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K-bZIP Mediated SUMO-2/3 Specific Modification on the KSHV Genome Negatively Regulates Lytic Gene Expression and Viral Reactivation.

Yang WS, Hsu HW, Campbell M, Cheng CY, Chang PC - PLoS Pathog. (2015)

Bottom Line: A rapid and widespread deposition of SUMO-2/3, compared with SUMO-1, modification across the KSHV genome upon reactivation was observed.These results indicate that SUMO-2/3 modification of viral chromatin may function to counteract KSHV reactivation.As induction of herpesvirus reactivation may activate cellular antiviral regimes, our results suggest that development of viral SUMO E3 ligase specific inhibitors may be an avenue for anti-virus therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China.

ABSTRACT
SUMOylation is associated with epigenetic regulation of chromatin structure and transcription. Epigenetic modifications of herpesviral genomes accompany the transcriptional switch of latent and lytic genes during the virus life cycle. Here, we report a genome-wide comparison of SUMO paralog modification on the KSHV genome. Using chromatin immunoprecipitation in conjunction with high-throughput sequencing, our study revealed highly distinct landscape changes of SUMO paralog genomic modifications associated with KSHV reactivation. A rapid and widespread deposition of SUMO-2/3, compared with SUMO-1, modification across the KSHV genome upon reactivation was observed. Interestingly, SUMO-2/3 enrichment was inversely correlated with H3K9me3 mark after reactivation, indicating that SUMO-2/3 may be responsible for regulating the expression of viral genes located in low heterochromatin regions during viral reactivation. RNA-sequencing analysis showed that the SUMO-2/3 enrichment pattern positively correlated with KSHV gene expression profiles. Activation of KSHV lytic genes located in regions with high SUMO-2/3 enrichment was enhanced by SUMO-2/3 knockdown. These findings suggest that SUMO-2/3 viral chromatin modification contributes to the diminution of viral gene expression during reactivation. Our previous study identified a SUMO-2/3-specific viral E3 ligase, K-bZIP, suggesting a potential role of this enzyme in regulating SUMO-2/3 enrichment and viral gene repression. Consistent with this prediction, higher K-bZIP binding on SUMO-2/3 enrichment region during reactivation was observed. Moreover, a K-bZIP SUMO E3 ligase dead mutant, K-bZIP-L75A, in the viral context, showed no SUMO-2/3 enrichment on viral chromatin and higher expression of viral genes located in SUMO-2/3 enriched regions during reactivation. Importantly, virus production significantly increased in both SUMO-2/3 knockdown and KSHV K-bZIP-L75A mutant cells. These results indicate that SUMO-2/3 modification of viral chromatin may function to counteract KSHV reactivation. As induction of herpesvirus reactivation may activate cellular antiviral regimes, our results suggest that development of viral SUMO E3 ligase specific inhibitors may be an avenue for anti-virus therapy.

No MeSH data available.


