Fluorescent labeling of SARS-CoV2 ORF products

From supplementary information of recent JACS paper

Figure 1 – Labeling of SARS-CoV-2 derived microproteins in fixed cells. 

COS-7PylRS-AF cells were transfected with the indicated constructs and incubated for 24 h with 50 µM TCO*K. Cells were subsequently fixed, permeabilized and labeled with met-tet-BDP-FL and analyzed by confocal microscopy. SARS-CoV-2 M (Membrane) protein was used as a control because of its known localization to the Golgi (Klumperman et al., 1994). ORF7b is a type I (luminal N-terminus) transmembrane protein implicated as virulence factor, generated by leaky ribosome scanning in an alternative frame within the main ORF7a (Pfefferle et al., 2009). The homologous microprotein in SARS1 is suggested to localize to the Golgi (Schaecher et al., 2008). Our observation is a wider distribution in Golgi, ER and plasma membrane. ORF9b is an alternative ORF within ORF9a and the SARS1 homolog is reported to localize to mitochondria (Shi et al., 2014). We also observe a distinct speckled pattern in the cytosol compatible with partial mitochondrial localization. ORF9c expression and function is currently unknown but we observe a distinct localization with cellular membranes. ORF3b of SARS-CoV-2 is truncated to a 22 amino acid fragment due to a premature stop codon as compared to the SARS1 homolog, but has been reported to potently suppress host cell antiviral interferon response (Konno et al., 2020). The truncation preserves a single predicted transmembrane domain and our imaging suggests an association with cellular membranes.

Movie 1: Widefield, live cell imaging of SARS-CoV-2-ORF6N-TCO*K. 24 h after transfection and TCO*K incubation, COS-7PylRS-AF cells were exposed for 30 min to 500 nM me-tet-BDP-FL. Right after removal of the dye, cells were imaged over a short period of time. In the movie, it is possible to see ORF6-containing vesicles travelling through the cytoplasm. Timestamps (min:sec) are displayed in the top-right corner of each frame. 

RESOURCES

IDNameReference/Repository
E407pAS1_4xh7SKPylT_EF1_PylRS AF_IRES_Puro Addgene 140023
K122.5pAS1_4x7SKPylT_EF1_Ub-*CoV2_MAddgene 162815
K124.1pAS1_4x7SKPylT_EF1_Ub-*CoV2_9bAddgene 162816
K125.2pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF6Addgene 162817
K126.1pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF7bAddgene 162818
K127.2pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF10Addgene 162819
K128.2pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF9c/14Addgene 162820
K132.2pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF3b_altAddgene 162821
K134.10pAS1_4x7SKPylT_EF1_Ub-*CoV2_ORF3b(CoV1h)Addgene 162822
Table 1 – Plasmids are available on request and via Addgene:

METHODS

COS-7 cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM), containing high glucose, GlutaMAXTM and pyruvate (Gibco). Medium was supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich). All cell lines were cultured in an ambient-controlled incubator at 37°C, 5% O2 and 5% CO2. Cells were forward transfected using Lipofectamine LTXTM with PLUSTM reagent (Invitrogen) according to manufacturer’s protocol. Axial trans-cyclooct-2-ene-l-lysine (TCO*K) was added at the time of transfection as indicated and cells were harvested after 24 h. TCO*K (SiChem, SC-8008) stock solution was prepared at 100 mM in 0.2 M NaOH/H2O, 15% DMSO. 6-Methyl-Tetrazine-BODIPY®-FL (me-tet-BDP-FL, Jena Bioscience) stocks were prepared in DMF and further diluted in either RIPA buffer (lysate labeling), TBS-T (fixed cells labeling) or the appropriate growth medium (live cell labeling). 

