Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet: A) Experimental design schematic for isolating small RNAs. HEK 293 cells +/- infection with WT or TMER1-only γHV68 were incubated for 24 hpi prior to total RNA collection and size fractionation for small RNAs under 300 nts. B) Experimental design schematic for 5’ end characterization of RNA. Small RNAs are treated with or without RNA 5’-polyphosphatase to convert 5’-triphosphate to 5’-monophosphate ends, and then treated with or without Terminator™ enzyme to degrade only RNAs with a 5’-monophosphate. Human tRNA valine (C) and human 5S rRNA (D) 5’ end characterization: enzymatic products indicated were resolved by SDS-PAGE and northern blots tested with probes specific to host tRNA or 5S rRNA transcripts (left). Densities of the resulting bands were normalized to an ethidium bromide stained 5S rRNA loading control. Relative band density was calculated as a fold change of the untreated RNA population, which was set to 1, and displayed as heat maps (right). Heat maps represent n = 4 for tRNA and n = 3 for 5S rRNA.

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Infection, Incubation, Fractionation, SDS Page, Northern Blot, Staining, Control

Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet: A) Schematic of the predicted secondary structure of TMER1 with sequence for northern TMER1 probe 1 indicated at the 3’ side of stem loop 1. B) TMER1 primary and processed forms: predicted structures in left column, name and length in center, and TMER1 probe 1 sequence in right column (“+” indicates sequence present; “-” indicates sequence not contained). The tRNA-like loop is shown in blue, and TMER1 miRNAs in purple. C) Following sequential enzymatic treatments as detailed in , small RNAs were resolved by SDS-PAGE then detected by northern blot with TMER1 probe 1. Blot is representative of three independent experiments. RNA bands detected only during infection and specific to TMER1 are marked with red arrows, in contrast to non-specific bands shared with mock infected samples. D) Densities of the TMER1 northern blot bands were normalized to an ethidium bromide stained 5S rRNA loading control. Relative density of bands were calculated as a fold change of the untreated RNA population, which was set to 1, and presented as heat maps. Band sizes were calculated as averages based on migration of a ladder included in each experiment. RNA bands not detectable in WT γHV68 infection are shown as gray boxes. Data is from three independent experiments.

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Sequencing, Northern Blot, SDS Page, Infection, Staining, Control, Migration

Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet: A) Schematic of features of TMER1 RNA. The TMER1 predicted structure consists of a tRNA- like loop (dark blue) and multiple stem loops that are processed into biologically active miRNAs (purple). B) Schematic of northern probe sequences used to detect TMER1. Different northern probes (light blue boxes) bind to various regions of TMER1 RNA, allowing detection of alternate, processed forms. C) Table showing the multiple possible alternate forms of TMER1 with varying lengths. The two probe sequences shown here (Probe 2 and Probe 3) are present in some TMER1 forms (+), but not others (-). Following sequential enzymatic treatments (P = RNA 5’-polyphosphatase, T = Terminator™), small RNAs were resolved by SDS-PAGE gel and northern blot was performed with TMER1 Probe 2 (D) or Probe 3 (E). The RNA bands specific to TMER1 are marked with red arrows. Band densities for the TMER1 RNAs detected by TMER1 probe 2 (F) and TMER1 probe 3 (G ). Densities of the TMER1 northern blot bands were normalized to a 5S rRNA loading control stained with ethidium bromide. Relative density of bands were calculated as a fold change of the untreated RNA population, which was set to 1, and presented as heat maps. Band sizes were calculated as averages based on migration of a ladder included in each experiment. RNA bands not consistently detectable in WT γHV68 infection are shown as gray boxes. Data for each probe is from two independent experiments.

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Northern Blot, SDS Page, Control, Staining, Migration, Infection

Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet: A) Experimental design to study ncRNA interactions with RIG-I and La. HEK 293 cells were transfected with FLAG-tagged proteins of interest; RIG-I or La. For EBER interaction analysis, cells were also transfected with a plasmid expressing both EBER1 and EBER2 (pSP73-EBERs). 24 hours after transfection, cells were infected with mock, WT, TMER1-only, or TMER-TKO γHV68 at an MOI of 5. 24 hpi (48 hours post-transfection), immunoprecipitation was performed, followed by “permissive wash” with TBS. An aliquot of beads was reserved for western blot analysis (B) and RNA was isolated from the remaining beads for RT-PCR (C). B) Proteins from whole cell lysate (W) or immunoprecipitation beads (IP) were resolved by SDS-PAGE and detected by western blot with a primary antibody targeting FLAG for RIG-I-FLAG (left) or La- FLAG (right) transfected samples. Ladder shows protein size in kDa. Ponceau red stained blots, below, demonstrate enrichment by IP. Blots are representative of two independent experiments with technical triplicates. C) RNA was isolated from whole cell lysate (W) or immunoprecipitated (IP) samples from cells transfected with RIG-I-FLAG (RIG) or La-FLAG (La). Primers targeting TMER1 (left) or EBER1 (right) were used for RT-PCR with 40 cycles. PCR without reverse transcription (“PCR”) was performed in conjunction with RT-PCR to test for DNA contamination. Data are representative of two independent experiments with technical duplicates or triplicates.