Related in: MedlinePlus

The SUMO E3 ligase activity of K-bZIP is necessary to alleviate the increase in gene expression and virus production during reactivation.(A) TCLs were collected from non-induced (0 hour) and 1 μg/ml Dox treated (for 24 and 48 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cell lines. Immunoblotting was used to confirm the induction of K-Rta and expression of K-bZIP. GAPDH was used as loading control. (B) Total RNA isolated from non-induced (0 hour) and 1 μg/ml Dox treated (24 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells was reverse transcribed using oligo-d(T)18 primer and analyzed by real-time qPCR using specific primer pairs representing genes in SUMO-2/3 enrichment and H3K9me3-rich regions. All reactions were run in triplicate and normalized against GAPDH. (C) Supernatants from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells were collected at 0 and 48 hours after Dox (1 μg/ml) treatment and filtered. Virion-associated DNA was purified and KSHV DNA levels were determined by TaqMan qPCR. Mean ± SD. **; P<0.005. (D) 293T cells were infected with filtered supernatants harvested from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells treated with or without 1 μg/ml Dox for 72 hours. GFP positive cells were analyzed by fluorescence microscopy (FITC, 10X magnification) 48 hours after infection. (E) The GFP positive cells were quantified using the average from >20 microscopic fields. Mean ± SD. **; P<0.005.
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ppat.1005051.g008: The SUMO E3 ligase activity of K-bZIP is necessary to alleviate the increase in gene expression and virus production during reactivation.(A) TCLs were collected from non-induced (0 hour) and 1 μg/ml Dox treated (for 24 and 48 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cell lines. Immunoblotting was used to confirm the induction of K-Rta and expression of K-bZIP. GAPDH was used as loading control. (B) Total RNA isolated from non-induced (0 hour) and 1 μg/ml Dox treated (24 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells was reverse transcribed using oligo-d(T)18 primer and analyzed by real-time qPCR using specific primer pairs representing genes in SUMO-2/3 enrichment and H3K9me3-rich regions. All reactions were run in triplicate and normalized against GAPDH. (C) Supernatants from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells were collected at 0 and 48 hours after Dox (1 μg/ml) treatment and filtered. Virion-associated DNA was purified and KSHV DNA levels were determined by TaqMan qPCR. Mean ± SD. **; P<0.005. (D) 293T cells were infected with filtered supernatants harvested from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells treated with or without 1 μg/ml Dox for 72 hours. GFP positive cells were analyzed by fluorescence microscopy (FITC, 10X magnification) 48 hours after infection. (E) The GFP positive cells were quantified using the average from >20 microscopic fields. Mean ± SD. **; P<0.005.

Mentions: To evaluate the potential role of K-bZIP SUMO E3 ligase, iSLK cells harboring different recombinant KSHV-BACmids were induced with Dox for 24 and 48 hours for gene expression and virus production analysis, respectively. Total protein lysates were collected for immunoblotting analysis to assess the successful induction of K-Rta and expression of K-bZIP (Fig 8A). Next, we measured the accumulation of KSHV orf46, K-bZIP, K8.1 and orf52 mRNA, representing the potential SUMO-2/3-regulated genes and orf19, orf20, orf23 and orf25 mRNA, representing non-SUMO-2/3-regulated genes. A significant higher increase in expression of the SUMO-2/3-regulated viral genes was found in iSLK-Puro-BAC16 K-ZIP-L75A mutant compared with K-bZIP-WT rev (Fig 8B; upper panel). Consistent with the significant increase of K-bZIP transcript by approximately 5-fold in iSLK-Puro-BAC16 K-ZIP-L75A mutant, a higher level of mutant K-bZIP protein was also observed during K-Rta-induced viral reactivation (Fig 8A). In line with our previous data, this result indicates that K-bZIP may mediate the SUMOylation of viral promoters in the low H3K9me3 region which results in a diminution of viral gene expression after reactivation. Unexpectedly, viral genes in the high H3K9me3 mark region showed defects in transactivation in the iSLK-Puro-BAC16 K-bZIP-L75A mutant (Fig 8B; lower panel). Since no SUMO enrichment was identified in the high H3K9me3 region, SUMO E3 ligase activity of K-bZIP should not influence the transactivation of genes in this region. As shown in our previous report, a high affinity direct interaction between K-bZIP and H3K9me3 was found [17]. The loss of gene transactivation in the high H3K9me3 region early after reactivation (24 hours) may relate to the direct binding of K-bZIP to this region and enhance late gene expression by recruiting SUMOylated transcription factors in a SIM-dependent manner. It should also be noted that K-bZIP is known to be required for lytic DNA replication [34–36], and that DNA replication induces high level expression of late genes, including orf19, orf20, orf23 and orf25. The SIM domain of K-bZIP may be required for KSHV replication and that is why the SIM mutation results in less expression of orf19, orf20, orf23 and orf25.


