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Class I histone deacetylases (HDACs) are cellular enzymes expressed in many tissues and play crucial roles in differentiation, proliferation, and cancer. HDAC1 and HDAC2 in particular are highly homologous proteins that show redundant or specific roles in different cell types or in response to different stimuli and signaling pathways. The molecular details of this dual regulation are largely unknown. HDAC1 and HDAC2 are not only protein modifiers, but are in turn regulated by post-translational modifications (PTMs): phosphorylation, acetylation, ubiquitination, SUMOylation, nitrosylation, and carbonylation. Some of these PTMs occur and crosstalk specifically on HDAC1 or HDAC2, creating a rational “code” for a differential, context-related regulation. The global comprehension of this PTM code is central for dissecting the role of single HDAC1 and HDAC2 in physiology and pathology.

We have previously demonstrated that Gam1, an avian adenoviral protein inhibits sumoylation. By counteracting the SUMO pathway, Gam1 has a significant impact on virus-infected cells, but in isolation the inhibitory effects of the Gam1 protein can be exploited to intentionally manipulate the SUMO system in vivo or in vitro. Here we discuss in detail the techniques we use to inhibit the SUMO pathway using the Gam1 protein.

Small ubiquitin-like modifier (SUMO)ylation is a key post-translational modification mechanism that controls the function of a plethora of proteins and biological processes. Given its central regulatory role, it is not surprising that it is widely exploited by viruses. A number of viral proteins are known to modify and/or be modified by the SUMOylation system to exert their function, to create a cellular environment more favorable for virus survival and propagation, and to prevent host antiviral responses. Since the SUMO pathway is a multi-step cascade, viral proteins engage with it at many levels, to advance and favor each stage of a typical infection cycle: replication, viral assembly and immune evasion. Here we review the current knowledge on the interplay between the host SUMO system and viral lifecycle.

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 HCMVinfected 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. peerReviewed