Role of the Specific Interaction of UL112-113 p84 with UL44 DNA Polymerase Processivity Factor in Promoting DNA Replication of Human Cytomegalovirus

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: 99%

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

“…UL112-113 encodes four phosphoproteins, which are supposed to recruit proteins involved in DNA replication, to viral RCs (128,131). These viral replication core proteins include the DNA polymerase UL54, the polymerase-associated processivity factor UL44, and the ssDNA binding protein UL57, as well as a heterotrimer consisting of the DNA helicase UL105, the primase UL70, and the primase-associated factor (132,133).…”

Section: Nuclear Dna Virus Replication Centersmentioning

confidence: 99%

DNA Virus Replication Compartments

Schmid

Speiseder

Dobner

et al. 2014

151

142

bViruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication.

“…IE2-p86 recruits DE proteins encoded by UL112-113 to specialized nuclear sites (18,19), which in turn recruits ppUL84 and ppUL44, the viral DNA Pol processivity (Proc) factor, forming a complex that recognizes the origin of viral DNA replication, oriLyt, to initiate DNA synthesis (20)(21)(22)(23). The IE2-p86/ppUL84/ ppUL44/UL112-113 protein complex recruits single-stranded DNA binding protein, DNA Pol, and helicase-primase (22,23), ensuring timely production of viral DNA starting about 24 h postinfection (hpi) and peaking at 48 hpi. Viral DNA is packaged into preformed capsids starting at 48 hpi and peaking at 72 to 96 hpi (1).…”

mentioning

confidence: 99%

Cytomegalovirus UL91 Is Essential for Transcription of Viral True Late (γ2) Genes

Omoto

Mocarski

2013

Human cytomegalovirus-encoded UL91 is a betagamma gene that is essential for viral replication. Here we show that the 111-amino-acid (aa) UL91 protein controls accumulation of true-late (␥2) viral transcripts. The primate betaherpesvirus conserved N-terminal region from aa 1 to 71 is sufficient to fully reconstitute function. Evaluation of viral DNA, RNA, and antigen revealed that UL91 protein is expressed with leaky-late (␥1) kinetics, localizes in the nucleus without influencing viral DNA synthesis, and must be present from 48 h postinfection to support full expression of late viral transcripts and proteins. In the absence of UL91, viral capsid assembly in the nucleus of infected cells is significantly reduced, and mature, cytoplasmic virions fail to form. Taken together, the evidence shows that UL91 regulates late viral gene expression by a mechanism that is apparently conserved in betaherpesviruses and gammaherpesviruses.