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, Mock-or LCMV-infected cells (24 hr pi) were simultaneously stained with smFISH probe sets 699 specific for either GPC mRNA and S genome (Cy5; green) or S genome only

, Representative LCMV infected cells with moderate (A) or high (B) levels of viral RNA as 701 well as a representative mock-infected cell (C) are displayed. Multiple Z stacks were acquired 702 spanning the thickness of the cell and max intensity projections are displayed. Boxed regions of 703 the cell are magnified and shown in rows labeled, p.704

, cytoplasmic (solid line) boundaries are shown in blue. (A to C) The same intensity levels for a 705 particular probe set were applied to all images of mock-and LCMV-infected cells to permit 706 comparisons

, FIG 3. smFISH probe sets recognizing viral mRNA species exhibit high signal-to-noise staining 709

, Signal-to-noise ratio of different smFISH probe sets labeled with the indicated fluorophores

, Signal-to-noise ratio was calculated as average amplitude of detected smFISH spots divided by 711 the standard deviation of signal in a region of the cell with no detected spots. The signal-to-noise 712 ratio of 20 cells per smFISH probe set labeled with the indicated fluorophore was calculated, and 713 the mean and standard deviation are graphed

, FIG 4 Automated detection and quantitation of LCMV RNAs labeled with spectrally distinct 716 fluorophores

, Cell nuclei and cytoplasms were automatically segmented using focus-based projections of 718

, Note that pixel intensities of the CellMask Green projection displayed here have been log 720 on March 6, 2020 at NYU LANGONE MED CTR-SCH OF MED http://jvi.asm.org/ Downloaded from transformed to aid visualization. Nuclear (hatched line) and cytoplasmic (solid line) boundaries 721 are shown in white

(. , C) Maximum intensity projections of LCMV-infected cells were fixed 24 hpi and stained 723 with smFISH probe sets to the NP mRNA/S antigenome (Cy5; green) and GPC mRNA/S

, Cells were segmented based on DAPI and CellMask Green staining (see panel A) and 726 spots were detected and localized in 3D using FISH-quant. Individually detected RNAs are 727 circled in green (NP mRNA/S antigenome) or red (GPC mRNA/S genome). The "Spots only" 728 column shows only the position of detected spots in relation to the cell boundaries defined by 729 segmentation. Nuclear (hatched line) and cytoplasmic

, The same intensity levels for a particular probe set were applied to both images of LCMV-731 infected cells to permit comparisons. The scale bar is 10 ?m

, Scatter plot shows the relationship between the fluorescence intensity in the smFISH channel 733 in the maximum intensity projection of smFISH images and the number of smFISH spots 734 detected by FISH-quant for LCMV-infected cells fixed 24 hpi and stained with the Cy5-labeled 735 smFISH probes specific for NP mRNA/S antigenome

, FIG 5 Transcription of NP and L genes is detectable soon following infection while GPC 738 transcription exclusively occurs after a several hour lag

N. Med, Cells were infected with LCMV at an MOI of 0.1, fixed at various times following infection, and 740 stained for NP mRNA (green) using a Cy5-labeled NP mRNA/S antigenome probe set, GPC 741 mRNA (red) using an A568-labled GPC mRNA/S genome probe set (A) or NP mRNA (green) 742 on March 6, 2020.

, Note that for the time points shown 744 (less than 8 hpi), genomic and antigenomic RNAs are not detectable by smFISH probe sets with 745 exclusive specificity for these RNAs (data not shown). Therefore, spots detected in this figure 746 are presumed to represent only the mRNAs, but not the genome or antigenome, targeted by each 747 respective probe set. Nuclear (hatched line) and cytoplasmic (solid line) boundaries as 748 determined by Cell Profiler are shown in blue. Identified spots are outline by circles that are 749 green for NP mRNA, red for GPC mRNA, and magenta for L mRNA. (A and B) The same 750 intensity levels for a particular probe set were applied to all images of mock-and LCMV-751, Quasar 670-labeled L mRNA/L antigenome probe set (B)

, FIG 6 Transcription of NP and L genes is detectable immediately upon infection while GPC 755 transcription exclusively occurs after a several hour lag

, A and B) Boxplots represent the number of viral RNAs detected in cells at 757 early time points following infection with LCMV (see Figure 5). (C and D) Stacked bar graphs 758 show the proportion of cells expressing RNAs detected by one, both, or neither viral RNA 759 smFISH probe set. Between 620 and 1316 cells were examined at each time point, p.760

, RNAs identified by specific probe sets are designated by color (green for NP mRNA/S 761 antigenome probes, red for GPC mRNA/S genome probes, and magenta for L mRNA/L 762 antigenome probes). Note that for time points prior to 8 hpi, genomic and antigenomic RNAs are 763 not detectable by smFISH probe sets with exclusive specificity for these RNAs

, Downloaded from genome or antigenome, recognized by each respective probe set. Spots detected at 8 hpi or later 766 are presumed to be a mixture of all RNAs recognized by a particular probe set, Therefore, spots detected before 8 hpi are presumed to represent only the mRNAs, but not the 765 on March 6, 2020 at NYU LANGONE MED CTR-SCH OF MED

, FIG 7 Peak viral RNA replication and transcription occurs 36 hpi and is slowly lost from 770 infected cells over the following days

, Cells were infected with LCMV at an MOI of 0.01, fixed at various times following infection, 772 and stained using smFISH probe sets specific for NP mRNA/S antigenome (Cy5; green) and 773 GPC mRNA/S genome (A568; red) (A) or NP mRNA

, Quasar 670; magenta) (B). (A and B) Representative maximum intensity 775 projections of fields of infected cells at various time points from 1 of 2 independent experiments 776 are shown. Each probe set is shown in its own row to highlight the difference in levels to which 777 these RNAs accumulate. The same intensity levels for a particular probe set were applied to all 778 images of mock-and LCMV

, FIG 8 Peak viral RNA replication and transcription occurs 36 hpi and is slowly lost from 782 infected cells over the following days

, A and B) Boxplots represent the number of mRNAs detected in cells at time 784 points during the peak period of LCMV infection. (C and D) Stacked bar graph shows the 785 proportion of cells expressing RNAs detected by one, both, or neither viral smFISH probe set

C. Langone-med and . Med, Downloaded from probe sets are designated by color (green for NP mRNA/S antigenome probes, red for GPC 788 mRNA/S genome probes, and magenta for L mRNA/L antigenome probes), Between 480 and 1659 cells were examined at each time point. RNAs identified by specific 787 on March 6, 2020 at NYU

, Cells were infected with LCMV at an MOI of 0.01. (A) Supernatants from infected cells were 793 collected at indicated time points post infection, titered, and the data are presented as mean 794