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

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 Promyelocytic Leukemia Protein facilitates human herpesvirus 6B chromosomal integration, immediate-early 1 protein multiSUMOylation and its localization at telomeres

“…It is tempting to speculate that PML might act a SUMO ligase for HHV-6B IE1, as was initially reported for the CMV IE1 protein [ 42 ]. However, recent results challenge these findings and rather propose that CMV IE1 SUMOylation occurs at the nucleosomes possibly via the PIAS1 E3 SUMO ligase [ 68 ]. Analysis of HHV-6B IE1 primary sequence revealed no homology with the CMV IE1 chromatin tethering domain [ 69 ].…”

Human Cytomegalovirus Genomes Survive Mitosis via the IE19 Chromatin-Tethering Domain

“…For Fig. 3C, viral DNA was isolated from culture supernatants with the DNeasy blood and tissue kit (69506; Qiagen) and quantitated as previously described (72). For all other experiments, total DNA was isolated using a genomic DNA minikit (IB47202; IBI).…”

Evidence for Tethering of Human Cytomegalovirus Genomes to Host Chromosomes

“…More likely, IE1 exhibits a supporting role in putative viral genome tethering with contributions from both CTD-dependent and CTD-independent mechanisms. The latter may involve cellular proteins that IE1 recruits to mitotic chromatin including PML, STAT2 or STAT3 (Paulus et al, 2006 , 2020 ; Huh et al, 2008 ; Krauss et al, 2009 ; Dimitropoulou et al, 2010 ; Shin et al, 2012 ; Reitsma et al, 2013 ; Harwardt et al, 2016 ). Our results are consistent with complex and partly redundant tethering mechanisms for hCMV episomes.…”