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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

Deposition of SUMO-2/3 modification on selected KSHV promoters representing high SUMO-2/3 enrichment or high H3K9me3 mark regions during reactivation.ChIP was performed as described in Fig 1 using anti-SUMO-1 or anti-SUMO-2/3 antibodies. SUMO-1 and SUMO-2/3 binding to orf46, K-bZIP, K8.1, and orf52 promoters in SUMO-2/3 enrichment region and orf19, orf20, orf23, and orf25 promoters in H3K9me3-rich region were analyzed by real-time qPCR. An enlarged view of the corresponding ChIP-seq mapping presented in Fig 1 is shown above each ChIP-qPCR plot. Gene promoters are solid boxes and the direction of transcription is shown by arrows. Rabbit IgG was used as negative antibody control and enrichment is not visible in the ChIP-qPCR plots. ***; P<0.001. NS; non-significant.
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ppat.1005051.g002: Deposition of SUMO-2/3 modification on selected KSHV promoters representing high SUMO-2/3 enrichment or high H3K9me3 mark regions during reactivation.ChIP was performed as described in Fig 1 using anti-SUMO-1 or anti-SUMO-2/3 antibodies. SUMO-1 and SUMO-2/3 binding to orf46, K-bZIP, K8.1, and orf52 promoters in SUMO-2/3 enrichment region and orf19, orf20, orf23, and orf25 promoters in H3K9me3-rich region were analyzed by real-time qPCR. An enlarged view of the corresponding ChIP-seq mapping presented in Fig 1 is shown above each ChIP-qPCR plot. Gene promoters are solid boxes and the direction of transcription is shown by arrows. Rabbit IgG was used as negative antibody control and enrichment is not visible in the ChIP-qPCR plots. ***; P<0.001. NS; non-significant.

Mentions: For further confirmation of the ChIP-seq results, we selected four genes from each of two viral genomic regions, one representing the region of high SUMO-2/3 enrichment with low H3K9me3 mark (orf46, K-bZIP, K8.1 and orf52) (Fig 1, red box) and the other one representing the region of high H3K9me3 mark with little SUMO-2/3 enrichment (orf19, orf20, orf23 and orf25) (Fig 1, blue box) during KSHV reactivation. Consistent with the ChIP-seq results, real-time qPCR data showed that the genes in SUMO-2/3 highly enriched region tested here displayed significant enrichment of SUMO-2/3 after viral reactivation when compared with the non-induced control cells (Fig 2, upper panel). In contrast, the genes in high H3K9me3 mark region showed little increase in SUMO-2/3 modification (Fig 2, lower panel). The reproducibility of this assay was obtained with independent repeat ChIP-qPCR experiment (S2B Fig). Expression of Flag-tagged SUMO-1 and SUMO-2 followed by ChIP using Flag antibody confirmed this phenomena (S3 Fig). These data indicate that the KSHV genome undergoes SUMO-2/3-specific modification following reactivation.


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)

Deposition of SUMO-2/3 modification on selected KSHV promoters representing high SUMO-2/3 enrichment or high H3K9me3 mark regions during reactivation.ChIP was performed as described in Fig 1 using anti-SUMO-1 or anti-SUMO-2/3 antibodies. SUMO-1 and SUMO-2/3 binding to orf46, K-bZIP, K8.1, and orf52 promoters in SUMO-2/3 enrichment region and orf19, orf20, orf23, and orf25 promoters in H3K9me3-rich region were analyzed by real-time qPCR. An enlarged view of the corresponding ChIP-seq mapping presented in Fig 1 is shown above each ChIP-qPCR plot. Gene promoters are solid boxes and the direction of transcription is shown by arrows. Rabbit IgG was used as negative antibody control and enrichment is not visible in the ChIP-qPCR plots. ***; P<0.001. NS; non-significant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4510548&req=5

ppat.1005051.g002: Deposition of SUMO-2/3 modification on selected KSHV promoters representing high SUMO-2/3 enrichment or high H3K9me3 mark regions during reactivation.ChIP was performed as described in Fig 1 using anti-SUMO-1 or anti-SUMO-2/3 antibodies. SUMO-1 and SUMO-2/3 binding to orf46, K-bZIP, K8.1, and orf52 promoters in SUMO-2/3 enrichment region and orf19, orf20, orf23, and orf25 promoters in H3K9me3-rich region were analyzed by real-time qPCR. An enlarged view of the corresponding ChIP-seq mapping presented in Fig 1 is shown above each ChIP-qPCR plot. Gene promoters are solid boxes and the direction of transcription is shown by arrows. Rabbit IgG was used as negative antibody control and enrichment is not visible in the ChIP-qPCR plots. ***; P<0.001. NS; non-significant.
Mentions: For further confirmation of the ChIP-seq results, we selected four genes from each of two viral genomic regions, one representing the region of high SUMO-2/3 enrichment with low H3K9me3 mark (orf46, K-bZIP, K8.1 and orf52) (Fig 1, red box) and the other one representing the region of high H3K9me3 mark with little SUMO-2/3 enrichment (orf19, orf20, orf23 and orf25) (Fig 1, blue box) during KSHV reactivation. Consistent with the ChIP-seq results, real-time qPCR data showed that the genes in SUMO-2/3 highly enriched region tested here displayed significant enrichment of SUMO-2/3 after viral reactivation when compared with the non-induced control cells (Fig 2, upper panel). In contrast, the genes in high H3K9me3 mark region showed little increase in SUMO-2/3 modification (Fig 2, lower panel). The reproducibility of this assay was obtained with independent repeat ChIP-qPCR experiment (S2B Fig). Expression of Flag-tagged SUMO-1 and SUMO-2 followed by ChIP using Flag antibody confirmed this phenomena (S3 Fig). These data indicate that the KSHV genome undergoes SUMO-2/3-specific modification following reactivation.

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