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

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

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics

Single-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution

Quantitative single-molecule localization microscopy reports on protein numbers in signaling protein complexes

Knowledge of how proteins organize into functional complexes is essential to understand their biological function. Optical super-resolution techniques provide the spatial resolution necessary to visualize and to investigate individual protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) builds on the detection of single fluorophore labels, which next to the generation of high-resolution images provides access to quantitative molecular information. We developed various tools for quantitative SMLM (qSMLM), an imaging method that both super-resolves individual protein clusters and reports on molecular numbers by analyzing the kinetics of single emitter blinking. This method is compatible with both fluorescent proteins and organic fluorophores. With qSMLM, we quantify protein copy numbers in single clusters, and we study how changes in the stoichiometry of protein complexes translates into function

Quantification of membrane receptor complexes with single-molecule localization microscopy

Knowledge of assembly, subunit architecture and dynamics of membrane proteins in a cellular context is essential to infer their biological function. Optical super-resolution techniques provide the necessary spatial resolution to study these properties of membrane protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) is particularly well suited, as next to high-resolution images, it provides quantitative information on the detection of single emitters. A challenge for current super-resolution methods is to resolve individual protein subunits within a densely packed protein cluster. For this purpose, we developed quantitative SMLM (qSMLM), which reports on molecular numbers by analyzing the kinetics of single emitter blinking. Next to theoretical models for various photophysical schemes, we demonstrate this method for a selection of fluorescent proteins and synthetic dyes and a selection of membrane proteins. We next applied this tool to toll-like receptor 4 (TLR4), and found a ligand-specific formation of monomeric or dimeric receptors. Next to fluorescent proteins, DNA-PAINT offers a novel and flexible approach for quantitative super-resolution microscopy. We demonstrate DNA-PAINT imaging of structurally defined DNA origami structures and robust quantification of target sites, as well as of membrane receptors. Molecular quantification, together with experiments following single receptor mobilities in live cells, will enlighten molecular mechanisms of receptor activation