Human Cytomegalovirus UL44 Concentrates at the Periphery of Replication Compartments, the Site of Viral DNA Synthesis

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Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Most biological processes require protein-protein interactions. We have been studying human cytomegalovirus (HCMV) UL44 with the hypothesis that association of viral and cellular proteins with this protein is required for viral DNA synthesis. UL44 is the presumptive processivity subunit of the HCMV DNA polymerase and shares notable structural homology with the eukaryotic DNA polymerase processivity factor PCNA (3, 4). Multiple proteins required for DNA synthesis and repair associate with PCNA as the need for the function of these proteins arises (31, 32). Based on the structure of UL44 (3) and the interaction of UL44 with the catalytic subunit of the HCMV DNA polymerase UL54 (4), we hypothesized that, like PCNA, UL44 might interact with multiple viral and cellular proteins during viral DNA synthesis (4). In several proteomic studies we catalogued a large number of viral and cellular proteins that associate with UL44 in infected cell lysate (47, 50). These included the cellular protein nucleolin (47) and the viral proteins IRS1, TRS1 (49), and UL84 (47, 50). UL44 has also been found to associate with UL84 in a proteomic screen for interaction partners of UL84 in infected cell lysate (15).UL84 has been described as necessary for virus replication of HCMV laboratory strains AD169 and Towne (13,16,55) and for viral DNA synthesis of AD169 (16). Chromatin immunoprecipitation and proteomic studies have shown that UL84 is present at the viral origin of replication (7,26). It has been reported that UL84 has UTPase activity, and it has been suggested that UL84 functions as a viral origin binding protein (7, 9). However, sequence analysis of UL84 and related proteins indicates that UL84 is more related to dUTPase, not UTPase (11). Additionally, UL84 can interact with the viral transcriptional transactivator IE2 (44), potentially to modulate IE2-mediated transcription (19). Interaction of UL84 with IE2, via amino acids 68 to 105 of UL84 is necessary to maintain UL84 protein levels in the infected cell (37, 38). However, a recent report has indicated that in at least one strain of HCMV (TB40/E), UL84 is not required for viral replication (45).It is unknown what purpose the association of UL44 and UL84 serves in the infected cell and ...

Section: Discussionmentioning

confidence: 87%

A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization

Strang

Bender

Sharma

et al. 2012

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“…Within these compartments, viral DNA synthesis occurs concomitant with the accumulation of DNA replication proteins. In cells infected with the betaherpesvirus human cytomegalovirus (HCMV), these replication proteins include the accessory subunit of the viral DNA polymerase UL44 and the viral single-stranded DNA (ssDNA) binding protein UL57 (1)(2)(3). The HCMV replication compartment architecture has several distinguishing features (2,4).…”

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confidence: 99%

“…In particular, UL44 concentrates at the periphery of replication compartments. Viral DNA synthesis occurs within this peripheral layer, and newly replicated DNA is subsequently localized within the interior of replication compartments, which is where most UL57 is found (2). In contrast, studies of the alphaherpesvirus herpes simplex virus 1 (HSV-1) have revealed little evidence of comparable replication compartment organization (5,6).…”

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confidence: 99%

“…One is viral DNA synthesis, as treatment of infected cells with an inhibitor of viral DNA synthesis leads to loss of compartment organization (1,2), and compartment formation is delayed in cells infected with a virus mutant in which viral genome replication is delayed (1). Notably, a host nucleolar component, nucleolin (Ncl), colocalizes with UL44 at the periphery of replication compartments (3).…”

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confidence: 99%

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Dynamic and Nucleolin-Dependent Localization of Human Cytomegalovirus UL84 to the Periphery of Viral Replication Compartments and Nucleoli

Bender

Coen

Strang

2014

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Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAGtagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. IMPORTANCEThe protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular proteins required for HCMV genome replication and replicating viral DNA. We found that UL84 localizes with viral proteins, viral DNA, and the cellular nucleolar protein nucleolin in the subnuclear replication compartments in which viral DNA replication occurs. Unexpectedly, we also found localization of UL84 with nucleolin in nucleoli and showed that the presence of nucleolin is involved in localization of UL84 to the nucleus. These results add to previous work showing the importance of nucleolin in replication compartment architecture and viral DNA synthesis and are relevant to understanding UL84 function.

“…Very recently, "click chemistry" detecting EdU incorporation into replicating DNA was introduced to visualize active viral DNA synthesis in the periphery and interior of hCMV replication compartments (51). We combined EdU labeling with immunofluorescence to find that H3K4me2 colocalizes with nascent viral DNA within late replication compartments (Fig.…”

Section: Resultsmentioning

confidence: 99%

Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

Nitzsche

Steinhäußer

Mücke

et al. 2012

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.