Barrier-to-autointegration factor 1 promotes gammaherpesvirus reactivation from latency

Abstract

Gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are DNA viruses that are globally associated with human cancers and establish lifelong latency in the human population. Detection of gammaherpesviral infection by the cGAS-STING innate immune DNA-sensing pathway is critical for suppressing viral reactivation from latency, a process that promotes viral pathogenesis and transmission. We report that barrier-to-autointegration factor 1 (BAF)-mediated suppression of the cGAS-STING signaling pathway is necessary for reactivation of KSHV and EBV. We demonstrate a role for BAF in destabilizing cGAS expression and show that inhibiting BAF expression in latently infected, reactivating, or uninfected cells leads to increased type I interferon-mediated antiviral responses and decreased viral replication. Furthermore, BAF overexpression resulted in decreased cGAS expression at the protein level. These results establish BAF as a key regulator of the lifecycle of gammaherpesviruses and a potential target for treating viral infections and malignancies.

Fig. 1. BAF is required for optimal KSHV reactivation from latency.

iSLK.219 cells were transfected with non-targeting control (NTC) siRNA or BANF1 targeting siRNA 48 h prior to the addition of 25 ng/mL doxycycline. TREx-BCBL1-RTA cells were transduced with lentiviral shRNA for 72 h prior to the addition of 1000 ng/mL doxycycline. A Fluorescent microscopy imaging of iSLK.219 cells for RFP and GFP signal at 72 h post-doxycycline treatment. B The fluorescence was quantified by plate reader. C Cells were harvested for RNA at 72 h (iSLK.219) or 96 h (TREx-BCBL1-RTA) post-doxycycline treatment and subsequent RT-qPCR was performed to quantify expression of viral mRNA transcripts. D iSLK.219 cDNA was prepared from cells harvested at 0 h and 72 h post-doxycycline treatment. Global KSHV gene expression profiling was performed. Data shown are the Z-score of the fold change (2^-ΔΔCt) over the geometric mean expression of three housekeeping genes averaged over two independent biological replicates. The heatmap was prepared using Partek Flow. E Cell lysates were prepared at 72 h (iSLK.219) or 96 h (TREx-BCBL1-RTA) post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. F Cells were harvested and RNA isolated at 48 h post-siRNA transfection and RT-qPCR was subsequently performed to quantify BANF1 mRNA transcripts. G Cell lysates were prepared at 48 h post-siRNA transfection or 72 h post-shRNA transduction and analyzed by western blotting with the indicated antibody. P values are the result of two-tailed Student’s T tests unless otherwise specified. Error bars indicate the standard error of the mean of three independent biological replicates. Source data are provided as a source data file.

Fig. 2. BAF antagonizes the cGAS-STING response to KSHV reactivation.

iSLK.219 cells were transfected with NTC siRNA or BANF1 targeting siRNA for 48 h prior to the addition of 25 ng/mL doxycycline. TREx-BCBL1-RTA cells were transduced with lentiviral shRNA for 72 h prior to the addition of 1000 ng/mL doxycycline. A iSLK.219 cell lysates were collected at 72 h post-doxycycline treatment and analyzed by 2’3’-cGAMP ELISA. B iSLK.219 cell lysates were collected at 48 h post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. C iSLK.219 culture supernatant was harvested at 72 h post-doxycycline treatment and analyzed by IFNβ ELISA. D Cells were harvested for RNA at 48 h (iSLK.219) or 96 h (TREx-BCBL1-RTA) post-doxycycline treatment and RT-qPCR was subsequently performed to determine ISG mRNA expression levels. E iSLK.219 cDNA as prepared in (D) was analyzed by Human Type I Interferon Response RT² Profiler PCR Array (Qiagen, GeneGlobe ID: PAHS-016Z). Data shown are the Z-score of the fold change (2^-ΔΔCt) over the geometric mean expression of four housekeeping genes averaged over two independent biological replicates. The heatmap was prepared using Partek Flow. F Culture supernatants were harvested at either 72 h (iSLK.219) or 96 h (TREx-BCBL1-RTA) post-doxycycline treatment and DNase treated prior to DNA extraction. DNase-resistant KSHV genomes were quantified by real-time qPCR. G iSLK.219 culture supernatants were transferred to naive HEK293 cells at 72 h post-doxycycline treatment. GFP + infected cells were measured at 48 h post-infection by fluorescent microscopy and (H) quantified by flow cytometry. P values are the result of two-tailed Student’s T tests unless otherwise specified. Error bars indicate the standard error of the mean of three independent biological replicates. Source data are provided as a source data file.