Immunofluorescence and live cell imaging

For immunofluorescence, cells were grown and transfected in 96-well µ-Plates (ibidi). After withdrawing the ncAA for 4 h, cells were fixed in 4% formaldehyde for 10 min at room temperature and permeabilized for 15 min with 0.1% (v/v) triton/PBS. Prior to incubation with the appropriate antibodies, cells were click-labeled with 500 nM 6-Methyl-tetrazine-BODIPY-FL (Jena Bioscience), washed 3 times with PBS and then blocked for 1 hour in 2% BSA in TBS supplemented with 0.1% Tween-20 (TBS-T). Cells were incubated with the HA-probe antibody (F-7, Santa Cruz Biotechnology) overnight at 4°C. After washing with TBS-T, cells were stained with Alexa555-conjugated secondary antibodies (Life Technologies) for 60 min at room temperature and counterstained with 1 mg/ml DAPI (Sigma-Aldrich). After washing, cells were imaged on a Zeiss LSM780 confocal laser scanning microscope using a 40x/1.3 oil objective.

For live cell imaging, cells were grown and transfected in 96-well imaging plates (BD Falcon). After withdrawing ncAA for 1 hour, cells were incubated 30 minutes at 37°C in presence of 500 nM me-tet-BDP-FL. Where stated, cells were co-stained with either 4 µM Hoechst, 10 mM ER-trackerTM Red or 250 nM MitoTrackerTM Orange CMTMRos (Invitrogen). After washing 2 times with PBS, cells were immediately imaged in Live Cell imaging Solution (Molecular Probes), on a Nikon eclipse Ti2 inverted widefield microscope equipped with a heated imaging chamber. Images were acquired using a 20×/0.75 air objective or a 40x/1.15 water objective. For the long-term imaging experiment shown in Supplementary Figure 3, cells were maintained in Leibovitz’s L-15 Medium (Gibco) and imaged using a 10x/0.45 air objective.

Bibliography

Klumperman, J., Locker, J.K., Meijer, A., Horzinek, M.C., Geuze, H.J., and Rottier, P.J. (1994). Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J. Virol. 68, 6523–6534.

Konno, Y., Kimura, I., Uriu, K., Fukushi, M., Irie, T., Koyanagi, Y., Sauter, D., Gifford, R.J., USFQ-COVID19 Consortium, Nakagawa, S., et al. (2020). SARS-CoV-2 ORF3b Is a Potent Interferon Antagonist Whose Activity Is Increased by a Naturally Occurring Elongation Variant. Cell Rep. 32, 108185.

Pfefferle, S., Krähling, V., Ditt, V., Grywna, K., Mühlberger, E., and Drosten, C. (2009). Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo. Virol. J. 6, 131.

Schaecher, S.R., Diamond, M.S., and Pekosz, A. (2008). The transmembrane domain of the severe acute respiratory syndrome coronavirus ORF7b protein is necessary and sufficient for its retention in the Golgi complex. J. Virol. 82, 9477–9491.

Shi, C.-S., Qi, H.-Y., Boularan, C., Huang, N.-N., Abu-Asab, M., Shelhamer, J.H., and Kehrl, J.H. (2014). SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome. J. Immunol. 193, 3080–3089.

STELLA protein labeling

Publication in JACS

Addgene plasmids

Proteins are the molecular machines of life, performing a myriad of functions inside every cell of our body. Proteins are assembled from small building blocks, the amino acids, by large protein factories called ribosomes. Understanding how proteins work is a quest of basic biology and medical research.

Postdoctoral researcher Lorenzo Lafranchi and PhD student Dörte Schlesinger. Not in the picture is Kyle Kimler, former master student, now at Broad Institute.

To study proteins inside of human cells, researchers have been using light microscopes for more than one hundred years. Thirty years ago, the cloning of the green fluorescent protein GFP, together with genetic engineering tools, revolutionized the field by enabling researchers to fuse a fluorescent ‘beacon’ to any protein of interest so that it can be directly observed in living cells using fluorescence microscopy. Fast forward, today’s microscopes achieve live imaging, at nanometer resolution, in multicolor, allowing researchers to resolve even the smallest subcellular structures and essentially watch protein at work.

Fluorescent proteins and other tools that are available to researchers have however one limitation: the size of the fluorescent tag is often equivalent to the size of a typical folded protein, thus adding a considerable molecular ‘cargo’ to the protein under study and potentially impacting its function. This can become a particular obstacle for the study of microproteins, a newly appreciated class of proteins that are much smaller than average. Such tiny proteins have often been overlooked in the past but seminal discoveries of microproteins with important biological functions have sparked growing interest by the research community. 

