EMBO/SourceData huggingface dataset:
f4(a) Atg7−/− livers display decreased ERK phosphorylation. Immunoblots for the indicated proteins in liver homogenate (Hom) and cytosolic (Cyt) fractions of control (Con) and liver-specific Atg7−/− mice are shown. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with Con; Student's t-test, n=4.(b) Atg7−/− livers display decreased ERK2 dimers. Immunoblots show the indicated proteins in Hom from Con and Atg7−/− livers. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared to Con; Student's t-test, n=4-5. Arrows indicate p42 monomers and dimers.(c) Atg7−/− brown adipose tissues (BAT) display decreased ERK phosphorylation. Immunoblots for indicated proteins in Hom from Con and Atg7−/−BAT. The bars represent mean±s.e.m. **P0.01 compared with Con; Student's t-test, n=4-6.(d) Atg7−/− livers exhibit decreased nuclear ERK phosphorylation. Immunoblots show the indicated proteins in homogenate (Hom, lane 1) and nuclear fractions (lanes 2-5) from Con and Atg7−/− livers. The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=4-5.(e) LC3 C terminus glycine-deleted (ΔG) mutants exhibit decreased ERK phosphorylation. Immunoblots for indicated proteins in total lysates from CFP-LC3- and CFP-LC3ΔG-transfected NIH/3T3 cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=8.
f5(a) Atg5−/− MEFs display reduced ERK phosphorylation during nutrient deprivation. Quantification of P-ERK levels normalized to total ERK (detected by immunoblotting) in WT and Atg5−/− MEFs cultured in the absence of serum for indicated times. The bars represent mean±s.e.m. *P0.05 compared with corresponding WT value; ***P0.001, ****P0.0001 compared with 10 min serum-starved Atg5−/− MEFs; ANOVA-Bonferroni post hoc test, n=3.(b) Serum-deprived Atg5−/− MEFs display decreased ERK and p90RSK phosphorylation. Immunoblots for the indicated proteins in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared with Con; Student's t-test, n=3.(c) Atg5−/− MEFs display decreased nuclear ERK activity. Immunoblots for phosphorylated HA (haemagglutinin)-tagged Elk1 in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min).(d) Atg5−/− MEFs display decreased Elk1-driven gene expression. Elk1-driven ZFP36 mRNA levels from 2 hserum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=3. ((e) Decreased ERK phosphorylation in Atg5−/− MEFs occurs independently of changes in ERK phosphatases. Immunoblots for the indicated proteins in 2 h serum-deprived WT MEFs transfected with scrambled siRNA (scr), and Atg5−/− MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF. The bars represent mean±s.e.m., n=3.
f6(a) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5-ATG12, LC3 and WIPI1. Immunofluorescence (IF) showing colocalization (depicted as white pixels by 'colocalization finder application') of WT-ERK2-HA, FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6), LC3 (panels 7-12) or WIPI1 (panels 13-18) in EGF-treated NIH/3T3 cells. ERK2 is stained in red, and autophagy proteins are stained in green. Scale bar, 10 μm. The bars represent mean±s.e.m. **P0.01, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. ((b) Mutations in FRS on ERK2 decrease colocalization of ERK2 with nuclearATG5-ATG12 or LC3. IF showing colocalization (white pixels) of WT-ERK2-HA (red), FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6) or LC3-II (panels 7-12) in EGF-treated NIH/3T3 cells. Scale bar, 5 μm. The bars represent mean±s.e.m. **P0.01, ***P0.001, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. Arrows depict colocalization.(c) ATG5-ATG12 conjugation is required for ERK phosphorylation. Immunoblots for P-ERK, total ERK and β-actin in NIH/3T3 cells transfected with WT ATG5 or the conjugation-defective ATG5 K130R mutant and treated with EGF (10 min). The bars represent mean±s.e.m. *P0.05 compared with WT ATG5-transfected cells; Student's t-test, n=3.