Fig. 3. Increased BAF expression promotes KSHV reactivation.

iSLK.219 cells were transfected with either pCMV6 (EV) or pCMV6-BANF1-HA (BAF) expression plasmid for 48 h prior to the addition of 25 ng/mL doxycycline. A Cell lysates were collected at 96 h post-doxycycline treatment and analyzed by 2’3’-cGAMP ELISA. B Cell lysates were collected at 72 h post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. C Cells were harvested and RNA was isolated at 72 h post-doxycycline treatment and subsequent RT-qPCR was performed to determine ISG mRNA expression levels. D Culture supernatant was harvested at 96 h post-doxycycline treatment and analyzed by IFNβ ELISA. E Fluorescent microscopy imaging of RFP and GFP signal was conducted at 96 h after doxycycline treatment and quantified (F) by plate reader. G Cells were harvested for RNA at 96 h post-doxycycline treatment and subsequent RT-qPCR was performed to quantify the expression of viral mRNA transcripts. H Cell lysates were prepared at 96 h post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. I Culture supernatants were harvested at 96 h post-doxycycline treatment and DNase treated prior to DNA extraction. DNase-resistant KSHV genomes were quantified by real-time qPCR. J At 96 h post-doxycycline treatment, culture supernatants from iSLK.219 cells were used to infect naive HEK293 cells. GFP + infected cells were measured at 48 h post-transfer by fluorescent microscopy and (K) quantified by flow cytometry. L Cell lysates were prepared at 48 h post-siRNA transfection and analyzed by western blotting with the indicated antibody. P values are the result of two-tailed Student’s T tests unless otherwise specified. Error bars indicate the standard error of the mean of three independent biological replicates. Source data are provided as a source data file.

Fig. 4. BAF promotes KSHV reactivation through a cGAS-dependent mechanism.

iSLK.219 cells were transfected with NTC, BANF1, cGAS, or BANF1 and cGAS targeting siRNA at a total siRNA concentration of 100 nM for 48 h prior to the addition of 25 ng/mL doxycycline. A Cell lysates were collected at 72 h post-doxycycline treatment and analyzed by 2’3’-cGAMP ELISA. B Cells were harvested for RNA at 48 h post-doxycycline treatment and subsequent RT-qPCR was performed to determine IFNB1 mRNA expression levels. C Culture supernatant was harvested at 72 h post-doxycycline treatment and analyzed by IFNβ ELISA. D Fluorescent microscopy imaging of RFP and GFP signal was conducted at 72 h after doxycycline treatment. E The fluorescence was quantified by a plate reader. F Cell lysates were prepared at 72 h post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. G Culture supernatants were harvested 72 h post-doxycycline treatment and DNase treated prior to DNA extraction. DNase-resistant KSHV genomes were quantified by real-time qPCR. H At 72 h post-doxycycline treatment, culture supernatants from iSLK.219 cells were used to infect naive HEK293 cells. At 48 h post-transfer, GFP + infected cells were quantified by flow cytometry. I The cells were also analyzed by fluorescent microscopy. J Cell lysates were prepared at 48 h post-siRNA transfection and analyzed by western blotting with the indicated antibody. P values are the result of two-tailed Student’s T tests unless otherwise specified. Error bars indicate the standard error of the mean of three independent biological replicates. Source data are provided as a source data file.

