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

This work was supported by a grant (MR/P022146/1) from the Medical Research Council (https://mrc.ukri.org) to MMN, a grant (T16/28) from Tenovus Scotland (https://tenovus-scotland.org.uk) to CP, a European Union Erasmus+ grant (https://www.erasmusplus.org.uk) to BW and the Wellcome Trust Institutional Strategic Support Fund (https://wellcome.ac.uk) to CP and MMN.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.Publisher PDFPeer reviewe

Human cytomegalovirus major immediate early 1 protein targets host chromosomes by docking to the acidic pocket on the nucleosome surface

The 72-kDa immediate early 1 (IE1) protein encoded by human cytomegalovirus (hCMV) is a nuclearly localized promiscuous regulator of viral and cellular transcription. IE1 has long been known to associate with host mitotic chromatin, yet the mechanisms underlying this interaction have not been specified. In this study, we identify the cellular chromosome receptor for IE1. We demonstrate that the viral protein targets human nucleosomes by directly binding to core histones in a nucleic acid-independent manner. IE1 exhibits two separable histone-interacting regions with differential binding specificities for H2A-H2B and H3-H4. The H2A-H2B binding region was mapped to an evolutionarily conserved 10-amino-acid motif within the chromatin-tethering domain (CTD) of IE1. Results from experimental approaches combined with molecular modeling indicate that the IE1 CTD adopts a β-hairpin structure, docking with the acidic pocket formed by H2A-H2B on the nucleosome surface. IE1 binds to the acidic pocket in a way similar to that of the latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus. Consequently, the IE1 and LANA CTDs compete for binding to nucleosome cores and chromatin. Our work elucidates in detail how a key viral regulator is anchored to human chromosomes and identifies the nucleosomal acidic pocket as a joint target of proteins from distantly related viruses. Based on the striking similarities between the IE1 and LANA CTDs and the fact that nucleosome targeting by IE1 is dispensable for productive replication even in "clinical" strains of hCMV, we speculate that the two viral proteins may serve analogous functions during latency of their respective viruses.PostprintPeer reviewe

Human cytomegalovirus immediate early 1 protein causes loss of SOX2 from neural progenitor cells by trapping unphosphorylated STAT3 in the nucleus

MHL was supported by the Ministry of Science and Technology of China (National Program on Key Basic Research Project 2015CB755600), the National Natural Science Foundation of China (81620108021, 31170155, and 81427801), the Sino-Africa Joint Research Centre (SAJC201605) and a seed grant from the University of Idaho (YDP-764). MN and CP were supported by the Wellcome Trust Institutional Strategic Support Fund, MN was supported by the Medical Research Council (MR/P022146/1) and Tenovus Scotland (T15/38), and CP was supported by the Deutsche Forschungsgemeinschaft (PA 815/2-1).The mechanisms underlying neurodevelopmental damage caused by virus infections remain poorly defined. Congenital human cytomegalovirus (HCMV) infection is the leading cause of fetal brain development disorders. Previous work has linked HCMV infection to perturbations of neural cell fate, including premature differentiation of neural progenitor cells (NPCs). Here, we show that HCMV infection of NPCs results in loss of the SOX2 protein, a key pluripotency-associated transcription factor. SOX2 depletion maps to the HCMV major immediate early (IE) transcription unit and is individually mediated by the IE1 and IE2 proteins. IE1 causes SOX2 downregulation by promoting the nuclear accumulation and inhibiting the phosphorylation of STAT3, a transcriptional activator of SOX2 expression. Deranged signaling resulting in depletion of a critical stem cell protein is an unanticipated mechanism by which the viral major IE proteins may contribute to brain development disorders caused by congenital HCMV infection.PostprintPeer reviewe

Human cytomegalovirus immediate-early 1 protein rewires upstream STAT3 to downstream STAT1 signaling switching an IL6-type to an IFNγ-like response

MN and CP were supported by the Wellcome Trust (www.wellcome.ac.uk) Institutional Strategic Support Fund and CP was supported by the Deutsche Forschungsgemeinschaft (PA 815/2-1; www.dfg.de).The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication.Publisher PDFPeer reviewe

Revisiting the role of PML protein targeting and disruption of PML bodies in human cytomegalovirus infection

Promyelocytic leukaemia (PML) bodies are nuclear organelles implicated in post-translational modification by small ubiquitin-like modifier (SUMO) proteins and in the antiviral host cell response to infection. The 72-kDa immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies encoded by the human cytomegalovirus, one of eight human herpesviruses. Previous work has suggested that the interaction between IE1 and PML proteins, the central organisers of PML bodies, and the subsequent disruption of these organelles serve a critical role in viral replication by counteracting intrinsic antiviral immunity and the induction of interferon (IFN)-stimulated genes. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be globally misfolded und metabolically unstable. We systematically screened for stable IE1 mutants by clustered charge-to-alanine scanning. We identified a mutant protein (IE1cc172-176) selectively defective for PML interaction. Functional comparisons between the mutant and wild-type protein revealed that IE1 can undergo modification by mixed polymeric SUMO chains and that it targets PML and Sp100, the two main constituents of PML bodies, via distinct mechanisms. Unexpectedly, IE1cc172-176 supported viral replication almost as efficiently as wild-type IE1. Moreover, lower instead of higher (as expected) levels of tumor necrosis factor alpha, IFN-beta, IFN-lambda and IFN-stimulated gene expression were observed with the mutant compared to the wild-type protein and virus. These results suggest that the disruption of PML bodies is linked to induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection