rabbit anti rap80  (Bethyl)

Journal: bioRxiv

Article Title: HSV-1 orchestrates host RAP80 ubiquitination by ICP0 and UL36USP to promote viral survival

doi: 10.1101/2025.06.10.658793

Figure Lengend Snippet: a. Endogenous RAP80 expression profiles across various post-HSV-1 infection stages were evaluated in HeLa cells exposed to HSV-1 at a multiplicity of infection (MOI) = 1. Cells were harvested at different time points post-infection, and RAP80 and ICP0 protein levels examined by immunoblotting with specific antibodies. b. Representative immunofluorescence images showing cellular RAP80 colocalization with the viral proteins ICP0, ICP4, and ICP8 in HeLa cells. Images were captured at 3 h post-infection with HSV-1 (MOI=1). Quantified RAP80 (red) and viral protein (green) fluorescence intensity values at colocalization sites were determined using ImageJ software. c. RAP80 knockdown (KD) effects on viral TK expression were assessed in HeLa cells with depleted RAP80 at 3 h or 20 h post-infection with HSV-1 (MOI=1). d. Specific RAP80 binding sites in the HSV-1 genome were investigated by ChIP-seq using ChIP DNA extracted from HeLa cells with a RAP80 antibody at 3 h post HSV-1 infection (MOI=1). Specific HSV-1 genomic regions, where ChIP-sequencing peaks aligned, are shown in a comprehensive plot. e. 3D-SIM was used to visualize RAP80 colocalization (red) with ICP4 (green) in HeLa cells infected with HSV-1 (MOI=1) for 3 h. f. RAP80 KD effects on RNA polymerase II recruitment to viral gene promoters were evaluated in HeLa cells by ChIP assays using the RNA polymerase II S2P antibody. g. Representative immunofluorescence images showing ICP4 in WT and RAP80 KD HeLa cells following infection with HSV-1 (MOI=1) for 3 h (left). ICP4 droplet diameters were measured in Imariz software (right). The graph represents data from three independent experiments and counts represent 50–100 infected cells. h, i . Immunofluorescence images showing RAP80 -depleted HeLa cells re-expressing WT RAP80 or its mutant ( 2AS or 3KR ), which were infected with HSV-1 (MOI=1) for 3 h. Cells were stained with DAPI and ICP4 and RAP80-specific antibodies. (h) ICP4 droplet diameters and colocalization between different RAP80 and ICP4 forms were measured (i). j. RAP80 effects on TK expression were evaluated in RAP80 -depleted HeLa cells plus reintroduced WT RAP80 or mutants ( 2AS or 3KR ). Cells were infected with HSV-1 (MOI=1) for 3 h and qRT-PCR assays performed to examine TK expression. k. RNA polymerase II recruitment to viral gene promoters was assessed in HeLa cells expressing WT RAP80 or mutants ( 2AS or 3KR ) in ChIP assays using a specific RNA polymerase II S2P antibody. l. The model shows multivalent interactions between RAP80 and ICP4 in phase separation formation (viral pre-replication foci). After HSV-1 infection, RAP80 binds to the viral transcription factor ICP4 and the viral genome through its UIM domain, driving phase separation formation. Data in panels c, f, g, i-k are presented as the mean ± standard error of the mean from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Data analysis was conducted using ordinary one-way and two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons tests. Exact P values are provided in source data. Scale bars, 5 μm.

Article Snippet: The following antibodies were used: Anti-Flag (M2, F3165, Sigma), anti-HA (HA-7, H9658, Sigma), anti-Myc (M047-3, MBL), anti-His (D291-3x, L), anti-β-actin (PM053, Abclonal), anti-RAP80 (A7244, Abclonal), anti-ICP0 (sc-53070, Santa Cruz), anti-ICP4 (sc-69809, Santa Cruz), anti-ICP8 (sc-53329, Santa Cruz), anti-RNA polymerase II S5P (ab5408, Abcam), anti-cGAS (sc-515777, Santa Cruz), and anti-CENP-A (A15995, Abclonal).

