawrence Corey

Keyword(s):

Human Cytomegalovirus ◽

Kaposi's Sarcoma ◽

Human Fibroblasts ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Lytic Cycle ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) andneo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.

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pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth

Virology ◽

10.1016/j.virol.2018.03.017 ◽

2018 ◽

Vol 518 ◽

pp. 414-422 ◽

Cited By ~ 2

Author(s):

Mark Slayton ◽

Tanvir Hossain ◽

Bonita J. Biegalke

Keyword(s):

Dna Replication ◽

Human Cytomegalovirus ◽

Lytic Replication ◽

Viral Growth

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Multiple Roles of Epstein-Barr Virus SM Protein in Lytic Replication

Journal of Virology ◽

10.1128/jvi.02665-06 ◽

2007 ◽

Vol 81 (8) ◽

pp. 4058-4069 ◽

Cited By ~ 24

Author(s):

Zhao Han ◽

Elessa Marendy ◽

Yong-Dong Wang ◽

Jing Yuan ◽

Jeffery T. Sample ◽

...

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Late Gene ◽

Late Gene Expression ◽

Barr Virus ◽

Ebv Dna ◽

Epstein Barr ◽

Sm Protein

ABSTRACT The effect of Epstein-Barr virus (EBV) SM protein on EBV gene expression was examined using a recombinant EBV strain with the SM gene deleted and DNA microarrays representing all known EBV coding regions. Induction of lytic EBV replication in the absence of SM led to expression of approximately 40% of EBV genes, but a block in expression of over 50% of EBV genes. Contrary to previous findings, several early genes were SM dependent, and lytic EBV DNA replication did not occur in the absence of SM. Notably, two genes essential for lytic EBV DNA replication, BSLF1 and BALF5, encoding EBV DNA primase and polymerase, respectively, were SM dependent. Lytic DNA replication was partially rescued by ectopic expression of EBV primase and polymerase, but virion production was not. Rescue of DNA replication only enhanced expression of a subset of late genes, consistent with a direct requirement for SM for late gene expression in addition to its contribution to DNA replication. Therefore, while SM is essential for most late gene expression, the proximate block to virion production by the EBV SM deletion strain is an inability to replicate linear DNA. The block to DNA replication combined with the direct effect of SM on late gene expression leads to a global deficiency of late gene expression. SM also inhibited BHRF1 expression during productive replication in comparison to that of cells induced into lytic replication in the absence of SM. Thus, SM plays a role in multiple steps of lytic cycle EBV gene expression and that it is transcript-specific in both activation and repression functions.

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Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

Journal of Virology ◽

10.1128/jvi.00049-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6641-6651 ◽

Cited By ~ 58

Author(s):

Ayumi Kudoh ◽

Satoko Iwahori ◽

Yoshitaka Sato ◽

Sanae Nakayama ◽

Hiroki Isomura ◽

...

Keyword(s):

Dna Replication ◽

Homologous Recombination ◽

Viral Genome ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Recombinational Repair ◽

Viral Dna ◽

Barr Virus ◽

Homologous Recombinational Repair ◽

Epstein Barr

ABSTRACT Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

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Murine gammaherpesvirus 68 ORF35 is required for efficient lytic replication and latency

Journal of General Virology ◽

10.1099/jgv.0.000310 ◽

2015 ◽

Vol 96 (12) ◽

pp. 3624-3634 ◽

Cited By ~ 2

Author(s):

Shin-ichi Hikita ◽

Yusuke Yanagi ◽

Shinji Ohno

Keyword(s):

Ex Vivo ◽

Essential Gene ◽

Coiled Coil ◽

Human Herpesvirus ◽

Virus Production ◽

Lytic Replication ◽

Genome Replication ◽

Murine Gammaherpesvirus ◽

Barr Virus ◽

C Terminus

Murine gammaherpesvirus (MHV) 68, a natural pathogen of field mice, is related to human gammaherpesviruses, Epstein–Barr virus (EBV; human herpesvirus 4) and Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8). The ORF35 of MHV-68 and its homologues of EBV and KSHV are located in the gene cluster composed of ORF34–ORF38 in which each gene overlaps with adjacent genes. Although MHV-68 ORF35 was reported to be an essential gene, its function during infection is presently unknown. In this study, we show, by analysing ORF35-transfected cells, that three serine residues in the C terminus are responsible for the phosphorylation and that the ORF35 protein forms homo-oligomers via a predicted coiled-coil motif. The ORF35 protein expressed by transfection was preferentially located in the cytoplasm of cells uninfected or infected with MHV-68. The recombinant virus lacking ORF35 (35S virus) exhibited genome replication and expression of lytic proteins comparable to those of the WT virus, but reduced levels of virus production, suggesting that the ORF35 protein acts at the virion assembly and/or egress step. Lytic replication in the lung after intranasal infection and the frequency of ex vivo reactivation from latency after intraperitoneal infection were lower in 35S virus-infected mice than in mice infected with the WT or marker-reverted virus. Our results indicate that ORF35 is not essential for MHV-68 lytic replication, but plays an important role in efficient viral replication and reactivation from latency.