We have made it a focus of our laboratory to tackle the challenges of discovering and characterizing microproteins. Here, we developed a method which allows fluorescent tagging of proteins with the smallest imaginable perturbation – a single amino acid – added genetically on either end of a (micro)protein of interest.

For the method, termed STELLA, a synthetic building block (a non-canonical “designer” amino acid, rather than one of the 21 canonical ones) is incorporated together with a larger tag using a technique termed genetic code expansion. The tag however is swiftly removed by the cell, leaving a single terminal designer amino acid on the protein of interest. As an advantage over existing labeling techniques relying on genetic code expansion, STELLA can thus be used to conveniently and universally label the termini of any proteins. While very similar to its natural counterpart, the designer amino acid introduces a peculiar chemical group into the protein that subsequently allows conjugation with a small organic fluorescent dye, now lighting up the protein of interest inside of the living cell.

Our research was funded by Karolinska Institutet SFO Molecular Biosciences, Ming Wai Lau Center for Reparative Medicine, Ragnar Söderbergs Stiftelse, Stiftelsen för Strategisk Forskning and Boehringer Ingelheim Fonds.

A dance of histones silences transposable elements in pluripotent stem cells.

Nature Communications Article
BioRxiv Article
GEO Dataset
GitHub
Mendeley Dataset

So-called transposons are abundant DNA-elements found in every eukaryotic organism as a consequence of their ability to jump and multiply within the host genome. Their activity represents a threat to the integrity of the host genome and thus the host cell engages a number of protective mechanisms to silence the expression of transposons. It is known that some of these mechanisms fail in cancer cells and also ageing cells, leading to a mobilization of transposons with largely unknown consequences. Histones, the proteins that package the genome in the eukaryotic nucleus, are key to the most fundamental line of defense to transposons. By forming a highly compacted array, so-called heterochromatin, they render the associated DNA sequence inert to being read and expressed. Heterochromatin is defined by characteristic modifications to histone proteins and DNA, such as histone H3 K9 trimethylation and DNA CpG methylation. 

Here, we studied endogenous retroviral elements (ERVs), a particularly active and abundant family of transposable elements in the mouse genome, which are in fact remnants of once-active viruses. Curiously, while we found all the hallmarks of heterochromatin to be employed in the silencing mechanism, ERV chromatin was highly enriched in a histone variant, termed histone H3.3, which has previously been invariably associated with active regions of the genome. Following up on this observation, wecould elucidate an unexpected mechanism involving a continuous loss of ‘old’ histones and replenishment with newly synthesized histones H3.3 molecules. By genetic manipulation, we were able to deduce a mechanism explaining this dynamic process: the ATP-dependent chromatin remodeler Smarcad1 evicts histones within heterochromatin, thus creating gaps in the chromatin fibre that could render parts of the ERV gene accessible. Following suit, the histone chaperone DAXX seals these gaps by facilitating reassembly of nucleosomes with histone variant H3.3. The concerted process of eviction of one and deposition of another histone is so smooth and efficient that it leaves no apparent trace of accessible DNA.

The result is puzzling because active remodeling and nucleosome eviction is expected to counteract a compacted chromatin structure, inert to transcriptional activation. But we believe that dynamic heterochromatin is an adaption of a ubiquitous silencing mechanism to the specific requirements of a pluripotent chromatin state. The highly transient opening of heterochromatin may allow sequence-specific co-repressors to find their target DNA sequence within the transposable element, in turn recruiting more repressive factors to propagate and amplify the silent state.

Adapted from Sean Taverna’s original 2010 cartoon

Cracking the histone code

Our paper in Nature Methods (“Genetic code expansion in stable cell lines enables encoded chromatin modification“, DOI:10.1038/NMETH.3701) is the first one to generate and characterize stable amber suppression cell lines for unnatural amino acid mutagenesis . The principle of genetic code expansion via amber suppression is shown below

