scholarly journals Host Cell Nucleolin Is Required To Maintain the Architecture of Human Cytomegalovirus Replication Compartments

mBio ◽  
2012 ◽  
Vol 3 (1) ◽  
Author(s):  
Blair L. Strang ◽  
Steeve Boulant ◽  
Tomas Kirchhausen ◽  
Donald M. Coen
Keyword(s):  
Viral Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Cellular Protein ◽  
Major Protein ◽  
Viral Dna ◽  
Infected Cells ◽  
Interfering Rna ◽  
Replication Compartment

ABSTRACTDrastic reorganization of the nucleus is a hallmark of herpesvirus replication. This reorganization includes the formation of viral replication compartments, the subnuclear structures in which the viral DNA genome is replicated. The architecture of replication compartments is poorly understood. However, recent work with human cytomegalovirus (HCMV) showed that the viral DNA polymerase subunit UL44 concentrates and viral DNA synthesis occurs at the periphery of these compartments. Any cellular factors involved in replication compartment architecture are largely unknown. Previously, we found that nucleolin, a major protein component of nucleoli, associates with HCMV UL44 in infected cells and is required for efficient viral DNA synthesis. Here, we show that nucleolin binds to purified UL44. Confocal immunofluorescence analysis demonstrated colocalization of nucleolin with UL44 at the periphery of replication compartments. Pharmacological inhibition of viral DNA synthesis prevented the formation of replication compartments but did not abrogate association of UL44 and nucleolin. Thus, association of UL44 and nucleolin is unlikely to be a nonspecific effect related to development of replication compartments. No detectable colocalization of 5-ethynyl-2′-deoxyuridine (EdU)-labeled viral DNA with nucleolin was observed, suggesting that nucleolin is not directly involved in viral DNA synthesis. Small interfering RNA (siRNA)-mediated knockdown of nucleolin caused improper localization of UL44 and a defect in EdU incorporation into viral DNA. We propose a model in which nucleolin anchors UL44 at the periphery of replication compartments to maintain their architecture and promote viral DNA synthesis.IMPORTANCEHuman cytomegalovirus (HCMV) is an important human pathogen. HCMV infection causes considerable rearrangement of the structure of the nucleus, largely due to the formation of viral replication compartments within the nucleus. Within these compartments, the virus replicates its DNA genome. We previously demonstrated that nucleolin is required for efficient viral DNA synthesis and now find that the nucleolar protein nucleolin interacts with a subunit of the viral DNA polymerase, UL44, specifically at the periphery of replication compartments. Moreover, we find that nucleolin is required to properly localize UL44 at this region. Nucleolin is, therefore, involved in the organization of proteins within replication compartments. This, to our knowledge, is the first report identifying a cellular protein required for maintaining replication compartment architecture.

scholarly journals Nucleolin Associates with the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 and Is Necessary for Efficient Viral Replication

2009 ◽  
Vol 84 (4) ◽  
pp. 1771-1784 ◽  
Author(s):  
Blair L. Strang ◽  
Steeve Boulant ◽  
Donald M. Coen
Keyword(s):  
Infected Cell ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Western Blotting ◽  
Human Cytomegalovirus ◽  
Major Protein ◽  
Cell Nuclei ◽  
Viral Dna ◽  
Infected Cells ◽  
Processivity Factor

ABSTRACT In the eukaryotic cell, DNA replication entails the interaction of multiple proteins with the DNA polymerase processivity factor PCNA. As the structure of the presumptive human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is highly homologous to that of PCNA, we hypothesized that UL44 also interacts with numerous proteins. To investigate this possibility, recombinant HCMV expressing FLAG-tagged UL44 was generated and used to immunoprecipitate UL44 and associated proteins from infected cell lysates. Unexpectedly, nucleolin, a major protein component of the nucleolus, was identified among these proteins by mass spectrometry and Western blotting. The association of nucleolin and UL44 in infected cell lysate was confirmed by reciprocal coimmunoprecipitation in the presence and absence of nuclease. Western blotting and immunofluorescence assays demonstrated that the level of nucleolin increases during infection and that nucleolin becomes distributed throughout the nucleus. Furthermore, the colocalization of nucleolin and UL44 in infected cell nuclei was observed by immunofluorescence assays. Assays of HCMV-infected cells treated with small interfering RNA (siRNA) targeting nucleolin mRNA indicated that nucleolin was required for efficient virus production, viral DNA synthesis, and the expression of a late viral protein, with a correlation between the efficacy of knockdown and the effect on virus replication. In contrast, the level of neither global protein synthesis nor the replication of an unrelated virus (reovirus) was reduced in siRNA-treated cells. Taken together, our results indicate an association of nucleolin and UL44 in HCMV-infected cells and a role for nucleolin in viral DNA synthesis.

scholarly journals Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

1997 ◽  
Vol 41 (12) ◽  
pp. 2680-2685 ◽  
Author(s):  
D J Tenney ◽  
G Yamanaka ◽  
S M Voss ◽  
C W Cianci ◽  
A V Tuomari ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Potent Inhibitor ◽  
Viral Dna ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv Thymidine Kinase

