Lytic DNA Replication Research Articles

Semisynthesis of (-)-Rutamarin Derivatives and Their Inhibitory Activity on Epstein-Barr Virus Lytic Replication.

(+)-Rutamarin inhibits EBV lytic DNA replication with an IC50 of 7.0 μM. (-)-Chalepin, a (-)-rutamarin derivative, was isolated from the whole plant of Ruta graveolens and used as a precursor of (-)-rutamarin. Altogether, 28 (-)-rutamarin derivatives were synthesized starting from (-)-chalepin. Of these, 16 compounds (2a-e, 3b-e, 3g, 4f, 4k, 4m-p) were found to be more potent against EBV lytic DNA replication than (-)-chalepin. Compounds 4m, 4n, and 4p exhibited IC50 values of 1.5, 0.32, and 0.83 μM and showed selectivity index values (SI) of 801, 211, and >120, respectively. Thus, compounds 4m, 4n, and 4p are considered promising leads for further laboratory investigation.

TRIM5α Promotes Ubiquitination of Rta from Epstein-Barr Virus to Attenuate Lytic Progression.

Replication and transcription activator (Rta), a key protein expressed by Epstein–Barr virus (EBV) during the immediate-early stage of the lytic cycle, is responsible for the activation of viral lytic genes. In this study, GST-pulldown and coimmunoprecipitation assays showed that Rta interacts in vitro and in vivo with TRIM5α, a host factor known to be involved in the restriction of retroviral infections. Confocal microscopy results revealed that Rta colocalizes with TRIM5α in the nucleus during lytic progression. The interaction involves 190 amino acids in the N-terminal of Rta and the RING domain in TRIM5α, and it was further found that TRIM5α acts as an E3 ubiquitin ligase to promote Rta ubiquitination. Overexpression of TRIM5α reduced the transactivating capabilities of Rta, while reducing TRIM5α expression enhanced EBV lytic protein expression and DNA replication. Taken together, these results point to a critical role for TRIM5α in attenuating EBV lytic progression through the targeting of Rta for ubiquitination, and suggest that the restrictive capabilities of TRIM5α may go beyond retroviral infections.

Chaetocin reactivates the lytic replication of Epstein-Barr virus from latency via reactive oxygen species.

Oxidative stress, regarded as a negative effect of free radicals in vivo, takes place when organisms suffer from harmful stimuli. Some viruses can induce the release of reactive oxygen species (ROS) in infected cells, which may be closely related with their pathogenicity. In this report, chaetocin, a fungal metabolite reported to have antimicrobial and cytostatic activity, was studied for its effect on the activation of latent Epstein-Barr virus (EBV) in B95-8 cells. We found that chaetocin remarkably up-regulated EBV lytic transcription and DNA replication at a low concentration (50 nmol L-1). The activation of latent EBV was accompanied by an increased cellular ROS level. N-acetyl-L-cysteine (NAC), an ROS inhibitor, suppressed chaetocin-induced EBV activation. Chaetocin had little effect on histone H3K9 methylation, while NAC also significantly reduced H3K9 methylation. These results suggested that chaetocin reactivates latent EBV primarily via ROS pathways.

An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription.

Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit “true” late kinetics–their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these “βγ” genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt) was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes.

Production and characterisation of Epstein-Barr virus helicase-primase complex and its accessory protein BBLF2/3.

The helicase-primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein-Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase-primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase-primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases.

A Herpesvirus Specific Motif of Epstein-Barr Virus DNA Polymerase Is Required for the Efficient Lytic Genome Synthesis.

Epstein-Barr virus (EBV) is associated with several malignancies, including Burkitt lymphoma and nasopharyngeal carcinoma. To overcome such disorders, understanding the molecular mechanisms of the EBV replication is important. The EBV DNA polymerase (Pol) is one of the essential factors for viral lytic DNA replication. Although it is well known that its C-terminal half, possessing DNA polymerase and 3’-5’ exonuclease activity, is highly conserved among Family B Pols, the NH2-terminal half has yet to be characterized in detail. In this study, we show that a stretch of hydrophobic amino acids within the pre-NH2-terminal domain of EBV Pol plays important role. In addition, we could identify the most essential residue for replication in the motif. These findings will shed light on molecular mechanisms of viral DNA synthesis and will help to develop new herpesviruses treatments.

DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

New Noncoding Lytic Transcripts Derived from the Epstein-Barr Virus Latency Origin of Replication, oriP, Are Hyperedited, Bind the Paraspeckle Protein, NONO/p54nrb, and Support Viral Lytic Transcription.