K-bZIP Mediated SUMO-2/3 Specific Modification on the KSHV Genome Negatively Regulates Lytic Gene Expression and Viral Reactivation.

Yang WS, Hsu HW, Campbell M, Cheng CY, Chang PC - PLoS Pathog. (2015)

The SUMO E3 ligase activity of K-bZIP is necessary to alleviate the increase in gene expression and virus production during reactivation.(A) TCLs were collected from non-induced (0 hour) and 1 μg/ml Dox treated (for 24 and 48 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cell lines. Immunoblotting was used to confirm the induction of K-Rta and expression of K-bZIP. GAPDH was used as loading control. (B) Total RNA isolated from non-induced (0 hour) and 1 μg/ml Dox treated (24 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells was reverse transcribed using oligo-d(T)18 primer and analyzed by real-time qPCR using specific primer pairs representing genes in SUMO-2/3 enrichment and H3K9me3-rich regions. All reactions were run in triplicate and normalized against GAPDH. (C) Supernatants from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells were collected at 0 and 48 hours after Dox (1 μg/ml) treatment and filtered. Virion-associated DNA was purified and KSHV DNA levels were determined by TaqMan qPCR. Mean ± SD. **; P<0.005. (D) 293T cells were infected with filtered supernatants harvested from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells treated with or without 1 μg/ml Dox for 72 hours. GFP positive cells were analyzed by fluorescence microscopy (FITC, 10X magnification) 48 hours after infection. (E) The GFP positive cells were quantified using the average from >20 microscopic fields. Mean ± SD. **; P<0.005.
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ppat.1005051.g008: The SUMO E3 ligase activity of K-bZIP is necessary to alleviate the increase in gene expression and virus production during reactivation.(A) TCLs were collected from non-induced (0 hour) and 1 μg/ml Dox treated (for 24 and 48 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cell lines. Immunoblotting was used to confirm the induction of K-Rta and expression of K-bZIP. GAPDH was used as loading control. (B) Total RNA isolated from non-induced (0 hour) and 1 μg/ml Dox treated (24 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells was reverse transcribed using oligo-d(T)18 primer and analyzed by real-time qPCR using specific primer pairs representing genes in SUMO-2/3 enrichment and H3K9me3-rich regions. All reactions were run in triplicate and normalized against GAPDH. (C) Supernatants from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells were collected at 0 and 48 hours after Dox (1 μg/ml) treatment and filtered. Virion-associated DNA was purified and KSHV DNA levels were determined by TaqMan qPCR. Mean ± SD. **; P<0.005. (D) 293T cells were infected with filtered supernatants harvested from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells treated with or without 1 μg/ml Dox for 72 hours. GFP positive cells were analyzed by fluorescence microscopy (FITC, 10X magnification) 48 hours after infection. (E) The GFP positive cells were quantified using the average from >20 microscopic fields. Mean ± SD. **; P<0.005.
Mentions: To evaluate the potential role of K-bZIP SUMO E3 ligase, iSLK cells harboring different recombinant KSHV-BACmids were induced with Dox for 24 and 48 hours for gene expression and virus production analysis, respectively. Total protein lysates were collected for immunoblotting analysis to assess the successful induction of K-Rta and expression of K-bZIP (Fig 8A). Next, we measured the accumulation of KSHV orf46, K-bZIP, K8.1 and orf52 mRNA, representing the potential SUMO-2/3-regulated genes and orf19, orf20, orf23 and orf25 mRNA, representing non-SUMO-2/3-regulated genes. A significant higher increase in expression of the SUMO-2/3-regulated viral genes was found in iSLK-Puro-BAC16 K-ZIP-L75A mutant compared with K-bZIP-WT rev (Fig 8B; upper panel). Consistent with the significant increase of K-bZIP transcript by approximately 5-fold in iSLK-Puro-BAC16 K-ZIP-L75A mutant, a higher level of mutant K-bZIP protein was also observed during K-Rta-induced viral reactivation (Fig 8A). In line with our previous data, this result indicates that K-bZIP may mediate the SUMOylation of viral promoters in the low H3K9me3 region which results in a diminution of viral gene expression after reactivation. Unexpectedly, viral genes in the high H3K9me3 mark region showed defects in transactivation in the iSLK-Puro-BAC16 K-bZIP-L75A mutant (Fig 8B; lower panel). Since no SUMO enrichment was identified in the high H3K9me3 region, SUMO E3 ligase activity of K-bZIP should not influence the transactivation of genes in this region. As shown in our previous report, a high affinity direct interaction between K-bZIP and H3K9me3 was found [17]. The loss of gene transactivation in the high H3K9me3 region early after reactivation (24 hours) may relate to the direct binding of K-bZIP to this region and enhance late gene expression by recruiting SUMOylated transcription factors in a SIM-dependent manner. It should also be noted that K-bZIP is known to be required for lytic DNA replication [34–36], and that DNA replication induces high level expression of late genes, including orf19, orf20, orf23 and orf25. The SIM domain of K-bZIP may be required for KSHV replication and that is why the SIM mutation results in less expression of orf19, orf20, orf23 and orf25.

