Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. The EBV critical latent antigens EBNA1 and EBNA3C interact with Nm23-H1, a known suppressor of cell migration and tumor metastasis. This interaction is critical for the regulation of downstream cellular genes involved in tumorigenesis and cell migration. The significance of these interactions was determined in nude mice using cancer cells expressing both EBV antigens and Nm23-H1. The EBV antigens promoted the growth of transformed cells in vivo, but their expression was less critical during the later stage of tumor development. The expression of Nm23-H1 affected the growth of cancer cells and suppressed their metastatic potential. This effect was effectively rescued by the expression of both EBV antigens. Interestingly, the prometastatic potential of EBNA3C was greater than that of EBNA1, which triggered a dramatic immune response, as indicated by increased spleen size and development of ascites in the mice. These studies now bridge the expression of the EBV antigens with tumorigenesis and metastasis and widen the range of potential targets for development of therapies for EBV-associated malignancies.
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27(KIP) proteins, by recruitment of the SCF(Skp2) E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S proteasomes. Surprisingly, in lymphoblastoid cell lines, EBNA3C is extremely stable, and the mechanism for this stability is unknown. In this report we show that EBNA3C can function as a deubiquitination enzyme capable of deubiquitinating itself in vitro as well as in vivo. Functional mapping using deletion and point mutational analysis showed that both the N- and C-terminal domains of EBNA3C contribute to the deubiquitination activity. We also show that EBNA3C efficiently deubiquitinates Mdm2, an important cellular proto-oncogene, which is known to be overexpressed in several human cancers. The data presented here further demonstrate that the N-terminal domain of EBNA3C can bind to the acidic domain of Mdm2. Additionally, the N-terminal domain of EBNA3C strongly stabilizes Mdm2. Importantly, EBNA3C simultaneously binds to both Mdm2 and p53 and can form a stable ternary complex; however, in the presence of p53 the binding affinity of Mdm2 toward EBNA3C was significantly reduced, suggesting that p53 and Mdm2 might share a common overlapping domain of EBNA3C. We also showed that EBNA3C enhances the intrinsic ubiquitin ligase activity of Mdm2 toward p53, which in turn facilitated p53 ubiquitination and degradation. Thus, manipulation of the oncoprotein Mdm2 by EBNA3C potentially provides a favorable environment for transformation and proliferation of EBV-infected cells.
ABSTRACT Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. Analysis of pathway-specific gene arrays showed that survivin expression was highly upregulated in BJAB cells expressing LANA. The mRNA levels of survivin were also upregulated in HEK 293 and BJAB cells expressing LANA. Similarly, protein levels of survivin were significantly higher in LANA-expressing, as well as KSHV-infected, cells. Survivin promoter activity assays identified GC/Sp1 and p53 cis -acting elements within the core promoter region as being important for LANA activity. Gel mobility shift assays revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 box of the survivin promoter. In addition, a LANA/p53 complex bound to the p53 cis -acting element within the survivin promoter, indicating that upregulation of survivin expression can also occur through suppression of p53 function. Furthermore, immunohistochemistry analyses revealed that survivin expression was upregulated in KSHV-associated Kaposi's sarcoma tissue, suggesting that LANA plays an important role in the upregulation of survivin expression in KSHV-infected endothelial cells. Knockdown of survivin expression by lentivirus-delivered small hairpin RNA resulted in loss of cell proliferation in KSHV-infected cells. Therefore, upregulation of survivin expression in KSHV-associated human cells contributes to their proliferation.
Despite the oncogenic potential of Merkel cell polyomavirus (MCPyV), it has been found in the normal skin of healthy individuals; however, little is known about geographical variations in the ecology of MCPyV in this tissue.This study included 284 Japanese participants. Sun-unexposed arm and sun-exposed forehead skin swab samples were obtained and analyzed for MCPyV infection, using quantitative polymerase chain reaction. Phylogenetic analyses were also conducted, based on the full-length genes encoding MCPyV large T antigen and viral protein 1.This study provides the first analyses of the age-specific prevalence and levels of MCPyV infection in normal skin. Steep increases in prevalence and viral load were observed in individuals aged >40 years. MCPyV infections with a high viral load were predominantly observed in the foreheads of subjects aged >60 years, among whom a high burden of MCPyV tended to persist. Phylogenetic analyses showed that all of the gene sequences obtained in this study clustered in a major clade, suggesting the existence of an Asian/Japanese genotype.This large study suggests that MCPyV infection with high viral loads is prevalent in the sun-exposed skin of elderly adults, making it necessary to follow up this cohort for possible transformation of MCPyV to a pathogenetic form.