We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5' untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive "hyperediting" at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade. Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication.

Molecular biology of KSHV lytic reactivation.

Kaposi’s sarcoma-associated herpesvirus (KSHV) primarily persists as a latent episome in infected cells. During latent infection, only a limited number of viral genes are expressed that help to maintain the viral episome and prevent lytic reactivation. The latent KSHV genome persists as a highly ordered chromatin structure with bivalent chromatin marks at the promoter-regulatory region of the major immediate-early gene promoter. Various stimuli can induce chromatin modifications to an active euchromatic epigenetic mark, leading to the expression of genes required for the transition from the latent to the lytic phase of KSHV life cycle. Enhanced replication and transcription activator (RTA) gene expression triggers a cascade of events, resulting in the modulation of various cellular pathways to support viral DNA synthesis. RTA also binds to the origin of lytic DNA replication to recruit viral, as well as cellular, proteins for the initiation of the lytic DNA replication of KSHV. In this review we will discuss some of the pivotal genetic and epigenetic factors that control KSHV reactivation from the transcriptionally restricted latent program.

Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

Kaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong latent infections. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. Infected cells are programmed to retain the incoming viral genomes during primary infection. Immediately after infection, KSHV transcribes many lytic genes that modulate various cellular pathways to establish successful infection. Analysis of the virion particle showed that the virions contain viral mRNAs, microRNAs, and other noncoding RNAs that are transduced into the target cells during infection, but their biological functions are largely unknown. We performed a comprehensive analysis of the KSHV virion packaged transcripts and the profiles of viral genes transcribed after de novo infections of various cell types (human peripheral blood mononuclear cells [PBMCs], CD14(+) monocytes, and telomerase-immortalized vascular endothelial [TIVE] cells), from viral entry until latency establishment. A next-generation sequence analysis of the total transcriptome showed that several viral RNAs (polyadenylated nuclear RNA, open reading frame 58 [ORF58], ORF59, T0.7, and ORF17) were abundantly present in the KSHV virions and effectively transduced into the target cells. Analysis of the transcription profiles of each viral gene showed specific expression patterns in different cell lines, with the majority of the genes, other than latent genes, silencing after 24 h postinfection. We differentiated the actively transcribing genes from the virion-transduced transcripts using a nascent RNA capture approach (Click-iT chemistry), which identified transcription of a number of viral genes during primary infection. Treating the infected cells with phosphonoacetic acid (PAA) to block the activity of viral DNA polymerase confirmed the involvement of lytic DNA replication during primary infection. To further understand the role of DNA replication during primary infection, we performed de novo PBMC infections with a recombinant ORF59-deleted KSHV virus, which showed significantly reduced numbers of viral copies in the latently infected cells. In summary, the transduced KSHV RNAs as well as the actively transcribed genes control critical processes of early infection to establish KSHV latency. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple human malignancies in immunocompromised individuals. KSHV establishes a lifelong latency in the infected host, during which only a limited number of viral genes are expressed. However, a fraction of latently infected cells undergo spontaneous reactivation to produce virions that infect the surrounding cells. These newly infected cells are primed early to retain the incoming viral genome and induce cell growth. KSHV transcribes a variety of lytic proteins during de novo infections that modulate various cellular pathways to establish the latent infection. Interestingly, a large number of viral proteins and RNA are encapsidated in the infectious virions and transduced into the infected cells during a de novo infection. This study determined the kinetics of the viral gene expression during de novo KSHV infections and the functional role of the incoming viral transcripts in establishing latency.

MiR-190 is upregulated in Epstein–Barr Virus type I latency and modulates cellular mRNAs involved in cell survival and viral reactivation

Epstein–Barr Virus (EBV) is a prevalent human pathogen infecting over 90% of the population. Much of the success of the virus is attributed to its ability to maintain latency. The detailed mechanisms underlying the establishment and maintenance of EBV latency remain poorly understood. A microRNA profiling study revealed differential expression of many cellular miRNAs between types I and III latency cells, suggesting cellular miRNAs may play roles in regulating EBV latency. mir-190 is the most differentially up-regulated miRNA in type I latency cells as compared with type III latency cells and the up-regulation appears to be attributed to EBER RNAs that express in higher levels in type I latency cells than type III cells. With the aide of a lentiviral overexpression system and microarray analysis, several cellular mRNAs are identified as potential targets of mir-190. By targeting TP53INP1, miR-190 enhances cell survival by preventing apoptosis and relieving G0/G1 cell cycle arrest. Additionally, miR-190 down-regulates NR4A3, a cellular immediate-early gene for EBV reactivation, and inhibits the expression of the viral immediate-early gene bzlf1 and viral lytic DNA replication. Taken together, our data revealed a mechanism that EBV utilizes a cellular microRNA to promote host cell survival and prevent virus from entering lytic life cycle for latency maintenance.

