wums  (ATCC)

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