Vol. 83, no. 14, p. 7129–7141, 2009, https://jvi.asm.org/content/83/14/7129.abstract. Page 7134, Fig. 4A to D: For the luciferase assay with results shown in the figure, the protein levels (WB:myc and WB:GAPDH) in the control lanes, pGL3B (empty vector), and the survivin promoter (SurP) are reshown in panels A through D. All of these lanes were derived from the same original blots and are reshown for easier comparison with the corresponding luciferase levels.
Previous studies have demonstrated the interaction between the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1 both in vitro and in vivo (C. Subramanian, M. A. Cotter II, and E. S. Robertson, Nat. Med. 7:350-355, 2001). Importantly EBNA3C can reverse the ability of Nm23-H1 to suppress migration of human cells in vitro. EBNA3C contributes to EBV-associated human cancers by regulating transcription of a number of cellular and viral promoters as well as targeting and altering the transcription activities of the metastasis suppressor Nm23-H1. Furthermore, Necdin is a cellular protein which is highly induced in terminally differentiated cells; it contributes to the regulation of cell growth and is also known to interact with viral oncoproteins. In this report, we show that Nm23-H1 and EBNA3C can modulate the biological functions of Necdin in the context of EBV infection and transformation. The levels of Necdin were consistently lower in EBV-positive cells, and EBNA3C could change the subcellular localization of Necdin as well as rescue cells from the antiangiogenic and antiproliferative effects mediated by Necdin. We also show that Necdin directly interacts with Nm23-H1, resulting in modulation of the biochemical function of Nm23-H1 as well as the biological function of Necdin. Both EBNA3C and Nm23-H1 were able to rescue not only Necdin-mediated transcriptional repression of the downstream vascular endothelial growth factor promoter but also Necdin-mediated growth suppression and antiangiogenic effects on cancer cells. The majority of this response was mediated through amino acid residues 191 to 222 of Necdin, which are also known to be important for nuclear matrix targeting. These studies suggest a role for Necdin in the regulation of downstream cellular targets in a hypoxic environment in virus-associated human cancers.
ABSTRACT Epstein-Barr virus (EBV)-encoded EBNA3C is one of the latent proteins essential for the efficient transformation of human primary B lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) in vitro through manipulation of a number of major cellular pathways. Although it does not have direct DNA-binding activity, EBNA3C plays a central role in the transcriptional modulation of a wide range of both viral and cellular genes during latent infection. Recently, we showed that EBNA3C can directly bind to the tumor suppressor protein p53 and repress its functions, in part by blocking its transcriptional activity as well as facilitating its degradation through stabilization of its negative regulator, Mdm2. In this study, we further showed that EBNA3C can negatively regulate p53-mediated functions by interacting with its regulatory proteins, the inhibitor of growth family proteins ING4 and ING5, shown to be frequently deregulated in different cancers. Functional mapping revealed that both ING4 and ING5 bound to N-terminal domain residues 129 to 200 of EBNA3C, which was previously demonstrated to associate with p53 and is also essential for LCL growth. In addition, we showed that a conserved domain of either ING4 or ING5 bound to both p53 and EBNA3C in a competitive manner, suggesting a potential role for EBNA3C whereby the ING4 or -5/p53 pathway is modulated in EBV-infected cells. Subsequently, we demonstrated that EBNA3C significantly suppresses both the ING4- and ING5-mediated regulation of p53 transcriptional activity in a dose-dependent manner. A colony formation assay as well as an apoptosis assay showed that EBNA3C nullified the negative regulatory effects on cell proliferation induced by coupled expression of p53 in the presence of either ING4 or ING5 in Saos-2 (p53 −/− ) cells. This report demonstrates a possible role for the candidate tumor suppressor ING genes in the biology of EBV-associated cancers.