Amber Suppression

We then apply the system to generate genetically encoded synthetic histone acetylation marks to directly test the function of this posttranslational modification in chromatin, one position at a time. This approach highlights the potential of the methodology to perform experiments with biochemical precision in living cells that could otherwise only be achieved in vitroIn vitro experiments can provide a clear link between molecular cause and effect, but are abstracted from the appreciable complexity of the cellular environment. In contrast, in vivo experiments typically provide a wealth of correlative information about changes of chromatin state in a native context, but it is commonly impossible to infer direct causation from these experiments. For example, all histone acetyl transferases (HATs) are known to acetylate a range of sites and substrates, including non-histone proteins, thus genetic knockout or enzymatic inhibition of HATs does not directly and exclusively test the function of histone acetylation. Employing a synthetic route to modulate  cognate posttranslational modifications has the power to show direct causality between the modifications and their downstream effects, abstracted from the  complexity of enzymes that set and erase the modifications. We believe that in the future such approaches to synthetic epigenetics will be very powerful for defining the function of posttranslational modifications, in particular the complex modification code present on histones.

Screen Shot 2016-01-02 at 8.13.12 PM

In a second publication, we have employed stable amber suppression in  HEK293 cell lines to synchronously activate a mutant Isocitrate-dehydrogenase enzyme (IDH2) in the entire populaiton of cells by light and followed changes in metabolic and epigenetic products:

http://pubs.acs.org/doi/abs/10.1021/jacs.5b07627

 

 

Short News

Simon Elsässer has been appointed to the Global Young Academy

Congratulations to Sigrid Lundin and Rahul Kumar for finishing the Berlin Marathon 2015!

Meet Simon Elsässer at the 65th Lindau Nobel Laureate Meeting, June 28th-July 3rd in Germany

Simon Elsässer will be presenting at the Eukaryotic Synthetic Biology Symposium in Heidelberg, 21-23rd June 2015 in Germany

Zakir Tnimov will give a talk at the International Synthetic and Systems Biology Summer School, 5-9th July in Italy

CONGRATULATIONS to Sigrid Lundin defending her Master thesis!

Good luck to Rahul Kumar running the Stockholm Marathon in torrential rain!

H3.3 silences endogenous retroviral elements

Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells.

Elsässer SJ, Noh K-M, Diaz N, Allis CD, Banaszynski LA Nature 2015 In press

The double-stranded DNA molecules that make up the human genome are present inside the nucleus of a cell in a highly condensed state – it requires a ~10000fold compaction to fit its 3 billion base pairs into the tiny available space. So-called histone proteins achieve this task by wrapping the DNA like strings on beads. Through this packaging mechanism, we think that histone proteins are the key to regulating access to the genetic information and provide a molecular basis for indexing or annotating the genome with so-called epigenetic information. Histone proteins carry a large number of distinct chemical modifications or ‘marks’, providing a verbose epigenetic language. As a field, we have only started to appreciate the intricate complexity of this histone code.  Our study investigated the mechanism of silencing transposable elements in a mouse embryonic stem cell system. These parasitic DNA elements can transpose or ‘jump’ and multiply within a host genome and have played an active role in animal evolution, facilitating genetic variation and adaptation. But their activity represents a threat to the host genome and thus they are almost always actively silenced. We have found a new factor that is used to mark specific DNA elements for silencing. It is a variant of one of the core histones, H3, called H3.3, which has been intensely studied in other processes. But no one has looked at transposable elements, possibly because they are often considered to be of no particular function to the cell. Unexpectedly, we found that a large fraction of the histone variant H3.3 occupies transposable elements in mouse embryonic stem cells and our genetic studies show that it is required to efficiently silence the underlying DNA elements. The combination of H3.3 with a known silencing mark, histone H3 lysine 9 trimethylation (which in no other instance are found together) provide an exceptionally strong signal to the cell to ‘not read from this genomic region’. Untitled But why is this mechanism so important? When we deleted all H3.3 genes, we found that the repressive histone modification H3K9me3 is significantly reduced and some previously silenced elements are reexpressed. While we conducted our experiment in mouse cells, the mechanism is very likely conserved to humans. Over the last few years a number of cancer types have been found to harbor frequent and recurrent mutations in the histone variant H3.3 and two other associated genes, DAXX and ATRX, notably pancreatic neuroendocrine tumors and a family of aggressive childhood gliomas. In our study, we found that DAXX and ATRX proteins, like histone H3.3, are required for the silencing mechanism. Thus it is possible that the new molecular detail we describe plays an important role in maintaining genomic stability in the affected human tissues, a hypothesis that can be tested in the future.