orf57 pr pgl4 16  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: The Kaposi’s sarcoma-associated herpesvirus TBP mimic uses a noncanonical DNA-binding mode to promote viral late gene transcription

doi: 10.1093/nar/gkaf1008

Figure Lengend Snippet: ORF24 requires interaction with Rpb1 and ORF34, as well as its CTD and vTBP to bind late promoters in infected cells. ( A ) ORF24 protein levels at 48 hpr in complemented iSLK cell lines. ( B ) ChIP-qPCR of indicated cell lines at 48 hpr was performed with HA-antibody, and IgG as a negative control. qPCR to detect the associated DNA was performed with promoter-specific primers for a representative late (K8.1) and early (ORF57) promoter. ( C ) Proposed model of ORF24 recognition of late promoters in comparison to eukaryotic mechanism. Created in BioRender. Llacsahuanga, L. (2025) https://BioRender.com/r23legx .

Article Snippet: Luciferase reporter plasmids K8.1 Pr pGL4.16 + Ori (Addgene 131038) and ORF57 Pr pGL4.16 (Addgene 120378) were used to measure late and early promoter activity, respectively.

Techniques: Infection, ChIP-qPCR, Negative Control, Comparison

Journal: Human molecular genetics

Article Title: Human herpesvirus 8 ORF57 protein is able to reduce TDP-43 pathology: network analysis identifies interacting pathways.

doi: 10.1093/hmg/ddad122

Figure Lengend Snippet: Figure 1. ORF57 protects against oxidative stress in TDP-43NLS overexpressing SH-SY5Y cells. Experiments conducted in mCherry and ORF57 mCherry express ing SH-SY5Y TDP-43NLS cells with and without SA stress. (A) Constructs for mCherry and ORF57::mCherry lentivirus. (B) Overexpression of ORF57 in SH-SY5Y cells by rt-qPCR. (C) Western blot for phosphorylated eIF2α and total eIF2α. (D) Quantification of C. (E) SUnSET of mCherry and ORF57 expressing TDP-43NLS cells with SA stress. (F) Quantification of E. (G) Western blot of cleaved caspase-3 in TDP-43NLS cells. (H) Quantification of G. (I) Western blot of cleaved caspase-3 in non-overexpressing SH-SY5Y cells. (J) Quantification of I.

Article Snippet: The ORF57 DNA plasmid was bought from Addgene (pCDNA4.TOORF57-2xCSTREP Plasmid #136217).

Techniques: Construct, Over Expression, Quantitative RT-PCR, Western Blot, Expressing

Journal: Human molecular genetics

Article Title: Human herpesvirus 8 ORF57 protein is able to reduce TDP-43 pathology: network analysis identifies interacting pathways.

doi: 10.1093/hmg/ddad122

Figure Lengend Snippet: Figure 2. Stress granule assembly and TDP-43 aggregation dysregulated by ORF57. (A) Immunocytochemistry in SH-SY5Y TDP-43NLS. DAPI (405 nm). mCherry or ORF57::mCherry (594 nm). TDP-43NLS::GFP (488 nm). G3BP1 (647 nm). Merge, overlay of all images. White arrows indicate overlap between ORF57 and TDP-43 aggregates. (B) Representation of small TDP-43NLS aggregates 10–200 Aggrecount units. (C) Representation of large TDP-43NLS aggregates 200–400 Aggrecount units. (D) Quantification of G3BP1 aggregates in each cell condition from A. (E) Mander’s coefficient of fraction of mCherry overlapping with TDP-43 signal. (F) Quantification of small TDP-43NLS aggregates. (G) Quantification of large TDP-43NLS aggregates. Two-way ANOVA with Bonferroni post hoc comparison, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001.

Article Snippet: The ORF57 DNA plasmid was bought from Addgene (pCDNA4.TOORF57-2xCSTREP Plasmid #136217).

Techniques: Immunocytochemistry, Comparison

Journal: Human molecular genetics

Article Title: Human herpesvirus 8 ORF57 protein is able to reduce TDP-43 pathology: network analysis identifies interacting pathways.

doi: 10.1093/hmg/ddad122

Figure Lengend Snippet: Figure 3. TDP-43 aggregates are more soluble with ORF57. Experiments conducted in mCherry and ORF57 mCherry expressing SH-SY5Y TDP-43NLS cells with SA stress. (A) Western blot of TDP-43NLS::GFP RIPA-soluble and in-soluble fractions. (B) Quantification of %RIPA-Soluble TDP-43NLS::GFP calculated as soluble TDP-43 divided by the sum of soluble and insoluble TDP-43. (C) Quantification of %RIPA-insoluble TDP-43NLS::GFP. (D) Time course experiment measuring TDP-43NLS::GFP aggregate recovery at 15, 30 min, 1 , 2 , 3.5 and 6.5 h after SA removal. (E) Imaging of TDP-43NLS using FRAP. (F) Quantification of D with exponential one-phase decay best fit curves. (G) Analysis of TDP-43NLS mobility using FRAP. mCherry controls cells had a mobile fraction of 0.03 and a t1/2 of 111 s; ORF57::mCherry expressing cells had a higher mobile fraction of 0.2 and a t1/2 of 68 s. Unpaired t-tests, ∗P < 0.05. Scale bars 20 μm.

Article Snippet: The ORF57 DNA plasmid was bought from Addgene (pCDNA4.TOORF57-2xCSTREP Plasmid #136217).

Techniques: Expressing, Western Blot, Imaging

Journal: Human molecular genetics

Article Title: Human herpesvirus 8 ORF57 protein is able to reduce TDP-43 pathology: network analysis identifies interacting pathways.

doi: 10.1093/hmg/ddad122

Figure Lengend Snippet: Figure 4. ORF57 interactome. (A) Outline of proteomic approach using SH-SY5Y cells expressing GFP::TDP-43NLS. (B) High-confidence ORF57-bound proteins observed under basal conditions and the GO-BP functional annotations terms associated with these enriched proteins. Functional annotation terms were determined using ShinyGO Version 0.76.3 (81) (C) High-confidence ORF57-bound proteins observed in presence of SA and the GO-BP functional annotations terms associated with these enriched proteins. Enriched proteins in basal and stress conditions were considered significant when they have a Log2 fold-change > 0.58 and an FDR adjusted P-value < 0.05.

Article Snippet: The ORF57 DNA plasmid was bought from Addgene (pCDNA4.TOORF57-2xCSTREP Plasmid #136217).

Techniques: Expressing, Functional Assay

Journal: Human molecular genetics

Article Title: Human herpesvirus 8 ORF57 protein is able to reduce TDP-43 pathology: network analysis identifies interacting pathways.

doi: 10.1093/hmg/ddad122

Figure Lengend Snippet: Figure 5. ORF57 interacts with the TDP-43 protein network. (A) Venn diagram using ORF57, ORF57-SA and the TDP-43 BioGRID-TARDBP) datasets. (B) Functional annotations terms for overlapping proteins between ORF57-SA and BioGRID-TARDBP datasets. (C) Multi-module representation of ORF57- bound protein networks (Using Cytoscape, STRING-db and ClusterMaker2. Node title describes gene names, node shape describes whether the protein was found in either ORF57 datasets (circle) or BioGRID-TARDBP (diamond), and color indicates whether the gene was only found in either ORF57, ORF57-SA or overlapped between them.

Article Snippet: The ORF57 DNA plasmid was bought from Addgene (pCDNA4.TOORF57-2xCSTREP Plasmid #136217).

Techniques: Functional Assay