f7a) ATG4B depletion increases steady-state LC3-II levels. Immunoblots for indicated proteins in NIH/3T3 cells transfected with scrambled siRNAs (scr) or siRNAs against ATG4B (siATG4B) in presence/absence of EGF. Bars represent mean±s.e.m. *P0.05, ***P0.001 compared with scr; Student's t-test, n=4.(b) siATG4B cells exhibit increased autophagic vesicles. Representative electron micrographs depicting autophagic vesicles (indicated by black arrows) in scr and siATG4B NIH/3T3 cells.(c) ATG4B depletion does not increase autophagic flux in serum-fed cells. Immunoblots for indicated proteins in lysates from scr or siATG4B NIH/3T3 cells in presence/absence of lysosomal inhibitors, ammonium chloride and leupeptin (Inh) for 2 h. Scale bars, 1 μm. Bars represent mean±s.e.m., n=3.(d) siATG4B cells display increased nuclear LC3-II. Images for LC3-II (red) in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.(e) ATG4B deficiency increases P-ERK/LC3-II colocalization. Immunofluorescence (IF) for P-ERK (green)/LC3 (red) colocalization in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bars, 10 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.(f) ATG4B-deficient cells display increased nuclear P-ERK/LC3 colocalization. IF depicting P-ERK (green)/LC3 (red) colocalization in nuclei of scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.(g) ATG4B deficiency augments EGF-induced MEK and ERK phosphorylation. Immunoblots for P- and total MEK and ERK, LC3 and GAPDH in total lysates from scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Bars represent mean±s.e.m., n=3.h) ATG4B deficiency increases nuclear P-ERK levels. Native (top)/inverted images (bottom) showing nuclear P-ERK content. P-ERK fluorescence in untreated (Con)/EGF-treated scr or siATG4B NIH/3T3 cells is shown. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2. Nuclei are blue (DAPI).
Figure 1Fate of Purkinje cells in mice with Purkinje-cell-specific knockout of Ccp1. Ccp1flox/flox L7-cre mice show a massive but incomplete degeneration of Purkinje cells (calbindin-stained; red) at 4 months, while all Purkinje cells are degenerated at 18 months. Scale bar: 500 µm.Fate of Purkinje cells in mice with combinatorial knockout alleles. Knockout of Ccp1 (pcd-mouse; Ccp1-/-) results in a complete degeneration of Purkinje cells (calbindin-stained; red), while the additional knockout of Ttll1 selectively in the Purkinje cells (L7-cre) protects the entire Purkinje cell layer from degeneration up to 18 months. Scale bar: 500 µm.Nissl staining of the Purkinje cell layer of the brains from various knockout mice shown in (C) confirms the absence of Purkinje cells in Ccp1-/-, and the presence of a wild-type-like Purkinje-cell density in Ccp1-/- Ttll1flox/flox L7-cre mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated.Immunoblot of tissue extracts from cerebella of Ttll1-/- and wild type mice, probed with the polyE, anti-∆2-tubulin and 12G10 antibodies. Increasing amounts of extracts were loaded for detection of polyglutamylated proteins with polyE.