Bottom Line: A rapid and widespread deposition of SUMO-2/3, compared with SUMO-1, modification across the KSHV genome upon reactivation was observed.These results indicate that SUMO-2/3 modification of viral chromatin may function to counteract KSHV reactivation.As induction of herpesvirus reactivation may activate cellular antiviral regimes, our results suggest that development of viral SUMO E3 ligase specific inhibitors may be an avenue for anti-virus therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China.

ABSTRACT
SUMOylation is associated with epigenetic regulation of chromatin structure and transcription. Epigenetic modifications of herpesviral genomes accompany the transcriptional switch of latent and lytic genes during the virus life cycle. Here, we report a genome-wide comparison of SUMO paralog modification on the KSHV genome. Using chromatin immunoprecipitation in conjunction with high-throughput sequencing, our study revealed highly distinct landscape changes of SUMO paralog genomic modifications associated with KSHV reactivation. A rapid and widespread deposition of SUMO-2/3, compared with SUMO-1, modification across the KSHV genome upon reactivation was observed. Interestingly, SUMO-2/3 enrichment was inversely correlated with H3K9me3 mark after reactivation, indicating that SUMO-2/3 may be responsible for regulating the expression of viral genes located in low heterochromatin regions during viral reactivation. RNA-sequencing analysis showed that the SUMO-2/3 enrichment pattern positively correlated with KSHV gene expression profiles. Activation of KSHV lytic genes located in regions with high SUMO-2/3 enrichment was enhanced by SUMO-2/3 knockdown. These findings suggest that SUMO-2/3 viral chromatin modification contributes to the diminution of viral gene expression during reactivation. Our previous study identified a SUMO-2/3-specific viral E3 ligase, K-bZIP, suggesting a potential role of this enzyme in regulating SUMO-2/3 enrichment and viral gene repression. Consistent with this prediction, higher K-bZIP binding on SUMO-2/3 enrichment region during reactivation was observed. Moreover, a K-bZIP SUMO E3 ligase dead mutant, K-bZIP-L75A, in the viral context, showed no SUMO-2/3 enrichment on viral chromatin and higher expression of viral genes located in SUMO-2/3 enriched regions during reactivation. Importantly, virus production significantly increased in both SUMO-2/3 knockdown and KSHV K-bZIP-L75A mutant cells. These results indicate that SUMO-2/3 modification of viral chromatin may function to counteract KSHV reactivation. As induction of herpesvirus reactivation may activate cellular antiviral regimes, our results suggest that development of viral SUMO E3 ligase specific inhibitors may be an avenue for anti-virus therapy.

No MeSH data available.


Related in: MedlinePlus