Ubiquitin-specific Protease 7 Plays a Critical Role in Control of Lytic Infection and Cell Transformation by Human Adenovirus Type 5

Abstract

The ubiquitin-specific protease 7 (Usp7), also known as herpesvirus-associated ubiquitin-specific protease (HAUSP) is a critical component of the p53-Mdm2 stress response pathway and acts as a specific de-ubiquitinase for both p53 and Mdm2 and thus is important for p53 regulation. In addition, Usp7 was shown to interact with immediate-early proteins from Herpes simplex virus type 1 (Icp0) and Epstein-Barr virus (EBNA1), indicating that Usp7 may play an important role in the life cycle of several DNA viruses.

In the present study, Usp7 was identified as a novel interaction partner of the adenovirus type 5 (Ad5) E1B-55K protein. Similar to Icp0 and EBNA1, E1B-55K is a key regulator of Ad5 replication, and is required for maximal virus production. In vitro and in vivo binding assays demonstrated that E1B-55K binds to Usp7 with high affinity. The binding region mapped to a highly conserved motif located in the amino-terminal region of the Ad5 protein. Phenotypic analyses and functional studies using an Usp7-binding deficient E1B mutant virus and stable Usp7 knockdown cell lines, showed that Usp7 regulates E1B-55K lytic functions and contributes to efficient virus replication by increasing the stability of the viral protein. Surprisingly, these studies also demonstrated that Usp7 additionally contributes to Ad5 replication by an E1B-55K-independent mechanism. This activity of Usp7 involves the binding to and stabilization of the Ad5 DNA-binding protein (DBP). It appeared that binding to DBP occurs in the context of Usp7-positive PML-containing nuclear bodies (PML bodies), which form the origin of viral microenvironments of viral DNA replication and transcription. Finally, one-step growth curve experiments demonstrated that efficient virus growth is directly proportional to the Usp7 steady-state concentrations of the host cell. Thus, Usp7 is a key factor that is required for maximal virus production in permissive host cells, by promoting the formation of viral transcription and replication centers through its interaction with E1B-55K and DBP in the context of PML bodies.

In addition, to its role in promoting virus replication, E1B-55K provides functions necessary for oncogenic transformation of primary mammalian cells in culture. It is generally considered that E1B-55K contributes to complete cell transformation by antagonizing apoptosis and growth arrest, which primarily result from the induction and metabolic stabilization of the tumor suppressor protein p53 by adenovirus E1A. These growth-promoting activities correlate with its ability to act as a transcriptional repressor that is targeted to p53-responsive promoters by binding to p53. Nonetheless, it has been hypothesized that the mode of action of E1B-55K during transformation may involve additional functions and other protein interactions. To test this model the role of Usp7 and p53 on E1A- plus E1B-mediated cell transformation was investigated by using an RNA interference approach. Similar to virus infected cells, the results of these experiments showed that Usp7 is required for efficient cell transformation of primary rat cells by affecting both p53 and E1B-55K steady-state concentrations indicating that Usp7 is an essential factor for Ad5 E1A and E1B-55K-mediated oncogenesis.