27 research outputs found
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
Π ΠΎΠ»Ρ Ρ Π΅ΠΌΠΎΠΊΠΈΠ½ΠΎΠ² Π² ΡΠ΅ΠΊΡΡΡΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ-ΠΏΡΠ΅Π΄ΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΈΠΊΠΎΠ² Π² ΠΎΠΏΡΡ ΠΎΠ»Π΅Π²ΡΡ Π½ΠΈΡΡ ΠΏΡΠΈ ΡΠ°ΠΊΠ΅ ΠΌΠΎΠ»ΠΎΡΠ½ΠΎΠΉ ΠΆΠ΅Π»Π΅Π·Ρ
Π Π°Π·Π²ΠΈΡΠΈΠ΅ ΠΏΠ΅ΡΠ²ΠΈΡΠ½ΠΎΠΉ ΠΎΠΏΡΡ
ΠΎΠ»ΠΈ ΡΠΎΠΏΡΠΎΠ²ΠΎΠΆΠ΄Π°Π΅ΡΡΡ ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ ΠΎΠΏΡΡ
ΠΎΠ»Π΅Π²ΠΎΠΉ Π½ΠΈΡΠΈ, ΠΊΠΎΡΠΎΡΠ°ΡΡΠΎΠ·Π΄Π°Π΅Ρ Π±Π»Π°Π³ΠΎΠΏΡΠΈΡΡΠ½ΡΠ΅ ΡΡΠ»ΠΎΠ²ΠΈΡ Π΄Π»Ρ Π²ΡΠΆΠΈΠ²Π°Π½ΠΈΡ ΠΈ ΠΏΡΠΎΠ»ΠΈΡΠ΅ΡΠ°ΡΠΈΠΈ ΡΠ°ΠΊΠΎΠ²ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ. ΠΠ΄Π½ΠΈΠΌ ΠΈΠ· ΠΊΠ»ΡΡΠ΅Π²ΡΡ
ΡΠ»Π΅ΠΌΠ΅Π½ΡΠΎΠ² ΡΠ²ΠΎΠ»ΡΡΠΈΠΈ ΠΎΠΏΡΡ
ΠΎΠ»Π΅Π²ΠΎΠΉ Π½ΠΈΡΠΈ ΡΠ²Π»ΡΠ΅ΡΡΡ ΡΠ΅ΠΊΡΡΡΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ ΠΊΠΎΡΡΠ½ΠΎΠΌΠΎΠ·Π³ΠΎΠ²ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ-ΠΏΡΠ΅Π΄ΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΈΠΊΠΎΠ², Π²ΠΊΠ»ΡΡΠ°Ρ ΠΊΠ»Π΅ΡΠΊΠΈ-ΠΏΡΠ΅Π΄ΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΈΠΊΠΈ ΠΌΠ°ΠΊΡΠΎΡΠ°Π³ΠΎΠ², ΠΌΠ΅Π·Π΅Π½Ρ
ΠΈΠΌΠ°Π»ΡΠ½ΡΠ΅ ΡΡΠΎΠ»ΠΎΠ²ΡΠ΅ ΠΊΠ»Π΅ΡΠΊΠΈ, ΡΠ½Π΄ΠΎΡΠ΅Π»ΠΈΠ°Π»ΡΠ½ΡΠ΅ ΠΈ Π³Π΅ΠΌΠΎΠΏΠΎΡΡΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΠΊΠ»Π΅ΡΠΊΠΈ-ΠΏΡΠ΅Π΄ΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΈΠΊΠΈ. ΠΠΈΠ³ΡΠ°ΡΠΈΡ ΡΠΏΠΎΠΌΡΠ½ΡΡΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ Π² ΠΎΠΏΡΡ
ΠΎΠ»Ρ ΡΠ΅Π³ΡΠ»ΠΈΡΡΠ΅ΡΡΡ ΡΡΠ΄ΠΎΠΌ Ρ
Π΅ΠΌΠΎΠΊΠΈΠ½ΠΎΠ², Π² ΡΠΎΠΌ ΡΠΈΡΠ»Π΅ CCL2, CXCL12, MSP (macrophage stimulating protein) ΠΈ MIF (macrophage inhibitory factor). Π¦Π΅Π»ΡΡ Π½Π°ΡΡΠΎΡΡΠ΅Π³ΠΎ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ ΡΠ²Π»ΡΠ»ΠΎΡΡ ΠΈΠ·ΡΡΠ΅Π½ΠΈΠ΅ ΠΏΠ°ΡΠ°ΠΌΠ΅ΡΡΠΎΠ² ΠΎΠΏΡΡ
ΠΎΠ»Π΅Π²ΠΎΠΉ Π½ΠΈΡΠΈ ΠΏΡΠΈ ΡΠ°ΠΊΠ΅ ΠΌΠΎΠ»ΠΎΡΠ½ΠΎΠΉ ΠΆΠ΅Π»Π΅Π·Ρ. ΠΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠ΅ Π²ΠΊΠ»ΡΡΠ°Π»ΠΎ 24 Π±ΠΎΠ»ΡΠ½ΡΡ
Ρ ΠΈΠ½Π²Π°Π·ΠΈΠ²Π½ΠΎΠΉ ΠΊΠ°ΡΡΠΈΠ½ΠΎΠΌΠΎΠΉ Π½Π΅ΡΠΏΠ΅ΡΠΈΡΠΈΡΠ΅ΡΠΊΠΎΠ³ΠΎ ΡΠΈΠΏΠ° ΠΌΠΎΠ»ΠΎΡΠ½ΠΎΠΉ ΠΆΠ΅Π»Π΅Π·Ρ. Π ΡΡΡΠΏΠ΅Π½Π·ΠΈΠΈ ΠΎΠΏΡΡ