Techniques: Expressing, Infection, Western Blot, Immunofluorescence, Fluorescence, Software, Knockdown, Binding Assay, ChIP-sequencing, Mutagenesis, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: HSV-1 orchestrates host RAP80 ubiquitination by ICP0 and UL36USP to promote viral survival

doi: 10.1101/2025.06.10.658793

Figure Lengend Snippet: a. ICP0 enzyme effects on the establishment of the HSV-1 replication compartment were evaluated in HeLa cells by immunofluorescence microscopy. Cells were infected with either WT HSV-1 or an ICP0-deficient mutant strain ( ΔICP0 HSV-1 ) (MOI=1) and incubated for 6 h. Subsequently, cells were stained with antibodies against ICP4 and RAP80. Representative immunofluorescence images are shown. b. ICP0 enzyme deficiency effects on RAP80 depletion-promoted TK expression were explored in HeLa cells infected with either WT HSV-1 or ΔICP0 HSV-1 (MOI=1) for 6 h. qRT-PCR was performed in triplicate to evaluate TK expression. c. Interactions between ICP0 and RAP80 mutants were examined using co-IP assays in HEK293T cells. Cells were co-transfected with Flag-tagged ICP0 and either Myc-tagged WT RAP80 or its mutants ( 2AS or 3KR ), and co-IP assays performed using indicated antibodies. d. Crystallographic analysis shows a significant binding pattern alteration in RAP80 following ubiquitination of its 3K sites (K75, 90, and 112). Post-ubiquitination, the RAP80 UIM domain binds to its own ubiquitination chains, attenuating its binding to ICP0. e. RAP80 ubiquitination effects on its association with viral proteins were examined in HEK293T cells by co-IP assays. Cells were co-transfected with Myc-RAP80 and either WT RAP80 or mutant variants ( 2AS and 3KR ) along with HA-tagged ubiquitin, and subsequently infected with HSV-1 for 6 h (MOI=1) before harvest. Cell lysates were subjected to IP with an anti-Flag antibody and analyzed by western blotting using anti-Flag, anti-ICP0, and anti-ICP4 antibodies. f. RAP80 and mutant effects on HSV-1 replication centers were observed by IF in HeLa cells infected with HSV-1 (MOI=1) for 6 h. g. The impact of UL36USP on ICP0-catalyzed ubiquitination was examined in HEK293T cells. Cells were co-transfected with Myc-tagged ICP0, HA-tagged ubiquitin, and Flag-tagged UL36USP, with cell lysates immunoprecipitated using an anti-Myc antibody, and analyzed by western blotting using anti-Myc and anti-HA antibodies. h. UL36USP effects on the interaction between RAP80 and ubiquitinated ICP0 were assessed in in vitro pull-down assays. Flag-tagged RAP80 immunoprecipitated from HEK293T cells was incubated in vitro with purified ubiquitinated ICP0 in the presence/absence of UL36USP protein, and then western blotting performed with indicated antibodies. i. The model shows the interplay of ubiquitination on RAP80 by ICP0 and UL36USP at different post-HSV-1 infection stages. During mid-stage, ICP0 catalyzes the K48- and K63-type ubiquitination of RAP80, resulting in RAP80 degradation and the dissolution of phase separation. In late infection stages, UL36USP deubiquitinates RAP80, thereby stabilizing RAP80 and facilitating viral propagation. Data in panels b and f are presented as the mean ± standard error of the mean from three independent experiments. **P < 0.01 and ***P < 0.001. Data analysis was conducted using ordinary one-way and two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons tests. Exact P values are provided in source data. Scale bars, 5 μm.

Article Snippet: The following antibodies were used: Anti-Flag (M2, F3165, Sigma), anti-HA (HA-7, H9658, Sigma), anti-Myc (M047-3, MBL), anti-His (D291-3x, L), anti-β-actin (PM053, Abclonal), anti-RAP80 (A7244, Abclonal), anti-ICP0 (sc-53070, Santa Cruz), anti-ICP4 (sc-69809, Santa Cruz), anti-ICP8 (sc-53329, Santa Cruz), anti-RNA polymerase II S5P (ab5408, Abcam), anti-cGAS (sc-515777, Santa Cruz), and anti-CENP-A (A15995, Abclonal).

Techniques: Immunofluorescence, Microscopy, Infection, Mutagenesis, Incubation, Staining, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Binding Assay, Ubiquitin Proteomics, Western Blot, Immunoprecipitation, In Vitro, Purification, Dissolution

Journal: bioRxiv

Article Title: HSV-1 orchestrates host RAP80 ubiquitination by ICP0 and UL36USP to promote viral survival

doi: 10.1101/2025.06.10.658793

Figure Lengend Snippet: a. RAP80 depletion effects on HSV-1 replication during late infection stages were monitored using plaque assays in HeLa cells with RAP80 knockdown (KD) at 20 h post HSV-1 infection (MOI=1). WT HeLa cells under identical conditions served as controls. b. WT HeLa and RAP80 KD cells were infected with HSV-1-GFP for 20 h, after which, viral GFP mean fluorescence intensity (MFI) was quantified using flow cytometry to determine RAP80 depletion effects on HSV-1 amplification. c. WT and RAP80 KD HeLa cells were infected with HSV-1 (MOI=1) for 20 h, after which RAP80 effects on apoptosis were evaluated by Annexin V and propidium iodide (PI) staining. WT HeLa cells under identical condition served as controls. d. RAP80 KD effects on viral replication were measured by infecting WT and RAP80 KD HeLa cells with HSV-1 (MOI=1). At 20 h post-infection, supernatants were harvested and incubated with BHK cells and finally subjected to plaque assays to measure HSV-1 replication. e. The impact of the apoptosis inhibitor Z-VAD on HSV-1 amplification in RAP80 KD HeLa cells was examined. WT and RAP80 KD cells were pretreated with 5 μM Z-VAD or control conditions before infection with HSV-1. Viral replication was quantified using plaque assays. f. WT and RAP80 KD HeLa cells were exposed to a Z-VAD concentration gradient (5–50 μM), after which plaque assays were conducted to assess varying Z-VAD concentration effects on HSV-1 replication efficiency. g. Gene enrichment analysis was conducted using RNA sequencing data to identify differentially expressed genes in RAP80 KD HeLa cells infected with HSV-1 at 20 h post-infection (MOI=1). h. WT and RAP80 KD HeLa cells were treated with 5 μM of RU.521 (cGAS inhibitor) prior to infection with HSV-1. HSV-1 replication was then measured using plaque assays. i. RAP80 KD effects on the interaction between cGAS and R-loops was assessed using PLA in WT and RAP80 KD HeLa cells. After infection with HSV-1 (MOI=1) for 20 h, PLA was performed to investigate in situ interactions between cGAS and R-loops (using the S9.6 antibody). j. PLA was performed to investigate RNase H effects on RAP80 depletion-promoted R-loops in HeLa cells. WT and RAP80 KD HeLa cells with/without overexpressed RNase H were infected with HSV-1 (MOI=1) for 20 h, and PLA conducted to examine interactions between cGAS and R-loops (S9.6 antibody). k, l. WT and RAP80 KD HeLa cells with/without overexpressed RNase H were infected with HSV-1 (MOI=1) for 20 h, after which flow cytometry was conducted to examine apoptosis levels (k), and plaque assays performed to quantify viral replication (l). m. The model shows disrupted cellular homeostasis by HSV-1 following RAP80 KD. RAP80 depletion leads to R-loop accumulation and transmission to the cytoplasm. In the cytoplasm, R-loops are recognized by cGAS, activating innate immunity pathways and inducing apoptosis, which attenuates the viral lytic cycle. Data are presented as the mean ± standard error of the mean from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Data analysis was conducted using ordinary one-way and two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons tests. Exact P values are provided in source data. Scale bars, 5 μm.

