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.

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

“…Therefore, there may be SUMOylation sites that cannot be predicted by bioinformatics software. For instance, mutation of the predicted SUMOylation sites in the human cytomegalovirus DNA polymerase processivity factor UL44 hardly attenuates the modification (25). Here, our finding of L150/D151/ L152 as a SIM site may expand the data pool for developing a higher-confidence algorithm.…”

“…Only EV71 3C protease is SUMOylated, while coxsackievirus B5 is involved in the host cell SUMOylation system in the family Picornaviridae (38,62,70). Current understanding of viral polymerase SUMOylation is limited to RdRp of Turnip mosaic virus (71), DNA polymerase subunit UL44 of HCMV (25), nonstructural protein 5 (NS5) of dengue virus (72), and polymerase basic protein 1 (PB1) of influenza virus (30). In the currently study, we reveal that the 3D protein of EV71, an RNA-dependent RNA polymerase, is SUMOylated.…”

“…Some viruses possess more than one SUMOylated protein, indicating the complexity of virus-host interactions. For instance, SUMOylation of human cytomegalovirus (HCMV) DNA polymerase subunit UL44 and immediate-early 1 and 2 (IE1 and IE2) proteins is required for efficient viral replication (23)(24)(25)(26). At least five influenza virus proteins are SUMO targets, and infection with influenza virus leads to a global increase in cellular SUMOylation (27).…”

SUMO-mediated regulation of DNA damage repair and responses

“…Recent characterization of additional substrates shows that this is not an isolated example; rather, the prerequisite of DNA association for sumoylation is a general trend. For instance, the non-homologous end joining (NHEJ) repair protein Yku70, the base excision repair protein Thymine DNA Glycosylase (TDG), the Rad1 nuclease, the viral polymerase processivity factor UL44, and the Fanconi anemia pathway proteins FANCI and FANCD2 all require DNA association for sumoylation [31–35]. In the case of the yeast Yku70 and human TDG proteins, which function upstream in their respective repair pathways, mutations abolishing their DNA binding prevent their sumoylation [34, 35].…”

“…In the case of the yeast Yku70 and human TDG proteins, which function upstream in their respective repair pathways, mutations abolishing their DNA binding prevent their sumoylation [34, 35]. Similarly, a DNA binding mutation in the PCNA-like UL44 protein also impairs its sumoylation [31]. In some cases, DNA repair engagement, rather than simply DNA association, is required for sumoylation, as seen for the yeast Rad1 nuclease and human FANCI and FANCD2 proteins [32, 33].…”