title: The human cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNA-dependent manner.
abstract: During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed 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 HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system.
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Sinigalia
authors.forename: Gualtiero
authors.initials: G
authors.lastname: Alvisi
authors.forename: Chiara V
authors.initials: CV
authors.lastname: Segré
authors.forename: Beatrice
authors.initials: B
authors.lastname: Mercorelli
authors.forename: Giulia
authors.initials: G
authors.lastname: Muratore
authors.forename: Michael
authors.initials: M
authors.lastname: Winkler
authors.forename: He-Hsuan
authors.initials: HH
authors.lastname: Hsiao
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ripalti
authors.forename: Susanna
authors.initials: S
authors.lastname: Chiocca
authors.forename: Giorgio
authors.initials: G
authors.lastname: Palù
authors.forename: Arianna
authors.initials: A
authors.lastname: Loregian
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D025841:Small Ubiquitin-Related Modifier Proteins; D014764:Viral Proteins; D004247:DNA; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9