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.DNA polymerase is essential for the replication of DNA. Most replicative DNA polymerases include a catalytic subunit, required for DNA polymerization, and a processivity subunit that holds the catalytic subunit of the polymerase on DNA to permit continuous DNA synthesis and, in some cases, to interact with other proteins required for DNA synthesis as the need arises. For example, proliferating cell nuclear antigen (PCNA), the processivity factor of eukaryotic DNA polymerases ␦ and ε, is capable of numerous interactions with proteins that aid and abet DNA synthesis (33,35). Human cytomegalovirus (HCMV) encodes a dimeric DNA polymerase, which includes the catalytic subunit UL54 and the presumptive processivity factor UL44. Previous studies of UL44 have revealed that UL44 forms a head-to-head homodimer (2) that has structural homology to PCNA (2, 3) and, like PCNA, can wrap around DNA (25). These results give rise to the hypothesis that UL44 can interact with multiple proteins involved in DNA synthesis.Other than UL54 (12), to date, three viral proteins have been reported to associate with UL44 in the infected cell: the viral kinase UL97 (26, 34), the uracil DNA glycosylase UL114 (39,40), and the DNA replication factor UL84 (14, 47). To investigate whether other viral and cellular proteins associate with UL44, ...

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For many years, the generally accepted model for the replication of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) incorporated initial circularization of linear molecules in the cell nucleus. Ensuing DNA synthesis resulted in the generation of head-to-tail concatemers which were subsequently cleaved into monomeric units and packaged into the nascent viral capsid. Recently, however, it has been proposed that circularization of HSV-1 genomes does not occur at the onset of lytic infection and moreover that this event is specifically inhibited by the HSV-1 transcriptional transactivator, ICP0 (S.A. Jackson and N.A. DeLuca, Proc. Natl. Acad. Sci. USA 100:7871-7876, 2003). To further investigate genome circularization, we have generated HSV-1 derivatives in which the viral a sequences, which contain the cleavage-packaging signals, have been replaced by a minimal packaging element located in the thymidine kinase gene. In contrast to wild-type HSV-1, fusion of the genomic termini of these viruses produces a novel fragment in circular or concatemeric DNA which can be detected by Southern blot hybridization. Utilizing these viruses, we demonstrate that fusion of the genomic termini occurred rapidly upon infection and in the presence of inhibitors of viral DNA or protein synthesis. We provide evidence indicating that the end joining represented circularization rather than concatemerization of input molecules and that circularized molecules functioned as templates for replication. Since the termini of these viruses lack direct repeats, our findings indicate that circularization can be mediated by direct end-to-end ligation of linear input genomes.Herpes simplex virus type 1 (HSV-1), the prototype virus of the family Herpesviridae, possesses a linear double-stranded DNA genome of approximately 153 kbp and is widely used as a model for the study of herpesvirus DNA replication and recombination. Like those of other members of the herpesvirus family, the HSV-1 genome is characterized by the presence of unique and repeated sequences (Fig. 1a). Two covalently joined segments, L and S, each comprise a unique region (U L and U S ) flanked by a set of inverted repeats (TR L and IR L , TR S and IR S , respectively). A region of approximately 400 bp, the a sequence, is present as a direct repeat at the genomic termini and in an inverted orientation at the junction between the L and S segments. The L and S segments of the genome have the capacity to invert relative to each other at high frequency, resulting in the appearance of four equimolar isomeric forms of virion DNA (reviewed in reference 35).Following infection, the HSV-1 genome is released into the nucleus and may either be retained in a latent state or enter into the lytic cycle (reviewed in reference 32). During the establishment of latency in both in vivo and tissue culture systems, the ends of the viral genome become joined, and there is now strong evidence for persistence as a nonreplicating circular episomal form (5,17,18,27,32,34,42). The lytic cyc...

The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In contrast, the viral single-stranded-DNA binding protein UL57 was distributed throughout replication compartments. Using "click chemistry" to detect 5-ethynyl-2=-deoxyuridine (EdU) incorporation into replicating viral DNA and pulse-chase protocols, we found that viral DNA synthesis occurs at the periphery of replication compartments and that replicated viral DNA subsequently localizes to the interior of replication compartments. The interiors of replication compartments also contain regions in which UL44 and EdU-labeled DNA are absent. The treatment of cells with a viral DNA polymerase inhibitor reversibly caused the dispersal of both UL44 and EdU-labeled viral DNA from replication compartments, indicating that ongoing viral DNA synthesis is necessary to maintain the organization of replication compartments. Our results reveal a previously unappreciated complexity of the organization of human cytomegalovirus replication compartments.T he replication of viral genomes takes place in discrete sites within the cell, which enables viruses to concentrate and organize factors required for genome replication. During herpesvirus infection, a drastic and dynamic reorganization of the nucleus is observed, including the partitioning of host cell chromatin and the rearrangement of cellular nuclear proteins due primarily to the development of viral replication compartments (20,23,26).The formation of human cytomegalovirus (HCMV) replication compartments in infected cells has been observed, as has the localization of several viral proteins within them (2, 10, 21). It is unclear how viral proteins are organized within replication compartments, and it is unknown where viral DNA synthesis occurs within compartments.In a previous report from our laboratory, we assayed the localization of the presumptive viral DNA polymerase processivity subunit UL44 (also known as ICP36) in infected cells as a marker for infected-cell nuclei (10). Although we did not comment upon it at the time, we observed that UL44 accumulates at the periphery of replication compartments. To our knowledge, no viral protein in any herpesvirus replication compartment had shown this distribution, so we wished to investigate this observation further, hypothesizing that it might signal how DNA synthesis is organized within replication compartments. We therefore examined the localization of UL44, another viral DNA replication protein, and viral DNA synthesis within replication compartments. MATERIALS AND METHODS Cells and viruses. Human foreskin fibroblast (HFF) cells (ATCC CRL-1684;...

