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.
Highlights
Most replicative DNA polymerases include a catalytic subunit, responsible for DNA polymerization, and a processivity factor that holds the catalytic subunit on DNA to allow continuous DNA synthesis
To identify cellular proteins that interact with UL44, Y2H screens were carried out with a bait consisting of full-length UL44 protein fused to the E. coli LexA protein
Control experiments demonstrated that the LexA-UL44 protein did not activate expression of either HIS3 or lacZ reporter gene by itself and, as expected [9,14], could both interact with UL54 and dimerize (Table 1 and data not shown)
Summary
Most replicative DNA polymerases include a catalytic subunit, responsible for DNA polymerization, and a processivity factor that holds the catalytic subunit on DNA to allow continuous DNA synthesis. One of the best-studied processivity factors is proliferating cell nuclear antigen (PCNA) of eukaryotic DNA polymerases d and e [1]. In addition to DNA replication, PCNA has been implicated in DNA recombination and repair, as well as in DNA methylation, chromatin remodeling, and cell cycle regulation [1]. The human cytomegalovirus (HCMV) DNA polymerase includes a catalytic subunit, UL54 (the UL54 gene product), and an accessory, homodimeric subunit, UL44 (the UL44 gene product), that binds DNA without the aid of clamp loaders [7] yet wraps around DNA akin to PCNA [8]. Its early-late kinetics of transcription and the high level of expression suggest that UL44 might play additional roles during the viral life cycle
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