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

Author summary Promyelocytic leukemia (PML) bodies are liquid droplet-like structures organized by the eponymous PML proteins in the nuclei of our cells. PML bodies have been implicated in the antiviral host cell response to infection. Consequently, viruses have evolved mechanisms that target the proteins composing PML bodies. Immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies produced by the human cytomegalovirus, one of eight human herpesviruses. Previous work suggested that the interaction between IE1 and PML and the consequent disruption of PML bodies serves a critical role in viral replication by counteracting the cellular antiviral response. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be unstable. We systematically screened for stable IE1 variants and identified a mutant protein selectively defective for PML interaction. Unexpectedly, the IE1 mutant supported viral replication almost as efficiently as the wild-type protein. Moreover, lower instead of higher (as expected) levels of antiviral gene expression were observed with the mutant compared to the wild-type. These results suggest that disruption of PML bodies is linked to the induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection. 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.