The central enzyme responsible for human cytomegalovirus (HCMV) DNA synthesis is a virally encoded DNA polymerase that includes a catalytic subunit, UL54, and a homodimeric accessory subunit, UL44, the presumptive HCMV DNA polymerase processivity factor. The structure of UL44 is similar to that of the eukaryotic processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous other proteins required for faithful DNA replication. We sought to determine whether, like PCNA, UL44 is capable of interacting with multiple DNA replication proteins and, if so, whether these proteins bind UL44 at the site corresponding to where multiple proteins bind to PCNA. Initially, several proteins, including the viral DNA replication factors UL84 and UL57, were identified by mass spectrometry in immunoprecipitates of UL44 from infected cell lysate. The association of UL44/UL84, but not UL44/UL57, was confirmed by reciprocal coimmunoprecipitation of these proteins from infected cell lysates and was resistant to nuclease treatment. Yeast two-hybrid analyses demonstrated that the substitution of residues in UL44 that prevent UL44 homodimerization or abrogate the binding of UL54 to UL44 do not abrogate the UL44/UL84 interaction. Reciprocal glutathione-S-transferase (GST) pulldown experiments using bacterially expressed UL44 and UL84 confirmed these results and, further, demonstrated that a UL54-derived peptide that competes with UL54 for UL44 binding does not prevent the association of UL84 with UL44. Taken together, our results strongly suggest that UL44 and UL84 interact directly using a region of UL44 different from the UL54 binding site. Thus, UL44 can bind interacting replication proteins using a mechanism different from that of PCNA.Protein-protein interactions orchestrate the process of DNA synthesis. Most replicative DNA polymerases include at least two interacting components, a catalytic subunit responsible for the polymerization of DNA and a processivity subunit. The processivity subunit of the polymerase holds the catalytic subunit on DNA while DNA polymerization takes place, thereby permitting long-chain DNA synthesis. One of the best-described processivity factors is proliferating cell nuclear antigen (PCNA) of eukaryotic DNA polymerases ␦ and ε (23, 26). PCNA is a head-to-tail homotrimer which, with the aid of clamp loader proteins, forms a toroidal ring around DNA, creating an internal channel of sufficient diameter to accommodate the DNA duplex (15). Studies of PCNA by numerous laboratories have described a large number of interactions between PCNA and proteins that participate in and abet DNA synthesis (reviewed in references 23 and 26). Of particular interest is that many of these proteins bind PCNA at a specific site, inserting a conserved hydrophobic domain (a PCNA-interacting protein [PIP] box) into a hydrophobic pocket lying beneath an interdomain connector loop of PCNA. PIP box proteins bind and dissociate from PCNA at different times during DNA replication when the need for their funct...

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.

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To identify new compounds with anti-human cytomegalovirus (HCMV) activity and new anti-HCMV targets, we developed a high-throughput strategy to screen a GlaxoSmithKline Published Kinase Inhibitor Set. This collection contains a range of extensively characterized compounds grouped into chemical families (chemotypes). From our screen, we identified compounds within chemotypes that impede HCMV protein production and identified kinase proteins associated with inhibition of HCMV protein production that are potential novel anti-HCMV targets. We focused our study on a top 'hit' in our screen, SB-734117, which we found inhibits productive replication of several HCMV strains. Kinase selectivity data indicated that SB-734117 exhibited polypharmacology and was an inhibitor of several proteins from the AGC and CMCG kinase groups. Using Western blotting, we found that SB-734711 inhibited accumulation of HCMV immediate-early proteins, phosphorylation of cellular proteins involved in immediate-early protein production (cAMP response element-binding protein and histone H3) and histone H3 lysine 36 trimethylation (H3K36me3). Therefore, we identified SB-734117 as a novel anti-HCMV compound and found that inhibition of AGC and CMCG kinase proteins during productive HCMV replication was associated with inhibition of viral protein production and prevented post-translational modification of cellular factors associated with viral protein production.