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.

scholarly journals Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

2012 ◽  
Vol 86 (18) ◽  
pp. 9817-9827 ◽  
Author(s):  
Alexandra Nitzsche ◽  
Charlotte Steinhäußer ◽  
Katrin Mücke ◽  
Christina Paulus ◽  
Michael Nevels
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Histone Modification ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Histone H3 ◽  
Viral Dna ◽  
The Impact ◽  
Preferential Incorporation

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using “click chemistry” revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.

scholarly journals The Human Cytomegalovirus IE2 and UL112-113 Proteins Accumulate in Viral DNA Replication Compartments That Initiate from the Periphery of Promyelocytic Leukemia Protein-Associated Nuclear Bodies (PODs or ND10)

1999 ◽  
Vol 73 (12) ◽  
pp. 10458-10471 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Won-Jong Jang ◽  
Gary S. Hayward
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Promyelocytic Leukemia Protein ◽  
The Core ◽  
Replication Compartment

ABSTRACT During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)-associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne)-infected HF cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the core replication machinery proteins in the initial stages of viral replication compartment formation.

scholarly journals Recruitment of Human Cytomegalovirus Immediate-Early 2 Protein onto Parental Viral Genomes in Association with ND10 in Live-Infected Cells

2007 ◽  
Vol 81 (18) ◽  
pp. 10123-10136 ◽  
Author(s):  
George Sourvinos ◽  
Nina Tavalai ◽  
Anja Berndt ◽  
Demetrios A. Spandidos ◽  
Thomas Stamminger
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Spatial Organization ◽  
Human Fibroblasts ◽  
Nuclear Bodies ◽  
Immediate Early ◽  
Viral Dna ◽  
Subnuclear Localization ◽  
Pml Nuclear Bodies ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise colocalizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.

scholarly journals Role of ATM in the Formation of the Replication Compartment during Lytic Replication of Epstein-Barr Virus in Nasopharyngeal Epithelial Cells

2014 ◽  
Vol 89 (1) ◽  
pp. 652-668 ◽  
Author(s):  
Pok Man Hau ◽  
Wen Deng ◽  
Lin Jia ◽  
Jie Yang ◽  
Tatsuya Tsurumi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Viral Replication ◽  
Epstein Barr Virus ◽  
Viral Dna ◽  
Barr Virus ◽  
Lytic Reactivation ◽  
Infected Cells ◽  
Epstein Barr ◽  
Replication Compartment

ABSTRACTEpstein-Barr virus (EBV), a type of oncogenic herpesvirus, is associated with human malignancies. Previous studies have shown that lytic reactivation of EBV in latently infected cells induces an ATM-dependent DNA damage response (DDR). The involvement of ATM activation has been implicated in inducing viral lytic gene transcription to promote lytic reactivation. Its contribution to the formation of a replication compartment during lytic reactivation of EBV remains poorly defined. In this study, the role of ATM in viral DNA replication was investigated in EBV-infected nasopharyngeal epithelial cells. We observed that induction of lytic infection of EBV triggers ATM activation and localization of DDR proteins at the viral replication compartments. Suppression of ATM activity using a small interfering RNA (siRNA) approach or a specific chemical inhibitor profoundly suppressed replication of EBV DNA and production of infectious virions in EBV-infected cells induced to undergo lytic reactivation. We further showed that phosphorylation of Sp1 at the serine-101 residue is essential in promoting the accretion of EBV replication proteins at the replication compartment, which is crucial for replication of viral DNA. Knockdown of Sp1 expression by siRNA effectively suppressed the replication of viral DNA and localization of EBV replication proteins to the replication compartments. Our study supports an important role of ATM activation in lytic reactivation of EBV in epithelial cells, and phosphorylation of Sp1 is an essential process downstream of ATM activation involved in the formation of viral replication compartments. Our study revealed an essential role of the ATM-dependent DDR pathway in lytic reactivation of EBV, suggesting a potential antiviral replication strategy using specific DDR inhibitors.IMPORTANCEEpstein-Barr virus (EBV) is closely associated with human malignancies, including undifferentiated nasopharyngeal carcinoma (NPC), which has a high prevalence in southern China. EBV can establish either latent or lytic infection depending on the cellular context of infected host cells. Recent studies have highlighted the importance of the DNA damage response (DDR), a surveillance mechanism that evolves to maintain genome integrity, in regulating lytic EBV replication. However, the underlying molecular events are largely undefined. ATM is consistently activated in EBV-infected epithelial cells when they are induced to undergo lytic reactivation. Suppression of ATM inhibits replication of viral DNA. Furthermore, we observed that phosphorylation of Sp1 at the serine-101 residue, a downstream event of ATM activation, plays an essential role in the formation of viral replication compartments for replication of virus DNA. Our study provides new insights into the mechanism through which EBV utilizes the host cell machinery to promote replication of viral DNA upon lytic reactivation.