Fig. 5. BAF suppresses the antiviral state in uninfected, latently infected, and lytically infected cells.

A iSLK.219 cells were transfected with EV or BANF1 expression plasmids for 48 h prior to the addition of 100 μg/mL cycloheximide. Cell lysates were prepared at the indicated timepoints and analyzed by western blotting. B HeLa cells were transfected with EV or BANF1 expression plasmids for 48 h prior to treatment with 10 μM MG132 or DMSO control. Cell lysates were prepared 12 h post-treatment and analyzed by western blotting. C Naive SLK cells were infected with equivalent units of concentrated cell-free KSHV or PBS (mock). Cell lysates were prepared 6 h post-infection and analyzed by western blotting. D Naive SLK cells were transfected with BANF1 or NTC siRNA for 48 h prior to infection with equivalent units of concentrated cell-free KSHV. Lysates were prepared pre-infection and analyzed by western blot. Blots are representative of three independent biological replicates. E GFP + infected cells were analyzed at 48 h post-infection by fluorescent microscopy. F Cells were also quantified by flow cytometry. G Cells were harvested and RNA isolated at 48 h post-siRNA transfection and RT-qPCR was subsequently performed to quantify BANF1 mRNA transcripts. iSLK.219 cells were transfected with NTC siRNA or BANF1 targeting siRNA for 48 h prior to the addition of 25 ng/mL doxycycline. Cells from two biological replicates were harvested for RNA (H) at 0 h and (I) at 48 h post-doxycycline treatment and subjected to RNA-Seq analysis. Interferon-stimulated genes were identified using Interferome (http://www.interferome.org). Data were visualized with VolcaNoseR (https://huygens.science.uva.nl/VolcaNoseR/). Adjusted P values were calculated using the two-tailed Wald test with adjustments for multiple comparisons, and significantly enriched GO terms were determined by the one-tailed Fisher exact test with adjustments for multiple comparisons. P values are the result of two-tailed Student’s T tests unless otherwise specified. Error bars indicate the standard error of the mean of three independent biological replicates. Source data are provided as a source data file.

Fig. 6. BAF facilitates EBV reactivation from latency in epithelial cells.

AGS-EBV cells were transfected with NTC or BANF1 targeting siRNA for 48 h prior to the addition of 5 ng/mL TPA. A Cell lysates were collected at 72 h post-TPA treatment and analyzed by 2’3’-cGAMP ELISA. B Cells were harvested and RNA was isolated at 48 h post-TPA treatment. RT-qPCR was subsequently performed to determine ISG mRNA expression levels. C Culture supernatant was harvested at 72 h post-TPA treatment and analyzed by IFNβ ELISA. D Cells were harvested and RNA was isolated at 72 h post-TPA treatment and RT-qPCR was performed to quantify viral mRNA transcripts. E Cell lysates were prepared at 72 h post-TPA treatment and analyzed by western blotting with the indicated antibodies. F Culture supernatants were harvested at 72 h post-TPA treatment and DNase treated prior to DNA extraction. DNase-resistant EBV genomes were quantified by real-time qPCR to assess viral load. G At 72 h post-TPA treatment, culture supernatants were used to infect naive HEK293 cells. Forty-eight h post infection, GFP + infected cells were analyzed by fluorescent microscopy. H Cells were also quantified by flow cytometry. I Cells were harvested for RNA at 48 h post-siRNA transfection and subsequent RT-qPCR was performed to quantify BANF1 mRNA transcripts. J Cell lysates were prepared at 48 h post-siRNA transfection and analyzed by western blotting with the indicated antibody. K Naive AGS cells were infected with equivalent units of concentrated cell-free EBV or PBS (mock). Cell lysates were prepared 6 h post-infection and analyzed by western blotting. P values are the result of two-tailed Student’s T tests unless otherwise specified. Error bars indicate the standard error of the mean of three independent biological replicates. Source data are provided as a source data file.