Structure of the herpes simplex virus 1 genome: manipulation of nicks and gaps can abrogate infectivity and alter the cellular DNA damage response.

The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA-modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or adding random sequences to the 3' ends of DNA, producing 3' flaps, did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5' flaps in the DNA following treatment resulted in a dramatic reduction (95 to 100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5' flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5'-flapped DNA was restored in cells that do not express DNA-PKcs and in cells cotransfected with the immediate early protein ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-dependent protein kinase (DNA-PK) and the nonhomologous end joining (NHEJ) repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5' flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection. For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest, and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes adopt a configuration suitable for lytic DNA replication. This study demonstrates that virion DNA activates the cellular DDR kinase, DNA-PK, and that this response is inhibitory to viral infection. Furthermore, we show that HSV-1 ubiquitin ligase, ICP0, plays an important role in counteracting the negative effects of DNA-PK activation. These findings support the notion that DNA-PK is antiviral and suggest that the fate of incoming viral DNA has important consequences for the progression of lytic infection. This study underscores the complex evolutionary relationships between HSV and its host.

Kaposi's Sarcoma-Associated Herpesvirus ORF6 Gene Is Essential in Viral Lytic Replication

Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposis's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs) that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BACΔ6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BACΔ6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BACΔ6 cell supernatants. DNA synthesis was defective in 293T-BACΔ6 cells. Expression of ORF6 in trans in BACΔ6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BACΔ6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV.

Antiviral activity of topoisomerase II catalytic inhibitors against Epstein–Barr virus

Herpesviruses require several cellular proteins for their lytic DNA replication including topoisomerase II (Topo II). Thus, Topo II could be an effective drug target against herpesviral infection. In this study, we examined several Topo II catalytic inhibitors for their potentials in blocking EBV replication and becoming efficacious antiviral agents. Topo II catalytic inhibitors in general exhibited marked inhibition of EBV lytic replication and minimal cytotoxicity. In particular, (+)-rutamarin, with the best selectivity index (SI>63) among the inhibitors tested in this study, is effective in inhibiting EBV DNA replication and virion production but shows little adverse effect on cell proliferation, suggesting its potential to become an efficacious and safe drug for the treatment of human diseases associated with EBV infection.

CTCF and Rad21 Act as Host Cell Restriction Factors for Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic Replication by Modulating Viral Gene Transcription

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular genome by acting as gene-specific activators of some promoters, but differ in acting as global negative regulators of transcription.

Antiviral Activity of (+)-Rutamarin against Kaposi's Sarcoma-Associated Herpesvirus by Inhibition of the Catalytic Activity of Human Topoisomerase II

Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of several AIDS-associated malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). Its lytic replication cycle has been proven to be critical for the pathogenesis of KSHV-associated diseases. In KS lesions, lytic viral replication, production of virion particles, and reinfection of endothelial cells are essential to sustain the population of infected cells that otherwise would be quickly lost as spindle cells divide. Thus, antivirals that block KSHV replication could be a strategy in the treatment of KSHV-associated diseases. However, there is no effective anti-KSHV drug currently available. Our previous work showed that human topoisomerase II (Topo II) is indispensable for KSHV lytic replication and is suggested to be an effective target for antiviral drugs. Here, we report the discovery and characterization of a novel catalytic inhibitor of human Topo IIα, namely, (+)-rutamarin. The binding mode of (+)-rutamarin to the ATPase domain of human Topo IIα was established by docking and validated by molecular dynamics (MD) simulations. More importantly, (+)-rutamarin efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with a half-maximal inhibitory concentration (IC50) of 1.12 μM and blocks virion production with a half-maximal antiviral effective concentration (EC50) of 1.62 μM. It possesses low cytotoxicity, as indicated by the selectivity index (SI) of 84.14. This study demonstrated great potential for (+)-rutamarin to become an effective drug for treatment of human diseases associated with KSHV infection.