scholarly journals Human cytomegalovirus miR-US33-5p inhibits viral DNA synthesis and viral replication by down-regulating expression of the host Syntaxin3

FEBS Letters ◽  
2015 ◽  
Vol 589 (4) ◽  
pp. 440-446 ◽  
Author(s):  
Xin Guo ◽  
Ying Qi ◽  
Yujing Huang ◽  
Zhongyang Liu ◽  
Yanping Ma ◽  
...  
Keyword(s):  
Viral Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Viral Dna

scholarly journals Sumoylation of the Carboxy-Terminal of Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Attenuates Viral DNA Replication

2021 ◽  
Vol 12 ◽  
Author(s):  
Jun Chen ◽  
Guanlie Li ◽  
Haiqing He ◽  
Xin Li ◽  
Wenjing Niu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Sumoylation Site ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Infected Cells ◽  
Processivity Factor ◽  
Carboxy Terminal

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV DNA polymerase processivity factor UL44 plays an essential role in viral DNA replication. To better understand the biology of UL44, we performed a yeast two-hybrid screen for host proteins that could interact with UL44. The most frequently isolated result was the SUMO-conjugating enzyme UBC9, a protein involved in the sumoylation pathway. The UBC9-UL44 interaction was confirmed by in vitro His-tag pull-down and in vivo co-immunoprecipitation assays. Using deletion mutants of UL44, we mapped two small regions of UL44, aa 11–16, and 260–269, which might be critical for the interaction with UBC9. We then demonstrated that UL44 was a target for sumoylation by in vitro and in vivo sumoylation assays, as well as in HCMV-infected cells. We further confirmed that 410lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although 410lysine had no effects on subcellular localization or protein stability of UL44, the removal of 410lysine sumoylation site enhanced both viral DNA synthesis in transfection-replication assays and viral progeny production in infected cells for HCMV, suggesting sumoylation can attenuate HCMV replication through targeting UL44. Our results suggest that sumoylation plays a key role in regulating UL44 functions and viral replication, and reveal the crucial role of the carboxy-terminal of UL44, for which little function has been known before.

scholarly journals Potent and Selective Inhibition of Human Cytomegalovirus Replication by 1263W94, a Benzimidazole l-Riboside with a Unique Mode of Action

2002 ◽  
Vol 46 (8) ◽  
pp. 2365-2372 ◽  
Author(s):  
Karen K. Biron ◽  
Robert J. Harvey ◽  
Stanley C. Chamberlain ◽  
Steven S. Good ◽  
Albert A. Smith ◽  
...  
Keyword(s):  
Viral Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Resistance Mutation ◽  
Selective Inhibition ◽  
Antiviral Effect ◽  
Viral Gene ◽  
Dependent Manner ◽  
Viral Dna

ABSTRACT Benzimidazole nucleosides have been shown to be potent inhibitors of human cytomegalovirus (HCMV) replication in vitro. As part of the exploration of structure-activity relationships within this series, we synthesized the 2-isopropylamino derivative (3322W93) of 1H-β-d-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural l-sugars corresponding to both compounds. One of the l derivatives, 1H-β-l-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94), showed significant antiviral potency in vitro against both laboratory HCMV strains and clinical HCMV isolates, including those resistant to ganciclovir (GCV), foscarnet, and BDCRB. 1263W94 inhibited viral replication in a dose-dependent manner, with a mean 50% inhibitory concentration (IC50) of 0.12 ± 0.01 μM compared to a mean IC50 for GCV of 0.53 ± 0.04 μM, as measured by a multicycle DNA hybridization assay. In a single replication cycle, 1263W94 treatment reduced viral DNA synthesis, as well as overall virus yield. HCMV mutants resistant to 1263W94 were isolated, establishing that the target of 1263W94 was a viral gene product. The resistance mutation was mapped to the UL97 open reading frame. The pUL97 protein kinase was strongly inhibited by 1263W94, with 50% inhibition occurring at 3 nM. Although HCMV DNA synthesis was inhibited by 1263W94, the inhibition was not mediated by the inhibition of viral DNA polymerase. The parent benzimidazole d-riboside BDCRB inhibits viral DNA maturation and processing, whereas 1263W94 does not. The mechanism of the antiviral effect of l-riboside 1263W94 is thus distinct from those of GCV and of BDCRB. In summary, 1263W94 inhibits viral replication by a novel mechanism that is not yet completely understood.

scholarly journals Impact of 2-Bromo-5,6-Dichloro-1-β-d-Ribofuranosyl Benzimidazole Riboside and Inhibitors of DNA, RNA, and Protein Synthesis on Human Cytomegalovirus Genome Maturation

2005 ◽  
Vol 79 (17) ◽  
pp. 11115-11127 ◽  
Author(s):  
Michael A. McVoy ◽  
Daniel E. Nixon
Keyword(s):  
Protein Synthesis ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Dna Polymerases ◽  
Viral Dna ◽  
Rna Transcription ◽  
Damage Repair ◽  
Genome Maturation ◽  
Herpesvirus Genome

ABSTRACT Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-β-d-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.

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