Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 and Its Isolated RING Finger Domain Act as Ubiquitin E3 Ligases In Vitro

Abstract

Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.Herpes simplex virus type 1 (HSV-1) is a significant human pathogen whose biological and clinical importance is emphasized by its ability to attain and reactivate from a latent state in sensory neurons (reviewed in reference 16). The mechanisms that control the balance between the lytic and latent states are of considerable interest yet are incompletely understood. Our past studies have concentrated on the functions and mechanisms of action of HSV-1 immediate-early regulatory protein ICP0, which is required for the efficient initiation of lytic cycle gene expression, reactivation of quiescent virus in cultured cells, and reactivation of latent virus in mouse models (for reviews, see references 5 and 15; see also references 18 and 19).ICP0 is being actively studied in a number of laboratories, and a wide spectrum of possible functions, interactions, and mechanisms of action are being revealed. Early transfection studies showed that ICP0 is able to increase the expression of a wide variety of genes in cotransfected cells, and this effect does not depend on specific promoter sequences. Since ICP0 does not bind directly to DNA (13), it is likely that it functions via interactions with other proteins. Recent studies have proposed a number of possible interactions, including USP7 (a ubiquitin-specific protease) (11), cyclin D3 (25), elongation factor EF-1␦ (23), the transcription factor BMAL1 (24), and the major HSV-1 transcriptional regulator ICP4 (42). ICP0 has also been suggested to activate cdk4 and to stabilize both cyclin D1 and cyclin D3 (40). Whatever the significance of these varied observations, it is now generally accepted that a major biological activity of ICP0 causes the disruption of specific nuclear structures known as ND10 or promyelocytic leukemia (PML) nuclear bodies in a process which correlates with the ability of ICP0 to stimulate viral infection and reactivation from quiescen...

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