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

Abstract

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an ␣ gene product required for viral replication at low multiplicities of infection. Upon entry, nuclear domain 10 (ND10) converges at the incoming DNA and represses viral gene expression. ICP0 contains a RING-type E3 ubiquitin ligase that degrades the ND10 organizer PML and disperses ND10 to alleviate the repression. In the present study, we focused on understanding the regulation of ICP0 E3 ligase activity in the degradation of different ICP0 substrates. We report the following. (i) A SUMO interaction motif located at ICP0 residues 362 to 364 is required for the degradation of PML isoforms II, IV, and VI but not isoform I. This differentiation mechanism exists in both HEp-2 and U2OS cells, regardless of the cell's permissiveness to the ICP0-null virus. (ii) Physical interaction between SIM 362-364 and PML II is necessary but not sufficient for PML II degradation. Both proximal sequences surrounding SIM 362-364 and distal sequences located at the ICP0 C terminus enhance the degradation of PML II. (iii) The ICP0 C terminus is dispensable for PML I degradation. Instead, bipartite PML I binding domains located in the N-terminal half of ICP0 coordinate to promote the degradation of PML I. (iv) The stability of ICP0, but not its ND10 fusion ability, affects the rate of PML I degradation. Taken together, our results show that ICP0 uses at least two regulatory mechanisms to differentiate its substrates. The disparate recognition of the ICP0 E3 substrates may be related to the different roles these substrates may play in HSV-1 infection. IMPORTANCEViruses have a limited genetic coding capacity but must encounter a multilayered comprehensive host defense. To establish a successful infection, viruses usually produce multifunctional proteins to coordinate the counteractions. Here we report that an HSV-1 protein, ICP0, can recognize individual host factors and target them differently for destruction. We identified elements that are important for the ICP0 E3 ubiquitin ligase to differentially recognize two of its substrates, PML I and PML II. This is the first study that has systematically investigated how ICP0 discriminates two similar molecules by very different mechanisms. This work lays the foundation for understanding the role of host defensive factors and the mechanisms viruses use to take advantage of some host proteins while destroying others. Herpes simplex virus (HSV) causes a wide range of mild to severe herpetic diseases, including cold sores, genital lesions, stromal keratitis, and encephalitis. Following infection, HSV establishes a lifelong latency in ganglion neurons. Its sporadic and sometimes asymptomatic reactivation nourishes a wide spread of the virus, making it one of the most prevalent opportunistic pathogens that cause severe problems in immunocompromised individuals (1).Upon HSV-1 infection, the incoming viral DNA encounters various host restrictive factors, namely, the intrinsic and innate antiviral responses. To establish ...

Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection

“…For this purpose, we infected U2OS cells with RHG103, in which ICP0 lacks C-terminal residues 669 to 775, or with RHG108 (18), in which ICP0 lacks residues 669 to 775 and at the same time contains the C116G/C156A substitutions. In previous studies, we showed that ICP0 ΔC maintained its ability to degrade PML but that ICP0 ΔC-RFm did not (18,23). Consistent to what we saw in RHG103-infected HEL cells (Fig.…”

“…We first took a recombinant virus, RHG120, in which ICP0 contains the C116G/C156A substitutions (ICP0 RFm) (18) and exhibits substantial defects in E3 ligase activity and viral replication during infection of nonpermissive HEL and HEp-2 cells (23). We compared the subcellular distributions of ICP0 RFm late during infection in both HEL and U2OS cells.…”

Immune Response to Herpes Simplex Virus Infection and Vaccine Development

“…Moreover, ICP0 utilizes different motifs of PML-NBs fusion and SUMO interaction [ 72 ], suggesting different molecular mechanisms of PML- ICP0 interaction and degradation. More so, among the PML isoforms, different methods of interactions and degradation were observed to be utilized by ICP0 in Hep-2 and U2OS cells [ 73 ]. Hembram and colleagues identified a bona fide SIM in ICP0 which is essential to target SUMOylated PML.…”

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

“…It is first detected at a dynamic nuclear structure termed nuclear domain 10 (ND10) 7 . The E3 ubiquitin ligase activity of ICP0 triggers the degradation of ND10 organizer proteins, promyelocytic leukemia (PML) protein, and speckled protein 100 kDa (Sp100) 8,9,10 . After the loss of organizer proteins, ND10 nuclear bodies are dispersed and ICP0 is diffused to fill the entire nucleus 4,11 .…”