Phosphorylation of Kaposi's Sarcoma-Associated Herpesvirus Processivity Factor ORF59 by a Viral Kinase Modulates Its Ability To Associate with RTA and ori Lyt

ORF59 of Kaposi's sarcoma-associated herpesvirus (KSHV) plays an essential role in viral lytic replication by providing DNA processivity activity to the viral DNA polymerase (ORF9). ORF59 forms a homodimer in the cytoplasm and binds and translocates ORF9 into the nucleus, where it secures ORF9 to the origin of lytic DNA replication (oriLyt) in order to synthesize long DNA fragments during replication. ORF59 binds to oriLyt through an immediate early protein, replication and transcription activator (RTA). Here, we show that viral kinase (ORF36) phosphorylates serines between amino acids 376 and 379 of ORF59 and replacement of the Ser378 residue with alanine significantly impairs phosphorylation. Although mutating these serine residues had no effect on binding between ORF59 and ORF9, viral polymerase, or ORF36, the viral kinase, it significantly reduced the ability of ORF59 to bind to RTA. The results for the mutant in which Ser376 to Ser379 were replaced by alanine showed that both Ser378 and Ser379 contribute to binding to RTA. Additionally, the Ser376, Ser378, and Ser379 residues were found to be critical for binding of ORF59 to oriLyt and its processivity function. Ablation of these phosphorylation sites reduced the production of virion particles, suggesting that phosphorylation is critical for ORF59 activity and viral DNA synthesis.

Epstein-Barr Virus Deubiquitinase Downregulates TRAF6-Mediated NF-κB Signaling during Productive Replication

Epstein-Barr virus (EBV), a human oncogenic herpesvirus that establishes a lifelong latent infection in the host, occasionally enters lytic infection to produce progeny viruses. The EBV oncogene latent membrane protein 1 (LMP1), which is expressed in both latent and lytic infection, constitutively activates the canonical NF-κB (p65) pathway. Such LMP1-mediated NF-κB activation is necessary for proliferation of latently infected cells and inhibition of viral lytic cycle progression. Actually, canonical NF-κB target gene expression was suppressed upon the onset of lytic infection. TRAF6, which is activated by conjugation of polyubiquitin chains, associates with LMP1 to mediate NF-κB signal transduction. We have found that EBV-encoded BPLF1 interacts with and deubiquitinates TRAF6 to inhibit NF-κB signaling during lytic infection. HEK293 cells with BPLF1-deficient recombinant EBV exhibited poor viral DNA replication compared with the wild type. Furthermore, exogenous expression of BPLF1 or p65 knockdown in cells restored DNA replication of BPLF1-deficient viruses, indicating that EBV BPLF1 deubiquitinates TRAF6 to inhibit NF-κB signal transduction, leading to promotion of viral lytic DNA replication.

Essential Role of Rta in Lytic DNA Replication of Epstein-Barr Virus

Two transcription factors, ZEBRA and Rta, switch Epstein-Barr virus (EBV) from the latent to the lytic state. While ZEBRA also plays an obligatory role as an activator of replication, it is not known whether Rta is directly required for replication. Rta is dispensable for amplification of an oriLyt-containing plasmid in a transient-replication assay. Here, we assessed the requirement for Rta in activation of viral DNA synthesis from the endogenous viral genome, a function that has not been established. Initially, we searched for a ZEBRA mutant that supports viral replication but not transcription. We found that Z(S186A), a mutant of ZEBRA unable to activate transcription of Rta or viral genes encoding replication proteins, is competent to bind to oriLyt and to function as an origin recognition protein. Ectopic expression of the six components of the EBV lytic replication machinery failed to rescue replication by Z(S186A). However, addition of Rta to Z(S186A) and the mixture of replication factors activated viral replication and late gene expression. Deletion mutagenesis of Rta indicated that the C-terminal 10 amino acids (aa) were essential for the function of Rta in replication. In vivo DNA binding studies revealed that Rta interacted with the enhancer region of oriLyt. In addition, expression of Rta and Z(S186A) together, but not individually, activated synthesis of the BHLF1 transcript, a lytic transcript required for the process of viral DNA replication. Our findings demonstrate that Rta plays an indispensable role in the process of lytic DNA replication.

Structure des enzymes de réplication du virus Epstein-Barr.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen which establishes life-long persistent infection in the large majority of the human population. During viral latency, the cellular machinery takes care of the replication of the viral episome. But EBV, as well as herpesviruses in general, codes for numerous enzymes required for lytic DNA replication which allow viral replication in resting cells. Recently, several tridimensional structures of these enzymes became available for EBV as well as for other herpesviruses so that structural information now exists for most of them. The replication process and the structures of the proteins involved in replication are reviewed in the light of potential drug development and of herpesvirus evolution. The structures of the proteins involved in lytic replication show the relationship between herpesviruses and tailed bacteriophages, furthermore they show that EBV proteins tend to be more complex than their counterparts in other organisms. In this review, we could show the phylogenetic position of the herpesvirus helicase close to the Dda helicases involved in initiation of replication of the caudovirales.