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Evidence for DNA Hairpin Recognition by Zta at the Epstein-Barr Virus Origin of Lytic Replication

Journal of Virology ◽

10.1128/jvi.02666-09 ◽

2010 ◽

Vol 84 (14) ◽

pp. 7073-7082 ◽

Cited By ~ 5

Author(s):

Andrew J. Rennekamp ◽

Pu Wang ◽

Paul M. Lieberman

Keyword(s):

Dna Replication ◽

Inverted Repeat ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Lytic Cycle ◽

Productive Infection ◽

Dna Hairpin ◽

Barr Virus ◽

Early Protein ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus immediate-early protein (Zta) plays an essential role in viral lytic activation and pathogenesis. Zta is a basic zipper (b-Zip) domain-containing protein that binds multiple sites in the viral origin of lytic replication (OriLyt) and is required for lytic-cycle DNA replication. We present evidence that Zta binds to a sequence-specific, imperfect DNA hairpin formed by an inverted repeat within the upstream essential element (UEE) of OriLyt. Mutations in the OriLyt sequence that are predicted to disrupt hairpin formation also disrupt Zta binding in vitro. Restoration of the hairpin rescues the defect. We also show that OriLyt DNA isolated from replicating cells contains a nuclease-sensitive region that overlaps with the inverted-repeat region of the UEE. Furthermore, point mutations in Zta that disrupt specific recognition of the UEE hairpin are defective for activation of lytic replication. These data suggest that Zta acts by inducing and/or stabilizing a DNA hairpin structure during productive infection. The DNA hairpin at OriLyt with which Zta interacts resembles DNA structures formed at other herpesvirus origins and may therefore represent a common secondary structure used by all herpesvirus family members during the initiation of DNA replication.

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Architecture of Replication Compartments Formed during Epstein-Barr Virus Lytic Replication

Journal of Virology ◽

10.1128/jvi.79.6.3409-3418.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3409-3418 ◽

Cited By ~ 61

Author(s):

Tohru Daikoku ◽

Ayumi Kudoh ◽

Masatoshi Fujita ◽

Yutaka Sugaya ◽

Hiroki Isomura ◽

...

Keyword(s):

Dna Replication ◽

Viral Replication ◽

Epstein Barr Virus ◽

Protein A ◽

Lytic Replication ◽

Viral Dna ◽

Barr Virus ◽

Epstein Barr ◽

Cellular Replication ◽

Cellular Dna

ABSTRACT Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.

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Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 K-bZIP Modulates Latency-Associated Nuclear Protein-Mediated Suppression of Lytic Origin-Dependent DNA Synthesis

Journal of Virology ◽

10.1128/jvi.00922-09 ◽

2009 ◽

Vol 83 (17) ◽

pp. 8492-8501 ◽

Cited By ~ 19

Author(s):

Cyprian Rossetto ◽

Irena Yamboliev ◽

Gregory S. Pari

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Nuclear Protein ◽

Human Herpesvirus ◽

Lytic Replication ◽

Human Herpesvirus 8 ◽

Dependent Manner ◽

Herpesvirus 8 ◽

Replication Assay ◽

Lytic Dna Replication

ABSTRACT The original cotransfection replication assay identified eight human herpesvirus 8 (HHV8)-encoded proteins required for origin-dependent lytic DNA replication. Previously, we demonstrated that under conditions where K-Rta is overexpressed, a K-bZIP knockout bacmid displayed an aberrant subcellular localization pattern for the latency-associated nuclear protein (LANA). Additionally, these same studies demonstrated that K-bZIP interacts with LANA in the absence of K-Rta and that K-bZIP does not directly participate in, but may facilitate, the initiation of lytic DNA synthesis. We developed a modification of the transient cotransfection replication assay wherein both lytic (oriLyt) and latent (terminal repeat) DNA replication are evaluated simultaneously. We now show that LANA represses origin-dependent lytic DNA replication in a dose dependent manner when added to the cotransfection replication assay. This repression was overcome by increasing amounts of a K-bZIP expression plasmid in the cotransfection mixture or by dominant-negative inhibition of the interaction of LANA with K-bZIP by the overexpression of the K-bZIP-LANA binding domain. Chromatin immunoprecipitation assays show that LANA interacts with oriLyt in the absence of K-bZIP expression, suggesting that suppression of lytic replication by LANA is mediated by direct binding. The interaction of K-bZIP with oriLyt was dependent upon the expression of LANA; however, LANA interacted with oriLyt independently of K-bZIP expression. These data suggest that the interaction of LANA with K-bZIP modulates lytic and latent replication and that K-bZIP facilitates lytic DNA replication and modulates the switch from the latent phase of the virus.

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