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, Immunoprecipitation, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, SDS Page, Staining, Reverse Transcription

Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet: HEK 293 cells were transfected with FLAG-tagged RIG-I or La, then infected with WT, TMER1-only (TMER1), or TMER-TKO (TKO) γHV68 as previously described. Whole cell lysates were collected 24 hpi and used for immunoprecipitation of FLAG-tagged RIG-I or La. RNA was isolated from immunoprecipitated complexes and analyzed by RT-PCR with primers targeting TMER1 (A) or TMER5 (B) . PCR without reverse transcription (“PCR”) was performed in conjunction with RT-PCR to test for DNA contamination. NT = non-template control, L = ladder. Data are representative of one experiment with technical triplicates (TMER1) or duplicates (TMER5).

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Transfection, Infection, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Control

Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet: A) Modified experimental design to detect ncRNA interactions with RIG-I and La. Experiment was performed as previously described with the following modifications. HEK 293 cells were transfected with FLAG-tagged RIG-I, La, or GFP as a non-specific binding control. 24 hours after transfection, cells were infected with WT or EBER-knock in (EBER-KI) γHV68. Immunoprecipitation was performed as before, followed with a “stringent” wash of beads (outlined in red box) prior to protein analysis and RNA isolation as before. B) Proteins from whole cell lysates (W) or immunoprecipitated beads (IP) were resolved by SDS-PAGE and western blot analysis was performed with a primary antibody targeting FLAG. Proteins were analyzed in mock, WT γHV68 infection (WT), or EBER-KI γHV68 infection (EBER). Protein ladder is indicated to the left of each blot (kDa). Expected approximate protein sizes: La = 47 kDa, RIG-I = 102 kDa, GFP = 27 kDa.

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Modification, Transfection, Binding Assay, Control, Infection, Knock-In, Immunoprecipitation, Isolation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet: A) Schematics representing genetic details of the γHV68 ncRNA recombinants. Line diagrams represent the first 6 kilobases of the γHV68 genome, including the M1 and M2 genes (black rectangles). Each intact TMER gene is depicted as a red triangle. Gray triangles represent TMERs that are not expressed due to promoter deletion as previously described . Orange diamonds represent the expression of EBERs in place of TMERs through knock-in of the EBER1 and EBER2 sequences into the left end of the γHV68 genome (EBER knock-in; EBER-KI). B) PCR of viral recombinant DNA. W = WT γHV68, 1 = TMER1-only γHV68, 4 = TMER4-only γHV68, 5 = TMER5-only γHV68, 8 = TMER8-only γHV68, E = EBER-KI γHV68, pK = pLE—TMER-TKO plasmid as previously described (; does not contain M3), “-” = no-template control. Targets listed to the right of PCR panels. C) RT-PCR of RNA collected from HEK 293 cells infected with γHV68 recombinants at an MOI of 1. Viruses indicated as in B, except M = mock and K = TMER-TKO γHV68 (expresses M3). Targets for B and C listed to the right of PCR panels. PCR without reverse transcription was run with the same conditions as each RT- PCR to confirm the absence of DNA contamination (not shown). Some product sizes differ than the same target in (B) due to the use of different primers better suited to RT-PCR analysis. D) Single step replication analysis with WT γHV68 (red squares) or recombinants in 3T12 cells at an MOI of 5. Other viral recombinants shown are TMER-TKO (gray circles, dashed line), TMER4-only (blue triangles), TMER5-only (flipped purple triangles), TMER8-only (half-filled green triangles), and EBER-KI (orange diamonds). Cells and supernatants were collectively harvested at the indicated times post-infection, then quantified by plaque assay. Data depict the mean of 3 biologic replicates within a single experiment. Error bars = SEM.

