Revisiting promyelocytic leukemia protein targeting by human cytomegalovirus immediate-early protein 1.

Christina Paulus,Edith Reuschel,Andrea Marxreiter,Marion Zenger,Thomas Harwardt,Michael M Nevels,Bernadette Walter

doi:10.1371/journal.ppat.1008537

Abstract

Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.

Highlights

Promyelocytic leukemia (PML) bodies, known as nuclear domain 10, are membrane-less nuclear organelles present in most cells
Promyelocytic leukemia (PML) bodies are liquid droplet-like structures organized by the eponymous PML proteins in the nuclei of our cells
Immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies produced by the human cytomegalovirus, one of eight human herpesviruses

Summary

Introduction

Promyelocytic leukemia (PML) bodies, known as nuclear domain 10, are membrane-less nuclear organelles present in most cells (reviewed in [1, 2]). PML bodies are heterogeneous and dynamic, ranging in size between 0.1 and 1.0 μm and typically displaying as five to 30 spherical structures per nucleus interspersed between chromatin. They are composed of the eponymous PML proteins, known as tripartite motif (TRIM) 19, that self-organize into a shell-like scaffold. Embedded in the scaffold or core are numerous unrelated proteins, most of which associate with PML bodies in a conditional and transient manner [11, 12]. PML bodies are considered to be SUMOylation hubs, and most proteins associated with these organelles are post-translationally modified by one or more SUMO paralogs [12, 15]. Aggregates referred to as mitotic accumulations of PML protein (MAPPs) are known to form following PML de-SUMOylation and breakdown of PML bodies at the onset of mitosis [19,20,21,22,23]

Methods

Results

Discussion

Conclusion