Article Snippet: The following antibodies were used: Anti-Flag (M2, F3165, Sigma), anti-HA (HA-7, H9658, Sigma), anti-Myc (M047-3, MBL), anti-His (D291-3x, L), anti-β-actin (PM053, Abclonal), anti-RAP80 (A7244, Abclonal), anti-ICP0 (sc-53070, Santa Cruz), anti-ICP4 (sc-69809, Santa Cruz), anti-ICP8 (sc-53329, Santa Cruz), anti-RNA polymerase II S5P (ab5408, Abcam), anti-cGAS (sc-515777, Santa Cruz), and anti-CENP-A (A15995, Abclonal).

Techniques: Infection, Knockdown, Fluorescence, Flow Cytometry, Amplification, Staining, Incubation, Control, Concentration Assay, RNA Sequencing, In Situ, Transmission Assay

Journal: bioRxiv

Article Title: HSV-1 orchestrates host RAP80 ubiquitination by ICP0 and UL36USP to promote viral survival

doi: 10.1101/2025.06.10.658793

Figure Lengend Snippet: a. ChIP-seq analysis was performed using an RAP80-specific antibody in HeLa cells, with ChIP DNA collected at 3 h and 20 h post-infection (MOI=1). Filtered peaks from ChIP-seq data were then annotated and analyzed for RAP80 binding patterns. b. R-loop accumulation at centromeres was evaluated in WT and RAP80 KD HeLa cells infected with HSV-1 (MOI=1) for 20 h post-infection, in the presence/absence of RNase H, using CUT-tag– quantitative PCR (CUT-tag-qPCR) assays with an anti-S9.6 antibody to analyze the binding profiles of various centromeric DNA, Cen1-like, Cen9, and mbox. c–j. Protein recruitment, including BRCA1(c), γH2AX (d); RAD51 (e), RAD52 (f) to centromeres, was evaluated in WT and RAP80 KD HeLa cells infected with HSV-1 (MOI=1) for 20 h. CUT-tag-qPCR assays were used to analyze protein binding profiles to different genes, including Cen1-like, Cen9, and mbox . g . Representative images showing chromosome spreads from HeLa cells with WT or depleted RAP80 in RNase H overexpressing cells infected with HSV-1 20 h post infection (MOI=1). White arrows indicate centromeric breaks. h. The model highlights a pivotal role for RAP80 in maintaining centromere stability via degraded R-loops. RAP80 degrades R-loops by recruiting BRCA1 and nucleases. RAP80 knockdown (KD) leads to R-loop accumulation and DNA damage at centromeres. This accumulation not only threatens centromere structural integrity but also disrupts finely tuned DNA damage repair processes. RAP80 plays a role in pathway selection for centromeric DSB repair. In its absence, DSBs are repaired by the RAD52-mediated pathway, and RAD51 is excluded from centromeric DSBs. The RAD52-mediated repair pathway results in uncontrolled homologous recombination, which is coupled to R-loop and DSB accumulation, and also prevents CENP-A deposition and promotes transcription. These combined effects lead to centromeric instability. Data in panels b-g are the mean ± standard error of the mean from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Ordinary one-way and two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons tests. Exact P values are provided in source data. Scale bars, 5 μm.

Article Snippet: The following antibodies were used: Anti-Flag (M2, F3165, Sigma), anti-HA (HA-7, H9658, Sigma), anti-Myc (M047-3, MBL), anti-His (D291-3x, L), anti-β-actin (PM053, Abclonal), anti-RAP80 (A7244, Abclonal), anti-ICP0 (sc-53070, Santa Cruz), anti-ICP4 (sc-69809, Santa Cruz), anti-ICP8 (sc-53329, Santa Cruz), anti-RNA polymerase II S5P (ab5408, Abcam), anti-cGAS (sc-515777, Santa Cruz), and anti-CENP-A (A15995, Abclonal).