Figure 2Analyses of tubulin polyglutamylation (antibody: polyE) in extracts of different brain regions of wild type, Ccp1-/-, Ccp6-/- and Ccp1-/-Ccp6-/- mice with immunoblot. In 3-weeks old mice, hyperglutamylation is observed in cerebral cortex, cerebellum and hippocampus of Ccp1-/- and Ccp1-/-Ccp6-/- mice. In 2- and 5-month-old mice, hyperglutamylation is restricted to the cerebellum in Ccp1-/- mice, however it persists in all three brain regions of the Ccp1-/-Ccp6-/- mice (complete analysis in Fig. EV1; α- and β-tubulin bands indicated).Nissl-stained frontal sections of wild type, Ccp1-/-, Ccp6-/- and Ccp1-/-Ccp6-/- mice at 1 and 5 months, showing age-dependent thinning (arrow heads) of the cortex in Ccp1-/-Ccp6-/- mice (complete analysis in Appendix Fig. S2A).Quantification of the thickness of motor and somatosensory cortex of 1- and 5-month-old mice shown in Appendix Fig. S2. The single values shown in Appendix Fig. S2B were averaged, and values from each genotype-specific group were tested against the average values of the remaining three genotypes (Appendix Fig. S2C). Only the cortical thickness of 5-month-old Ccp1-/-Ccp6-/- mice is significantly decreased (mean ±SEM; Mann-Whitney t-test).Immunohistochemistry of brain sections with anti-MAP2 antibody for visualization of neurons (layer V) and apical dendritic (layer IV) and with anti-GFAP for reactive astrocytes (layer V). The complete analysis is shown in Fig. EV2A, EV3A. Scale bars: 50 µm. Quantifications of number of MAP2-positive neurons in layer V, and of number of apical dendrites in layer IV (Fig. EV2B) reveal reduced neuron number in layer V, and reduced dendritic density in layer IV of 5-month-old Ccp1-/-Ccp6-/- mice. Quantification of the number of reactive astrocytes in layer V (Fig. EV3B) shows a significant increase over wild type in Ccp1-/- and in Ccp6-/- mice, however a much stronger increase is seen in Ccp1-/-Ccp6-/- mice. Complete analyses are shown in Fig. EV2C, EV3C (mean ±SEM; student's t-test).Immunohistochemistry of brain sections stained with SMI-32 antibody (a representative section of one brain per genotype group is shown, complete analysis in Appendix Fig. S3). Neuronal SMI-32 labelling indicates increased axonal damage in the cortex of Ccp1-/-Ccp6-/- mice at 5 months. Scale bar: 100 µm.
Figure 4Fate of Purkinje cells in 1-month-old wild type, spastin-/-, Ccp1-/- and Ccp1-/-spastin-/- mice visualized with anti-calbindin antibody. Ccp1-/- and Ccp1-/-spastin-/- mice show almost complete loss of the Purkinje-cell layer. Scale bar: 500 µm.Nissl staining shows the complete loss of Purkinje cells in Ccp1-/- and Ccp1-/-spastin-/- mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated.Calbindin staining of sections showing axons of Purkinje cells as in (A). While wild type and spastin-/- show normal axons, Ccp1-/- and Ccp1-/-spastin-/- mice show axonal swellings (arrow heads) in the remaining Purkinje cells. Scale bars: 20 µm.
Figure 5Immunoblot of cell extracts from Ccp1flox/floxCcp6flox/flox hippocampal neurons without, or after transduction with lentivirus expressing either GFP or GFP-2A-cre after 4 days in culture. Note the specific increase of polyglutamylation (polyE) in neurons transduced with GFP-2A-cre lentivirus.All kymographs of one representative set of experiments. Mitochondria movements (Movie S1) were plotted in kymographs and analyzed as described in (A). Scale bars: 60 sec, 20 µm. Analyses of (D) run length and (E) speed distribution, represented as scatter dot plots with each point representing a single run extracted from the kymographs shown in (C). Each group represents the pool of measurement of multiple neurons made in the experiment. The black line indicates the median of the distribution with whiskers at interquartile range. Numerical value of the medians is indicated below each plot. (F) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s. Values (below plots) from one set of experiment (C) show a decrease of about 50% in Ccp1-/-Ccp6-/- conditions, while (G) mitochondria density is unaltered. (H-K) Statistical analyses of mitochondria transport parameters of experiments shown in (D-G; Fig. EV4A-D). Each bar represents the mean (±SEM) of medians of single experiments for run length (H) and speed (I) distributions, and average (±SEM) mitochondria motility (J) and density (K) of all seven experimental sets. Values of fold change between control and Ccp1-/-Ccp6-/- conditions are indicated below the graphs. Green bars are for anterograde, and red for retrograde transport events. Significance was tested using unpaired t-test. Average run length (H) as well as speed (I) showed no significant differences in these two parameters between control and Ccp1-/-Ccp6-/- conditions. (J) The average of overall motility reveals an about 50% decrease in overall mitochondria motility, while mitochondria density (K) is unaltered between control and Ccp1-/-Ccp6-/- conditions.
