Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells

  title={Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells},
  author={Elisa Sinigalia and Gualtiero Alvisi and Beatrice Mercorelli and Donald M Coen and Gregory S. Pari and David Andrew Jans and Alessandro Ripalti and Giorgio Pal{\`u} and Arianna Loregian},
  journal={Journal of Virology},
  pages={12574 - 12579}
ABSTRACT The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV oriLyt… 

The Carboxy-Terminal Segment of the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 Is Crucial for Viral Replication

Although the amino-terminal domain was sufficient for origin-dependent synthesis in a transient-transfection assay, the carboxy- terminal segment was crucial for virus replication and for the formation of DNA replication compartments in infected cells, even when this segment was replaced with a simian virus 40 NLS that ensured nuclear localization.

The Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Is Modified by SUMO in a DNA-Dependent Manner

It is shown that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in H CMV life cycle and UL44 function(s).

Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

Results strongly suggest that UL44 and UL84 interact directly using a region of UL44 different from the UL54 binding site, which means UL44 can bind interacting replication proteins using a mechanism different from that of PCNA.

The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells

ULTS indicate that ppUL44Δloop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type pUL44.

Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods

Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppul44 homodimer and in the DNA polymerase holoenzyme.

Towards the identification of small molecules inhibiting he dimerization of HCMV DNA polymerase processivity factor UL44

It is confirmed that UL44 forms dimers in cells, as well as gaining insights relative to the formation of HMCV DNA polymerase holoenzyme, suggesting conformational changes within UL44 upon DNA binding in complex to UL54.

Regulated Transport into the Nucleus of Herpesviridae DNA Replication Core Proteins

Mechanisms controlling the nuclear import of herpesviral DNA replication machinery are summarized and potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle are discussed.

Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

It is shown that full length UL44 dimerizes in a cellular context with high affinity and that such interaction could be targeted by small molecules, thus inhibiting the replication of several HCMV strains, including a drug-resistant mutant.



The cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer.

Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus.

It is shown that pUL44 dimerizes in the cytoplasm via its N-terminal domain, before translocating to the nucleus, and that nuclear translocation of differently tagged ppUL44 heterodimers can occur even when one subunit carries a nonfunctional nuclear localization signal.

Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication

Preliminary deletion analysis suggests that multiple components within the boundaries of oriLyt cooperate to enable initiation of HCMV lytic-phase DNA synthesis.

Residues of Human Cytomegalovirus DNA Polymerase Catalytic Subunit UL54 That Are Necessary and Sufficient for Interaction with the Accessory Protein UL44

It is observed that deletion of the C-terminal segment or substitution of alanine for Leu1227 or Phe1231 in UL54 greatly impaired both the UL54-UL44 interaction in pulldown assays and long-chain DNA synthesis without affecting basal polymerase activity, identifying these residues as important for subunit interaction.

Human Cytomegalovirus DNA Polymerase Catalytic Subunit pUL54 Possesses Independently Acting Nuclear Localization and ppUL44 Binding Motifs

Two distinct sites within the HCMV DNA polymerase are identified, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import, which is identified and established.

Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA

The data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

Physical and functional interaction of human cytomegalovirus DNA polymerase and its accessory protein (ICP36) expressed in insect cells

Expression of the human cytomegalovirus (HCMV) (AD169) DNA polymerase gene under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus in Spodoptera frugiperda

The Human Cytomegalovirus UL44 Protein Is a Substrate for the UL97 Protein Kinase

It is indicated that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL 44 in infected cells, which strongly suggests that UL44 is a natural substrate of UL97.

Specific Residues in the Connector Loop of the Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 Are Crucial for Interaction with the UL54 Catalytic Subunit

The crystal structure of UL44 revealed the presence of a connector loop analogous to that of the processivity subunit of herpes simplex virus DNA polymerase, UL42, which is crucial for interaction with its cognate catalytic subunit, UL30.