Techniques: ChIP-sequencing, Infection, Binding Assay, Real-time Polymerase Chain Reaction, Protein Binding, Knockdown, Selection, Homologous Recombination

Journal: bioRxiv

Article Title: HSV-1 orchestrates host RAP80 ubiquitination by ICP0 and UL36USP to promote viral survival

doi: 10.1101/2025.06.10.658793

Figure Lengend Snippet: a. Interactions between HSV-1 and host chromosome 1 (chr.1) near the centromere region. Tracks from the top to the bottom are (1–2) RAP80 enrichment signals in HeLa cells from ChIP-seq data at 3 h (1) and 20 h (2) post-infection. (5–6) Contacts between each 50 kb genomic bin and the HSV-1 genome in WT-HeLa cells are shown at 6 h, 9 h, 12 h, 18 h, and 24 h post-infection. Contacts without mapping quality filters are used. The centromere region is salmon-colored. b. The odds ratio for interaction enrichment between HSV-1 and the host genome in the centromere region. An odds ratio of 1 indicates no HSV-1 preference for the centromere region when compared to other regions. A ratio > 1 indicates enrichment in the centromere region, while a ratio < 1 indicates a reduction in that region. Data measured at 6 h, 9 h, 12 h, 15 h, 18 h, and 24 h post-infection are presented. c. Similar to a , but Hi-C tracks display data from RAP80 knockdown (KD) HeLa cells. d. CUT-tag-qPCR on RNA polymerase II was performed at different centromeric regions in HeLa cells infected with HSV-1 for 20 h (MOI=1). e. RAP80 KD and WT HeLa cells, as well as HeLa cells stably overexpressing RNase H, were infected with HSV-1. Samples were harvested at 20 h post-infection to analyze cenRNA transcripts using RT-qPCR. Data in panels d, e are the mean ± standard error of the mean from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Ordinary one-way and two-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons tests. Exact P values are provided in source data. f. Similar to a , but Hi-C track data come from the following 24 h post-infection cells: RAP80 KD HeLa cells overexpressing RNase H, WT HeLa cells overexpressing RNase, RAP80 KD HeLa cells, and WT HeLa cells. g. The model highlights multifaceted roles of RAP80 in regulating the process of Herpes simplex virus 1 life cycle. RAP80 inhibits HSV-1 transcription in early post-invasion stages by interacting with the viral genome to hinder ICP4 binding to transcription factors. This interaction, mediated by phase separation influenced by multivalent interactions between RAP80’s UIM domain and ICP0/ICP4, is crucial for transcription inhibition. As infection progresses to mid-stages, phase separation dissipates. ICP0 targets RAP80 for proteasomal degradation, disrupting the RAP80-ICP0/ICP4 interaction, dissolving phase separation and facilitating the formation of virus replication compartments, which are essential for viral propagation. In late infection stages, a pivotal change occurs as RAP80 is deubiquitinated by UL36USP, allowing it to play critical roles preserving cellular homeostasis and promoting HSV-1 survival by modulating the R-loop-cGAS-apoptosis axis. This comprehensive analysis of RAP80’s multifaceted roles underscores the dynamic interplay between viral and host factors during HSV-1 infection

Article Snippet: The following antibodies were used: Anti-Flag (M2, F3165, Sigma), anti-HA (HA-7, H9658, Sigma), anti-Myc (M047-3, MBL), anti-His (D291-3x, L), anti-β-actin (PM053, Abclonal), anti-RAP80 (A7244, Abclonal), anti-ICP0 (sc-53070, Santa Cruz), anti-ICP4 (sc-69809, Santa Cruz), anti-ICP8 (sc-53329, Santa Cruz), anti-RNA polymerase II S5P (ab5408, Abcam), anti-cGAS (sc-515777, Santa Cruz), and anti-CENP-A (A15995, Abclonal).

Techniques: ChIP-sequencing, Infection, Hi-C, Knockdown, Stable Transfection, Quantitative RT-PCR, Virus, Binding Assay, Inhibition, Preserving