Figure 1(A) Optically evoked auditory brainstem response in an AAV-CatCh-injected gerbil in response to a 1 ms laser pulse of ~35 mW (mean of 1000 stimulus presentations at 10 Hz repetition rate). (B-D) Catch-eYFP-expressing SGNs in the apical cochlear turn identified by parvalbumin expression (B) and CatCh-eYFP (C). The inlay (D´) of the merged immunostaining (D) indicates the lack of CatCh-EYFP signal in inner hair cells (dashed white line). Scale bar: 20 µm.  (I) oCI inserted into the cochlea (dashed black lines) via a cochleostomy in the basal cochlear turn (black, solid line). The round window niche is highlighted by a dashed white line, the round window by a solid white line. SA: stapedial artery.  (J) 3D X-ray tomography reconstruction of a 16-channel oCI implanted in a gerbil cochlea via the round window. Cables and μLEDs are marked in blue, the most apical µLED is indicated by the black arrowhead; the basilar membrane is marked in green. µLEDs have a size of 60×60 µm.
Figure 3 (A) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via the round window, from apical to basal direction (A-D). White markers define the best electrode (BE), and measurement of the spread of excitation (SoE) is indicated in the left panel. In STCs where more than one peak was detected, the dorsal- and ventral-most electrodes have been considered as the boundaries of the STC in order to avoid underestimation of the SoE.  (C) Spectral spread of excitation upon SGN stimulation with µLED-based oCIs (blocks of 4 µLEDs, solid lines; mean ± standard error of the mean), acoustic (black dashed line), laser-coupled optical fibers (blue dashed line), as well as mono- and bipolar electrical stimulation with a clinical-style eCI (dashed and dotted red lines, respectively). Average data from stimulation via optical fibers and eCIs are replotted from Dieter et al (Dieter et al, 2019).
Figure 1 Figure 1b. Genotyping of AS and WT CDK12 clones. Ethidium bromide-stained agarose gel visualizing PCR products from genomic DNA of AS (AS-PCR) and WT (WT-PCR) CDK12 HCT116 cells and their digest with BslI enzyme (indicated as AS- BslI and WT- BslI). Primer positions and BslI restriction sites are depicted at Expanded View Fig. 1a. Numbers on the left and right indicate DNA marker and DNA fragment sizes, respectively.  Figure 1d. Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII. Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 µM 3-MB-PP1 for indicated times. Long and short exp.= long (4-14 min) and short (10-60 s) exposures, respectively. FUS and tubulin are loading controls. A representative image from 3 replicates is shown.  Figure 1e, f. Inhibition of CDK12 in AS CDK12 HCT116 cells results in down-regulation of CDK12-dependent HR genes. Graph shows RT-qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 (e) and WT CDK12 HCT116 (f) cells treated for indicated times with 3-MB-PP1. mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control (CTRL) cells were set to 1. n=3 replicates, error bars indicate standard error of the mean (SEM).
Figure 2 Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation . Flow cytometry profiles of control (-3-MB-PP1) or inhibitor (+3-MB-PP1) treated cells from the experiment depicted in Fig. 2a. The red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. To better visualize the G1/S delay in the presence of the inhibitor the 24 h time point is also shown. n=3 replicates; representative result is shown. Figure 2d. CDK12 protein levels peak in the G0/G1 phase of the cell cycle. Western blots show levels of proteins at indicated time points after the release of serum starved AS CDK12 HCT116 cells. Corresponding cell cycle phases are depicted above time points. A representative Western blot from 3 replicates is shown.  Figure 2f, g. Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (f) and BrdU (g) labeled cells upon addition of 3-MB-PP1 at indicated time points after serum addition in the experiment depicted in Fig. 2e. CTRL in Fig. 2g = control sample without 3-MB-PP1. n=3 replicates, representative result is shown.  Figure 2h. Short-term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 µM camptothecin. A representative Western blot from 3 replicates is shown. FUS is a loading control.