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Expressing, Knock-In, Recombinant, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Infection, Reverse Transcription, Plaque Assay

Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet: BALB/c IFNγ -/- mice were infected with a panel of γHV68 ncRNA recombinants. At 8 days p.i., lung tissue was collected for viral titer analysis by (A) qPCR for viral DNA (gB gene) and (B) plaque assay quantitation of infectious virus. Limit of detection (LOD) is indicated by a horizontal dashed line on each graph. Virus was not detected in mock-infected tissue samples in each analysis. Individual symbols represent the value from an individual mouse. Three mice were analyzed for WT and TMER-TKO γHV68, and five mice were analyzed for all other viruses. Horizontal black lines indicate the mean of each group. One-way ANOVA analysis with multiple comparisons of each γHV68 recombinant to WT γHV68 detected no significant difference. C) Analysis of BALB/c IFNγ -/- mice following infection with WT or recombinant γHV68 monitored for signs of morbidity over the course of 15 days. The number of mice in each group is indicated. Statistical analysis of survival curves was done by log-rank (Mantel-Cox) test with pairwise comparisons of recombinant viruses and WT γHV68.βla. P-values for survival following infection with each recombinant except TMER-TKO compared to WT virus are all greater than 0.05 (not significantly different, “ns”); TMER-TKO = 0.025, TMER4-only = 0.47, TMER5-only = 0.21, TMER8-only = 0.14, EBER-KI = 0.79.

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Infection, Plaque Assay, Quantitation Assay, Virus, Recombinant

Journal: bioRxiv

Article Title: Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

doi: 10.1101/2022.05.09.491269

Figure Lengend Snippet:

Article Snippet: All viruses and recombinants were derived from γHV68 strain WUMS (ATCC VR-1465) ( ).

Techniques: Recombinant, Sequencing

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: High-dimensional single-cell protein analysis of MHV68 infection by CyTOF. (A) 3T12 fibroblasts were either mock or MHV68-infected with WT MHV68.LANAβlac (MOI=0.5), with indicated populations FACS purified according to LANA::β-lactamase expression at 16 hours pi. (B) PhenoGraph analysis of mock, LANA- and LANA+ cells subjected to CyTOF analysis and visualized by tSNE-based dimensionality reduction identified 14 cell clusters (colored by cluster ID). (C) Comparison of the frequency (left) and protein expression profile (right) of PhenoGraph-defined clusters in LANA- and LANA+ samples. Clusters (in rows), ranked from those exclusively in LANA- samples (top) to those exclusively in LANA+ samples (bottom); asterisks indicate clusters statistically significantly different between conditions, defined by unpaired t-test corrected for multiple comparisons using the Holm-Sidak method, p<0.05. Data show mean ± SEM with individual symbols indicating individual sample values (n=4 per group). Relative protein expression for each cluster (right panel) is indicated by heatmap intensity, with protein markers divded by functional categories. (D) tSNE plot of LANA- and LANA+ cells colored according to LANA- and LANA+ exclusivity (left), vRCA expression (middle), and pH2AX expression (right panel). Red and blue lines identify events exclusive to LANA+ or LANA- clusters (left panel). Ruler defines range of expression with values calculated using the equation arcsinh (x/5) where x is raw expression value. Live, DNA+ cells ( 191 Ir+ 193 Ir+ 195 Pt-) were imported into PhenoGraph with 9,162 events total analyzed (1,018 events from each sample; n=1, mock, n=4 each for LANA+ and LANA- samples), clustered on 9 cell surface proteins (vRCA, BST2, CD9, CD29, CD44, CD63, Ly6A/E, Ly6C, and MHCI). See also Figure S1.

Article Snippet: All experiments used WT MHV68 (strain WUMS; ATCC VR-1465) , using either bacterial artificial chromosome–derived WT MHV68 ( ) or WT MHV68.LANAβlac, which encodes a fusion between ORF73 and the beta-lactamase gene ( ).

Techniques: Infection, Purification, Expressing, Comparison, Functional Assay

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: CyTOF analysis of LANA+ MHV68-infected cells as in . (A) PhenoGraph analysis of 40,000 LANA+ cells (10,000 cells/sample, 4 samples) clustered by 9 cell surface proteins (vRCA, BST2, CD9, CD29, CD44, CD63, Ly6A/E, Ly6C, and MHCI) identified 15 unique cellular clusters colored by cluster ID and displayed on a tSNE plot. (B) LANA+ cells can be stratified into six categories based on expression of pH2AX, vRCA, Ly6C, and BST2, with (C) LANA+ cells colored by expression levels for each protein marker. Ruler defines range of expression with values calculated using the equation arcsinh (x/5) where x is raw expression value. (D) LANA+ cells were divided into pH2AX Lo and pH2AX Hi events, and compared for raw median expression. Data depict proteins whose expression was statistically significantly different between pH2AX Lo and pH2AX Hi events. Data depict mean ± SEM with individual symbols indicating values from independent samples (n=4/group). All samples were analyzed for statistical significance using unpaired t tests, corrected for multiple comparisons using the Holm-Sidak method, with statistical significance denoted as *p<0.05, ** p<0.01, ***p<0.001. See also Figure S2.

