Journal: bioRxiv

Article Title: Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

doi: 10.1101/2021.12.10.472177

Figure Lengend Snippet: HeLa cells were transduced with lentiviruses encoding gene specific or non-silencing control shRNAs. Cells were treated with 10 IU/mL of IFN-β for 6 hours, infected with VEEV TC83 at a MOI of 1, and fixed and immunolabled for VEEV E2 and analyzed by flow cytometry 24 hours post-infection (hpi). Lentivirus (shRNA) positive cells are indicated by GFP, and E2-labled cells were counterlabeled with Alexaflour 647 secondary antibody. Summary of shRNA validation screen (53 genes) results expressed as the fold increase in VEEV positive cells relative to the shNSC control (A) . Each individual point represents a unique shRNA for the indicated gene. Mean fold change of all shRNAs against each target is represented by bars. (B) Representative flow plots of positive hits of known alphavirus restriction factors and IFN regulatory molecules detected in the validation screen. (C) 16-week-old WT (n=10) and Ifit2 -/- (n=10) mice were infected with 10 2 ffu of VEEV ZPC738 and monitored for survival (P = 0.0138). Statistical significance was calculated using Log-rank test.

Article Snippet: An expression vector encoding human IFIT2 (hIFIT2) (pET28a(+)-6xHis-TEV-IFIT2, Addgene; [ ]) was modified to express mouse IFIT2 (mIFIT2).

Techniques: Transduction, Control, Infection, Flow Cytometry, shRNA, Biomarker Discovery

Journal: bioRxiv

Article Title: Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

doi: 10.1101/2021.12.10.472177

Figure Lengend Snippet: The VEEV TC83 genome nucleotide content was analyzed using sliding window analysis (window = 50 nucleotide, step = 1 nucleotide), and the Z-score of the AU content graphed (A) . A Z-score cutoff of 2.32635 (representing the 99 th percentile) was used to determine significance, and regions with Z-scores > 2.32635 are indicated by asterisk (*). Z-scores below this cutoff are shaded in grey. A schematic of the VEEV genome is depicted below. Gene boundaries correspond with the approximate nucleotide number represented in the Z-score analysis. 5’ and 3’UTRs are indicated by grey lines, and open reading frames and poly A tail are annotated. (B) Z-score analysis of the AU-content of the 3’ end of the VEEV TC83 genome comprising part of E1 and the full length 3’UTR, with the corresponding genomic region depicted below. (C, D) Determination of IFIT2 binding specificity using DRaCALA. P32 5’ end-labeled RNA corresponding to the 3’ terminal 200 nucleotide was incubated with serial 2-fold dilutions of rIFIT2 (green) or bovine serum albumin (BSA; black) for 15 min at 37°C, then spotted onto nitrocellulose and visualized after exposure to phosphor screen. A representative blot of a single replicate from one experiment is shown in (C) . The fraction of bound RNA was calculated and graphed in relation to protein concentration (D) . (E, F) Determination of IFIT2 binding specificity to different RNAs using DRaCALA. Binding of rIFIT2 to 100bp RNAs corresponding to the VEEV 3’UTR (black), VEEV nsp3 (purple), and a synthetic RNA (blue) was performed as described for (C) and (D). A representative blot of a single replicate from one experiment is shown in (E) . The fraction of bound RNA was calculated and graphed in relation to protein concentration (F) . (G) Sequence of 3’, nsp3, and synthetic RNAs assayed in E and F. AU content (%) for each RNA is indicated and A/U nucleotides highlighted in grey. (H) Sequence alignment of VEEV 3’UTR consensus sequences from IAB, IC, ID, and IE subtypes (see also Supplementary Figure 1 ). Full-length and partial VEEV genomes were aligned using Geneious Prime (MAFFT), and the 3’UTR region of the alignment extracted (including the ORF2 stop codon). Consensus sequences were generated from individual IAB, IC, ID, and IE alignments, and an alignment of these consensus sequences was performed. The ncbi accession number for each subtype analysis is listed in Supplementary Figure 1 , and strains were grouped by subtype as indicated on the left of the alignment. Consensus sequence is shown at the top of the alignment in IUPAC-IUB notation (R=purine, Y=pyrimidine, K=keto (G/U), W=weak interaction (2-hydrogen bonds; A/U), H=not G). Identical sequences are represented as periods (.) and sequence gaps are represented as dashes (-).

Article Snippet: An expression vector encoding human IFIT2 (hIFIT2) (pET28a(+)-6xHis-TEV-IFIT2, Addgene; [ ]) was modified to express mouse IFIT2 (mIFIT2).