Figure 3 Figure 3a. CDK12 inhibition results in differential expression of a subset of genes. Comparison of log2 fold-changes versus log2 mean expression in 3'end RNA-seq data shows differentially regulated genes after inhibition of CDK12. Down- (log2 fold-change < -1) and up-regulated (log2 fold-change >1) genes are shown in blue and red, respectively.  Figure 3d. Validation of RNA-seq for select DNA replication genes by RT-qPCR. Graph shows relative levels of mRNAs of described genes in serum arrested and released (0 h G0/G1) AS CDK12 HCT116 cells either treated (3-MB-PP1) or not (CTRL) with the inhibitor for indicated times after the release. mRNA levels were normalized to B2M mRNA expression and mRNA levels for each gene at the time of release (0 h) was set as 1. n=3 replicates, error bars indicate SEM.  Figure 3e. Protein levels of core DNA replication factors are dependent on the CDK12 kinase activity. Western blot analyses of protein expression by the depicted antibodies in serum synchronized and released (0 h) cells either treated or not with 3-MB-PP1 for the indicated times after the release. FUS is a loading control. A representative Western blot of 3 replicates is shown.  Figure 3f. CDK12 inhibition affects loading of CDC6 and CDT1 DNA replication factors to chromatin. Western blotting analyses of chromatin association of the indicated DNA replication factors in serum synchronized and released AS CDK12 HCT116 cells treated or not with 3-MB-PP1 for the indicated times. Histone H2A serves as a loading control of chromatin fractions. A=asynchronous cells, 0 h=time of release. A representative Western blot of 3 replicates is shown.
Figure 4 Removal of CDK12 inhibitor in early G1/S rescues replication gene expression and cell cycle progression. RT-qPCR of replication gene mRNA RT-qPCR were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.  Removal of CDK12 inhibitor in early G1/S rescues cell cycle progression. Western blotting of , protein levels Western blotting were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.  Removal of CDK12 inhibitor in early G1/S rescues replication and cell cycle progression. flow cytometry analyses of cell cycle progression, flow cytometry analyses were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.  Figure 4e. Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor. Western blot analyses of chromatin fractions of serum starved AS CDK12 HCT116 cells treated with 3-MB-PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment. CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition. All cells were harvested either 6 or 9 h after the serum addition. Histone H2A serves as a loading control of chromatin fractions and studied DNA replication factors are indicated. A representative image of three replicates is shown.  Figure 4f. Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3-MB-PP1 and replicating BrdU-stained cells were quantified by FACS analyses. CTRL=control samples without the 3-MB-PP1. A representative image of 3 replicates is shown. Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).
Figure 7 Figure 7a. Longer genes tend to have a larger fraction of differentially used exons. Box plot shows the fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles based on the genomic length (including exons and introns) of their longest transcripts. n=3 replicates.
Figure 8 Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs. Figure 8c. Proposed model. Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes. The situation in cells with normal and aberrant CDK12 kinase activity is depicted. CDK12 (green oval) phosphorylates (P) unknown substrate(s) (orange oval), possibly including the CTD (blue line), which results in optimal elongation and processivity (blue arrow) of RNAPII (blue oval) for CDK12-sensitive genes. Full length, functional mRNAs are synthesized (upper panel). Inhibition of CDK12 leads to hyperphosphorylation (capital P) of Ser2 (S2) in bodies of CDK12-sensitive genes which is associated with slower elongation and premature termination. Shorter, aberrant mRNAs are made (lower panel). mRNAs are depicted as black lines.
Fig. 2. Comparison of SARS-CoV-2 and SARS-CoV replication kinetics in Vero E6 cells.(a) Growth curve showing the release of infectious viral progeny into the medium of infected Vero E6 cells (m.o.i. 3), as determined by plaque assay (n = 4; mean ± sd is presented). (b). Comparison of SARS-CoV-2 Australia/VIC01/2020 and SARS-CoV Frankfurt-1 plaque phenotype in Vero E6 cells.