Article Snippet: All experiments used WT MHV68 (strain WUMS; ATCC VR-1465) , using either bacterial artificial chromosome–derived WT MHV68 ( ) or WT MHV68.LANAβlac, which encodes a fusion between ORF73 and the beta-lactamase gene ( ).

Techniques: Infection, Expressing, Marker

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: Flow cytometric analysis of protein and RNA expression in 3T12 fibroblasts infected with MHV68 (MOI=0.5, harvested 16 hpi). (A) Analysis of vRCA and pH2AX protein expression in mock and MHV68-infected cultures identified four populations denoted by numbers and color (1=Gray, 2=Blue, 3=Orange, 4=Red). (B) Analysis of viral ORF18 and host Actin (Actb) mRNA expression across four populations of MHV68-infected cultures, stratified by vRCA and pH2AX expression. Border color of biaxial plots corresponds to the parent population stratified by protein expression in panel A. (C) Quantitaiton of the frequency of cells based on RNA expression profile as in panel B, depicting mean ± SEM from 3 independent experimental samples. Flow cytometric analysis was done on singlets, following gating to remove doublets.

Article Snippet: All experiments used WT MHV68 (strain WUMS; ATCC VR-1465) , using either bacterial artificial chromosome–derived WT MHV68 ( ) or WT MHV68.LANAβlac, which encodes a fusion between ORF73 and the beta-lactamase gene ( ).

Techniques: RNA Expression, Infection, Expressing

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: scRNA-seq analysis of MHV68 viral gene expression in LANA+ 3T12 fibroblasts as in . (A) LANA+ cells show a wide range in how many viral genes are expressed on a per-cell basis. Expression of a viral gene was defined conservatively as any viral gene with ≥1 UMI detected per cell, with data showing the distribution of cells based on number of viral genes expressed per cell. (B) Viral genes vary widely in mean UMI per cell and the frequency of cells expressing an individual viral gene. Mean viral UMI per cell was calculated from positive cells that expressed ≥1 UMI per gene, with five representative genes identified by unique shading. Each dot represents expression for a single viral gene product for all 80 annotated MHV68 open reading frames. (C) The distribution of cells based on total viral UMIs reveals a bimodal distribution of virus low and virus high cells. (D) Total viral UMIs per cell as a function of the number of viral genes detected per cell (as in panel A). Cells demonstrate a bimodal distribution of total viral UMIs with virus high and low cells, with a positive Spearman correlation coefficient, rs, as indicated. (E) Frequency of cells expressing each viral gene, with viral gene expression defined as ≥1 UMI for each viral gene. Dashed line indicates 50% of events. (F) Mean UMI count per viral gene, defined by calculating mean value only from positive cells (≥1 UMI for each viral gene), depicting mean ± SD. Data represent scRNA-seq data from all LANA+ cells (n=1605 cells). nd, genes that were not detected. See also Figure S3 and Table S1.

Article Snippet: All experiments used WT MHV68 (strain WUMS; ATCC VR-1465) , using either bacterial artificial chromosome–derived WT MHV68 ( ) or WT MHV68.LANAβlac, which encodes a fusion between ORF73 and the beta-lactamase gene ( ).

Techniques: Gene Expression, Expressing, Virus

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: scRNA-seq analysis of MHV68 gene expression in LANA+ 3T12 fibroblasts as depicted in . Comprehensive visualization of viral gene expression on a single cell basis. Each row depicts expression pattern for a different viral gene. Each column depicts expression within an individual cell. Cells are rank ordered from the cell with the least (left) to the greatest total viral UMI count (right), with corresponding total viral (filled orange line) and host (black dots) UMIs per cell depicted in the bottom panel. Cells are further bisected into Virus low cells (< 500 viral UMIs per cell) and Virus high cells (> 500 viral UMIs per cell) by a thick vertical black line, from a total of 1605 cells. UMI counts are stratified based on the indicated heatmap scale. See also Figure S4.