Techniques: Binding Assay, Labeling, Incubation, Protein Concentration, Sequencing, Generated

Journal: bioRxiv

Article Title: Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

doi: 10.1101/2021.12.10.472177

Figure Lengend Snippet: (A) Sequence alignment of 3’UTR sequences from TC83, IC (TC83/IC-3’UTR), ID (TC83/ID-3’UTR), and IE (TC83/IE-3’UTR) 3’UTR chimeras. Viruses encoding epizootic 3’UTRs (IAB and IC) are shaded in red, and viruses encoding enzootic 3’UTRs (ID and IE) are shaded in blue. Consensus sequence is shown at the top of the alignment in IUPAC-IUB notation (R=purine, Y=pyrimidine, K=keto (G/U), W=weak interaction (2-hydrogen bonds; A/U), H=not G). Nucleotide numbering is indicated on the right side. Identical sequences are represented as periods (.) and sequence gaps are represented as dashes (-). (B-G) Growth kinetics of VEEV 3’UTR mutants in WT and Ifit2 -/- primary MEF. WT (black) and Ifit2 -/- (red or blue) cells were mock (solid lines) of IFN-β pre-treated (dotted lines) for 12 hours, then infected with indicated viruses at a MOI of 0.01. Cell culture supernatant was serially harvested at 1, 6, 12, 24, 36, and 48 hpi and infectious virus titered using focus forming assay (FFA). Red lines indicate mutant viruses encoding epizootic 3’UTR sequences, and blue indicate mutant viruses encoding enzootic 3’UTR sequences. Infections with each virus were performed simultaneously and displayed individually for ease of viewing. Each experiment was performed in triplicate three times independently and the mean and SEM graphed. Statistical analysis was performed by calculating the area under the curve (AUC) for each replicate and experiment, and AUC values from WT and KO cells analyzed by unpaired t-test. (F, G) Data from B-E plotted by treatment (no IFN, +IFN). Statistical significance comparing all viruses in WT cells +IFN and KO cells +IFN was calculated on the AUC by one-way ANOVA with multiple comparisons and is indicated on the right. **, P ≤ 0.01; ns, not significant.

Article Snippet: An expression vector encoding human IFIT2 (hIFIT2) (pET28a(+)-6xHis-TEV-IFIT2, Addgene; [ ]) was modified to express mouse IFIT2 (mIFIT2).

Techniques: Sequencing, Infection, Cell Culture, Virus, Focus Forming Assay, Mutagenesis

Journal: bioRxiv

Article Title: Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

doi: 10.1101/2021.12.10.472177

Figure Lengend Snippet: Validation of Ifit2 CRISPR KO macrophages (Raw264.7); (A) generated using CRISPR. Cell lysates were electrophoresed, blotted onto nitrocellulose, and probed using monoclonal IFIT2-specific antibodies, as well as IFIT1 antibodies to determine specificity of gene KO for IFIT2. Growth kinetics of 3’UTR mutants in WT (empty; black) and Ifit2 KO (g13; red/blue) Raw264.7 (B-I) . Red lines indicate mutant viruses encoding epizootic 3’UTR sequences, and blue lines indicate mutant viruses encoding enzootic 3’UTR sequences. Each experiment was performed in triplicate three times independently and the mean and SEM graphed. Statistical analysis was performed by calculating the area under the curve (AUC) for each replicate and experiment, and AUC values from WT and KO cells analyzed by unpaired t-test. (J,K) Data from B-I plotted by cell line (Raw264.7-empty, Raw264.7-g13). Statistical significance comparing all viruses in WT or KO cells was calculated on the AUC by one-way ANOVA with multiple comparisons. Comparison of all non-ID viruses to all ID viruses was significant. No significant difference was observed between any of the ID viruses. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant.

Article Snippet: An expression vector encoding human IFIT2 (hIFIT2) (pET28a(+)-6xHis-TEV-IFIT2, Addgene; [ ]) was modified to express mouse IFIT2 (mIFIT2).

Techniques: Biomarker Discovery, CRISPR, Generated, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

doi: 10.1101/2021.12.10.472177

Figure Lengend Snippet: WT and Ifit2 -/- primary MEF were mock (A, B) or IFN-β pre-treated (C, D) , then electroporated with 2ug of viral reporter RNAs encoding TC83, IC, ID, or IE 3’UTR sequences. Cell lysates were harvested at 30, 60, 120, and 240 minutes post-electroporation, luciferase activity measured, and normalized to total protein. Measurements are given as relative light units (RLU)/ug of protein. Each data point represents three independent experiments. (E and F) WT (Empty) and Ifit2 -/- CRISPR (g13) Raw264.7 cells were electroporated with 10μg viral reporter RNAs and lysates harvested and analyzed for luciferase activity as for MEF.

Article Snippet: An expression vector encoding human IFIT2 (hIFIT2) (pET28a(+)-6xHis-TEV-IFIT2, Addgene; [ ]) was modified to express mouse IFIT2 (mIFIT2).

Techniques: Electroporation, Luciferase, Activity Assay, CRISPR