Sequences Related to SUMO Interaction Motifs in Herpes Simplex Virus 1 Protein ICP0 Act Cooperatively To Stimulate Virus Infection

Abstract

Herpes simplex virus type 1 immediate-early protein ICP0 is an E3 ubiquitin ligase of the RING finger class that degrades several cellular proteins during infection. This activity is essential for its functions in stimulating efficient lytic infection and productive reactivation from latency. ICP0 targets a number of proteins that are modified by the small ubiquitin-like SUMO family of proteins, and it includes a number of short sequences that are related to SUMO interaction motifs (SIMs). Therefore, ICP0 has characteristics that are related to those of cellular SUMO-targeted ubiquitin ligase enzymes. Here, we analyze the impact of mutation of a number of SIM-like sequences (SLSs) within ICP0 on HSV-1 replication and gene expression and their requirement for ICP0-mediated degradation of both sumoylated and unmodified promyelocytic leukemia (PML) and other sumoylated cellular proteins. One SLS in the central portion of the ICP0 sequence (SLS4) was found to be absolutely required for targeting cellular sumoylated species in general and sumoylated forms of PML other than those of PML isoform I. Mutation of a group of SLSs in the C-terminal quarter of ICP0 also reduced ICP0-mediated degradation of sumoylated PML in a cooperative manner. Although mutation of individual SLSs caused only modest decreases in viral replication, combined mutation of SLS4 with SLS sequences in the C-terminal quarter of the protein reduced plaque formation efficiency by up to two orders of magnitude. These results provide further evidence that the biological activities of ICP0 are connected with host cell sumoylation events. IMPORTANCEHerpes simplex virus type 1 protein ICP0 plays important roles in regulating the initial stages of lytic infection and productive reactivation from latency. ICP0 mediates its effects through inducing the degradation of cellular proteins that have repressive effects on viral gene expression. An increasing number of cellular proteins are known to be sensitive to ICP0-mediated degradation; therefore, it is important to understand how ICP0 selects its substrates for degradation. This study identifies sequence motifs within ICP0 that are involved in targeting cellular proteins that are modified by the SUMO family of ubiquitin-like proteins and describes how mutation of combinations of these motifs causes a 100-fold defect in viral infectivity. Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0, a member of the RING finger class of E3 ubiquitin ligases, is required for efficient lytic infection and productive reactivation from latency (1-3). These biological functions of ICP0 require its biochemical ubiquitin ligase activity that results in the degradation of several cellular proteins. While these basic aspects of ICP0 have been firmly established, several questions remain concerning the mechanisms by which ICP0 selects its substrates for degradation and the significance of individual substrates for the outcome of infection. It has long been established that ICP0 initially localizes to cellular...

A Tale of Two PMLs: Elements Regulating a Differential Substrate Recognition by the ICP0 E3 Ubiquitin Ligase of Herpes Simplex Virus 1

“…4B, b and e). Consistent with the RHG113 infection and the previous report (29), ICP0 containing mutated SIM 362-364 (RHG130) was able to degrade mycPML I to the same extent as the wild type (Fig. 4B, a and c), showing a half-life shorter than 2 h (Fig.…”

“…One of them, SLS4, located at residues 362 to 364, is responsible for the interaction with SUMO-2/3 and the ubiquitination of a poly-SUMO-2 substrate in vitro. Another report from the same group showed that a recombinant virus containing a mutant SLS4 degraded PML isoform I but not isoform II in the absence of all endogenous PMLs (29). SLS4 (we named it SIM 362-364 ) resides in the center of segment C. To test whether SIM 362-364 was responsible for the differential PML degradation observed above, we used the recombinant virus RHG130, in which ICP0 contains the I362G, V363A, and I364G substitutions, and examined the halflives of mycPML I and mycPML II (Fig.…”

Identification of Three Redundant Segments Responsible for Herpes Simplex Virus 1 ICP0 To Fuse with ND10 Nuclear Bodies

“…1B, panel 5). Figure 7B shows that at either 2 or 20 PFU/cell, both ICP0 lacking ND10-FS2 (RHG113) and ICP0 containing I362G/V363A/I364G substitutions (RHG130) were unable to completely degrade PML, suggesting that defective PML degradation is caused by mutations in SLS4 located within residues 343 to 391, consistent with data from a previous report by Everett et al (20). The fact that increasing the MOI from 2 PFU/ cell to 20 PFU/cell did not substantially enhance PML degradation (Fig.…”

“…Various reports have shown that the E3 ligase activity of ICP0 is tightly regulated by multiple factors located in different domains of ICP0. For example, point mutations of D671A/E673A in the CoREST binding site can negatively affect PML degradation by ICP0 (35), and mutations in SLSs (SUMO interaction motif [SIM]-like sequences) of ICP0 also cooperatively influence the ability of ICP0 to degrade PML (20). Although, at this point, we cannot rule out the possibility that deletion of the ICP0 central region may change the RING structure, it is safe to postulate that even without structural changes in the RING finger, deletion of the ICP0 central region can have significant impacts on the E3 ligase activity of ICP0.…”

“…Although the relationships among ICP0, USP7, and their substrates are not yet clear, it is justifiable to postulate that fine-tuning of the levels of these checkpoint proteins can have a pivotal role in the tug-of-war between HSV-1 and the host. Recently, SUMO (small ubiquitin-like modifier) interaction motifs (SIMs) have also been identified in ICP0 (19,20), suggesting that ICP0 may regulate more cellular factors via SUMO-SIM interactions.…”