Article Snippet: All experiments used WT MHV68 (strain WUMS; ATCC VR-1465) , using either bacterial artificial chromosome–derived WT MHV68 ( ) or WT MHV68.LANAβlac, which encodes a fusion between ORF73 and the beta-lactamase gene ( ).

Techniques: Gene Expression, Expressing, Virus

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: scRNA-seq analysis of MHV68-infected LANA+ cells for viral and host RNAs. (A-E) 3D tSNE-based dimensionality reduction was used to visualize gene expression within individual cells. (A) K-means clustering identified 5 unique cell clusters, as labelled. (B) Viral and host RNAs show different patterns of expression, comparing viral immediate early (IE), early-late (E-L) and late (L) genes with host beta actin (Actb). UMI counts visualized in individual cells (symbols) as defined by key. (C) The ratio of host Actb to ORF37 UMIs, calculated for each cell and overlaid onto tSNE-based visualization, varies widely across cells. (D) Percent of viral (left) and host UMIs (right) per cell was calculated for each cell, portrayed by shade of color overlaid onto tSNE visualization. (E) Cells were separated into two groups: virus biased (red) and host biased (black) based on percent viral and host UMIs, and K-means clustering. Host biased cells included clusters A and B with <20% virus UMIs on a per cell basis. (F) Characteristics of virus and host biased cells based on scRNA-seq data. (G-H) Number of (G) viral or (H) host genes with mean UMI expression ≥1, among either virus (open red symbol) or host biased cells (dashed black line). (I) Violin plots depict the distribution of mean gene expression among expressed genes (mean UMI expression ≥1), comparing viral genes (left) or host genes (right) in either virus biased (open red symbol) or host biased cells (open black line). Quartiles and median are depicted in each violin plot. Statistical comparisons compared viral or host genes and their difference between virus and host biased cells. **** p <0.0001, one way ANOVA with Tukey’s multiple comparisons test. ns, not significant. (J-K) Biaxial analysis of LANA+ cells (n=1605 cells) comparing total host UMI count (y axis) versus total viral UMI count (x axis) identified three major cell populations, stratified by differential expression of viral or host genes. (K) Identification of cells that are Virus low Host high (blue), Virus high Host high (orange), and Virus high Host low (red). (L-P) Histogram overlays comparing relative expression of (L-O) viral and (P) host genes for the three identified cell populations. Each gene is denoted by its IE, E, or L class and by its kinetic class defined by . See also Figure S5-6, Table S2.

Article Snippet: All experiments used WT MHV68 (strain WUMS; ATCC VR-1465) , using either bacterial artificial chromosome–derived WT MHV68 ( ) or WT MHV68.LANAβlac, which encodes a fusion between ORF73 and the beta-lactamase gene ( ).

Techniques: Infection, Gene Expression, Expressing, Virus, Quantitative Proteomics

Journal: bioRxiv

Article Title: Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

doi: 10.1101/2020.08.11.203117

Figure Lengend Snippet: The inter-relationship of viral gene expression was interrogated using scRNA-seq data from MHV68-infected LANA+ cells. (A) Correlation matrix of viral genes among Virus high cells (>500 viral UMIs/cell), depicting Spearman correlation coefficient, rs, for each gene pair according to the indicated heatmap. Positive correlation is indicated by blue, negative correlation indicated by red. Correlation matrix depicts all viral genes except for M10b and M10c (X indicates gene for which there were no values) and M12, M13 and M14 (excluded due to negligible values). (B) A comparison of correlation coefficients between viral gene pairs when analyzing all cells (top), Virus high (middle) or Virus low (bottom) cells. Data identify discrepant correlations observed in different cell subsets. (C) Examples of host and virus gene pairs with positive correlations, demonstrated by plotting scRNA-seq gene expression values on biaxial plots across all LANA+ cells. (D) Examples of host and virus gene pairs with a lack of correlation or negative correlation. Panels in C-D depict scRNA-seq values for all LANA+ cells (n=1,605 cells), including both Virus high and Virus low events. Each plot includes the Spearman correlation coefficient for the indicated gene pair defined across all cells. See also Figure S7.

Article Snippet: All experiments used WT MHV68 (strain WUMS; ATCC VR-1465) , using either bacterial artificial chromosome–derived WT MHV68 ( ) or WT MHV68.LANAβlac, which encodes a fusion between ORF73 and the beta-lactamase gene ( ).

Techniques: Gene Expression, Infection, Virus, Comparison