ΠΎΠ»Π΅Π²ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ ΠΌΠ΅ΡΠΎΠ΄ΠΎΠΌ ΠΏΡΠΎΡΠΎΡΠ½ΠΎΠΉ ΡΠΈΡΠΎΡΠ»ΡΠΎΡΠΈΠΌΠ΅ΡΡΠΈΠΈ ΠΎΠΏΡΠ΅Π΄Π΅Π»ΡΠ»ΠΈ ΡΠΎΠ΄Π΅ΡΠΆΠ°Π½ΠΈΠ΅ ΠΊΠ»Π΅ΡΠΎΠΊ-ΠΏΡΠ΅Π΄ΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΈΠΊΠΎΠ². ΠΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΡ Ρ
Π΅ΠΌΠΎΠΊΠΈΠ½ΠΎΠ² CCL2, CXCL12, MSP ΠΈ MIF Π² Π²Π΅Π½ΠΎΠ·Π½ΠΎΠΉ ΠΊΡΠΎΠ²ΠΈ Π±ΠΎΠ»ΡΠ½ΡΡ
ΠΎΡΠ΅Π½ΠΈΠ²Π°Π»ΠΈ Ρ ΠΏΠΎΠΌΠΎΡΡΡ ΡΠ²Π΅ΡΠ΄ΠΎΡΠ°Π·Π½ΠΎΠ³ΠΎ ΠΈΠΌΠΌΡΠ½ΠΎΡΠ΅ΡΠΌΠ΅Π½ΡΠ½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π°. ΠΠΎΡΡΠΎΠ²Π΅ΡΠ½ΡΡ
ΡΠ°Π·Π»ΠΈΡΠΈΠΉ Π² ΡΠΎΠ΄Π΅ΡΠΆΠ°Π½ΠΈΠΈ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½Π½ΡΡ
ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
ΠΏΠΎΠΏΡΠ»ΡΡΠΈΠΉ, Π° ΡΠ°ΠΊΠΆΠ΅ ΠΊΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΠΈ ΠΈΠ·ΡΡΠ΅Π½Π½ΡΡ
Ρ
Π΅ΠΌΠΎΠΊΠΈΠ½ΠΎΠ² ΠΌΠ΅ΠΆΠ΄Ρ ΠΏΠ°ΡΠΈΠ΅Π½ΡΠ°ΠΌΠΈ, ΡΠ°Π·Π΄Π΅Π»Π΅Π½Π½ΡΠΌΠΈ Π½Π° Π³ΡΡΠΏΠΏΡ Π²Π·Π°Π²ΠΈΡΠΈΠΌΠΎΡΡΠΈ ΠΎΡ Π½Π°Π»ΠΈΡΠΈΡ ΠΈΠ»ΠΈ ΠΎΡΡΡΡΡΡΠ²ΠΈΡ Π»ΠΈΠΌΡΠΎΠ³Π΅Π½Π½ΡΡ
ΠΌΠ΅ΡΠ°ΡΡΠ°Π·ΠΎΠ² ΠΈ Π½Π΅ΠΎΠ°Π΄ΡΡΠ²Π°Π½ΡΠ½ΠΎΠ³ΠΎ Π»Π΅ΡΠ΅Π½ΠΈΡ, ΠΎΠ±Π½Π°ΡΡΠΆΠ΅Π½ΠΎ Π½Π΅ Π±ΡΠ»ΠΎ. Π ΡΠΎ ΠΆΠ΅ Π²ΡΠ΅ΠΌΡ, ΡΡΡΠ°Π½ΠΎΠ²Π»Π΅Π½Π° ΠΏΡΡΠΌΠ°Ρ ΠΊΠΎΡΡΠ΅Π»ΡΡΠΈΠΎΠ½Π½Π°Ρ ΡΠ²ΡΠ·Ρ ΠΌΠ΅ΠΆΠ΄Ρ ΡΠΎΠ΄Π΅ΡΠΆΠ°Π½ΠΈΠ΅ΠΌ Π³Π΅ΠΌΠΎΠΏΠΎΡΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΊΠ»Π΅ΡΠΎΠΊ-ΠΏΡΠ΅Π΄ΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΈΠΊΠΎΠ² Π² ΠΎΠΏΡΡ
ΠΎΠ»ΠΈ ΠΈ ΠΊΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΠ΅ΠΉ CXCL12 ΠΈ MIF Π² ΠΊΡΠΎΠ²ΠΈ
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 IFNΞ³-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 IFNΞ³. 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 IFNΞ³-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 IFNΞ³-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
Characterization of metal matrix composites by synchrotron refraction computed topography
Activation and Cross-Interaction of Receptor Tyrosine Kinases Studied by Single-Particle Tracking
Single-Molecule Super-Resolution Microscopy Reveals Heteromeric Complexes of MET and EGFR upon Ligand Activation
Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors