Research Article: Genipin Enhances Kaposi’s Sarcoma-Associated Herpesvirus Genome Maintenance

Date Published: October 13, 2016

Publisher: Public Library of Science

Author(s): Miyeon Cho, Seok Won Jung, Soomin Lee, Kuwon Son, Gyu Hwan Park, Jong-Wha Jung, Yu Su Shin, Taegun Seo, Hyojeung Kang, Shou-Jiang Gao.

http://doi.org/10.1371/journal.pone.0163693

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a Gammaherpesvirus that causes acute infection and establishes life-long latency. KSHV causes several human cancers, including Kaposi’s sarcoma, an acquired immune deficiency syndrome (AIDS)-related form of non-Hodgkin lymphoma. Genipin, an aglycone derived from geniposide found in Gardenia jasminoides, is known to be an excellent natural cross-linker, strong apoptosis inducer, and antiviral agent. Although evidence suggests antiviral activity of genipin in several in vitro viral infection systems, no inhibitory effect of genipin on KSHV infection has been reported. Thus, our aim was to determine, using the iSLK-BAC16 KSHV infection system, whether genipin has inhibitory effects on KSHV infection. For this purpose, we evaluated biological effects of genipin on KSHV infection and finally determined the underlying mechanisms responsible for the bioactive effects of genipin. A cytotoxicity assay revealed that genipin caused 50% cytotoxicity at 49.5 μM in iSLK-puro (KSHV-negative) cells and at 72.5 μM in iSLK-BAC16 (KSHV-positive) cells. Caspase 3/7 activities were slightly suppressed by genipin treatment in iSLK-BAC16 cells while significantly induced in iSLK-puro cells. Production of the KSHV latency-associated nuclear antigen (LANA), but not that of the R-transactivator (RTA) protein, was significantly induced by genipin treatment at lower concentration. Consistent with the LANA upregulation, KSHV LANA transcripts, but not RTA transcripts, were expressed at a higher level. Furthermore, KSHV intracellular copy numbers were slightly increased at lower concentration of genipin, while KSHV extracellular copy numbers were significantly increased at higher concentration of genipin. Interestingly, genipin treatment at a lower concentration did induce the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1); however, a co-immunoprecipitation assay showed that the DNMT1 and LANA induced by genipin did not co-precipitate from iSLK-BAC16 cells. Moreover, a chromatin immunoprecipitation assay demonstrated that genipin treatment enhanced the binding of CCCTC-binding factor (CTCF) to the CTCF-binding site in the KSHV latency control region but suppressed the binding of structural maintenance of chromosomes protein 3 (SMC3) to this site. Genipin treatment also led to the recruitment of additional RNA polymerase to the majority of binding sites of some interesting proteins in the KSHV latency control region, which might be related to the extension of S phase in iSLK-BAC16 cells by genipin treatment. Finally, genipin treatment at lower concentration could promote the KSHV latent replication. In contrast, the treatment at higher concentration could induce the KSHV lytic replication. In conclusion, genipin was shown to be an interesting reagent, which we used to manipulate KSHV life cycle in KSHV latently infected cells.

Partial Text

Members of the Herpesviridae family are well-known viruses that can be found in many different species across the animal kingdom. Herpesviruses have a double-stranded DNA genome (124–230 kb) enclosed in an icosahedral capsid (~125 nm in diameter), which is composed of 162 capsomeres. Based on their biological properties, such as a host range, replication cycle, and cell tropism, these viruses are classified into the alpha, beta, and gamma herpesvirus subfamilies [1]. Kaposi’s sarcoma-associated herpesvirus (KSHV, also known as HHV-8) is the eighth human herpesvirus, and it belongs to Gammaherpesviruses [2]. KSHV infection is associated with Kaposi’s sarcoma (KS) and some B-cell malignancies such as an acquired immune deficiency syndrome (AIDS)-related form of non-Hodgkin lymphoma, called primary effusion lymphoma, and multicentric Castleman’s disease [2]. Chemotherapy has been recommended for invasive KSHV-related diseases, and ganciclovir targeting KSHV replication has been used to inhibit KS development, despite the fact that the drug becomes useless once KS develops [3]. So far, the most effective therapy has been highly active antiretroviral therapy (HAART) that reduces HIV infection in AIDS–KS patients [4]. Although KSHV causes a wide range of human cancers, there are still not enough antiviral agents that specifically and effectively target KSHV.

We evaluated the biological effects of genipin on KSHV infection and determined the underlying mechanisms involved in the bioactive effects of genipin. A cytotoxicity assay revealed that the genipin treatment resulted in a 50% level of cytotoxicity at 49.5 μM in iSLK-puro (KSHV-negative) and 72.5 μM in iSLK-BAC16 (KSHV-positive) cells. The caspase 3/7 activities were slightly suppressed by the genipin treatment in iSLK-BAC16 cells while significantly induced in iSLK-puro cells. The KSHV LANA protein, but not the RTA protein, was produced at a higher level with genipin treatment, although both latent and lytic KSHV gene transcripts were upregulated. Furthermore, KSHV intracellular copy numbers were in slice increased by genipin treatment at lower concentration, while KSHV extracellular copy numbers were significantly increased by genipin treatment at higher concentration. Interestingly, the genipin treatment did induce the DNMT1 expression, besides that of LANA, but a co-IP assay showed that the proteins did not co-precipitate in iSLK-BAC16 cell lysates. Moreover, the chromatin IP assay demonstrated that the genipin treatment enhanced CTCF binding to the CTCF-binding site in the KSHV latency control region but suppressed SMC3 binding to this site. In addition to CTCF and SMC3, the genipin treatment recruited more RNA polymerase to most binding sites of some interesting proteins in the KSHV latency control region, which could be related to the extension of S phase in iSLK-BAC16 cells by the genipin treatment. Finally, it was suggested that the genipin treatment at lower concentration might accelerate the KSHV latent replication but the treatment at higher concentration might accelerate the KSHV lytic replication.

 

Source:

http://doi.org/10.1371/journal.pone.0163693