Suberoylanilide hydroxamic acid induces viral lytic cycle in Epstein‐Barr virus‐positive epithelial malignancies and mediates enhanced cell death

In Epstein‐Barr virus (EBV)‐associated malignancies, the virus is harbored in every tumor cell and persists in tightly latent forms expressing a very limited number of viral latent proteins. Induction of EBV lytic cycle leads to expression of a much larger number of viral proteins, which may serve as potential therapeutic targets. We found that 4 histone deacetylase inhibitors, trichostatin A (TSA), sodium butyrate (SB), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), all significantly induced EBV lytic cycle in EBV‐positive gastric carcinoma cells (AGS/BX1, latency II) but only weakly induced in Burkitt lymphoma cells (AK2003, latency I) and did not induce in lymphoblastoid cells (LCLs, latency III). Interestingly, SAHA potently induced viral lytic cycle in AGS/BX1 cells at micromolar concentrations (evidenced by 8‐fold increase in viral DNA replication, strong expression of viral lytic proteins and production of infectious virus particles) and mediated enhanced cell death of EBV‐positive AGS/BX1 cells when compared with that of EBV‐negative AGS cells, possibly related to cell cycle arrest at G2/M phase. Furthermore, SAHA effected strong induction of EBV lytic cycle in nasopharyngeal carcinoma but not in NK lymphoma cells (both expressing EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent, which warrants further investigation into its potential application as a novel virus‐targeted drug for treatment of EBV‐associated epithelial malignancies.     

RBP－Jκ在KSHV复制中的作用研究进展

KSHV被称为人类疱疹病毒8型,是卡波氏肉瘤,原发性渗出性淋巴瘤和多中心Castleman病等疾病的病原。KSHV存在潜伏和裂解两种复制状态,但是其机制尚不清楚。现有研究证实病毒的复制和转录激活因子（RTA）能促进多种病毒基因的表达,激活病毒进入裂解期;同时RTA也可以激活相关抗原（LANA）基因表达,而LANA又反过来抑制RTA的表达,在KSHV潜伏感染中发挥关键作用。RBP－Jκ是Notch通路的主要转录抑制因子,也是RTA激活病毒裂解复制所必需的因子;RBP－Jκ也可与LANA结合,发挥抑制RTA表达的功能。本文将对RBP－Jκ在KSHV复制中的重要作用作一综述。     

Gammaherpesvirus and lymphoproliferative disorders in immunocompromised patients

Two lymphotropic human gamma herpesviruses can cause lymphoproliferative disorders: Epstein Barr virus (EBV, formally designated as human herpesvirus 4) and Kaposi sarcoma herpesvirus (KSHV, also called human herpesvirus 8). Individuals with inherited or acquired immunodeficiency have a greatly increased risk of developing a malignancy caused by one of these two viruses. Specific types of lymphoproliferations, including malignant lymphomas, occur in individuals with HIV infection, transplant recipients and children with primary immunodeficiency. Some of these diseases, such as Hodgkin's and non-Hodgkin lymphoma resemble those occurring in immunocompetent patients, but the proportion of tumors in which EBV is present is increased. Others, like primary effusion lymphoma and polymorphic post-transplant lymphoproliferative disorder are rarely seen outside the context of a specific immunodeficient state. Understanding the specific viral associations in selected lymphoproliferative disorders, and the insights into the molecular mechanisms of viral oncogenesis, will lead to better treatments for these frequently devastating diseases.     

Induction of lytic Epstein-Barr virus (EBV) infection in EBV-associated malignancies using adenovirus vectors in vitro and in vivo

The consistent presence of EBV genomes in certain tumor types (in particular, AIDS-related central nervous system lymphomas and nasopharyngeal carcinomas) may allow novel, EBV-based targeting strategies. Tumors contain the latent (transforming) form of EBV infection. However, expression of either of the EBV immediate-early proteins, BZLF1 and BRLF1, is sufficient to induce lytic EBV infection, resulting in death of the host cell. We have constructed replication-deficient adenovirus vectors expressing the BZLF1 or BRLF1 immediate-early genes and examined their utility for killing latently infected lymphoma cells in vitro and in vivo. We show that both the BZLF1 and BRLF1 vectors efficiently induce lytic EBV infection in Jijoye cells (an EBV-positive Burkitt lymphoma cell line). Furthermore, lytic EBV infection converts the antiviral drug, ganciclovir (GCV), into a toxic (phosphorylated) form, which inhibits cellular as well as viral DNA polymerase. When Jijoye cells are infected with the BZLF1 or BRLF1 adenovirus vectors in the presence of GCV, viral reactivation is induced, but virus replication is inhibited (thus preventing the release of infectious EBV particles); yet cells are still efficiently killed. Finally, we demonstrate that the BZLF1 and BRLF1 adenovirus vectors induce lytic EBV infection when they are directly inoculated into Jijoye cell tumors grown in severe combined immunodeficiency mice. These results suggest that induction of lytic EBV infection in tumors, in combination with GCV, may be an effective strategy for treating EBV-associated malignancies.     

Therapies based on targeting Epstein‐Barr virus lytic replication for EBV‐associated malignancies

In recent years, Epstein‐Barr virus (EBV) lytic infection has been shown to significantly contribute to carcinogenesis. Thus, therapies aimed at targeting the EBV lytic cycle have been developed as novel strategies for treatment of EBV‐associated malignancies. In this review, focusing on the viral lytic proteins, we describe recent advances regarding the involvement of the EBV lytic cycle in carcinogenesis. Moreover, we further discuss 2 distinct EBV lytic cycle‐targeted therapeutic strategies against EBV‐induced malignancies. One of the strategies involves inhibition of the EBV lytic cycle by natural compounds known to have anti‐EBV properties; another is to intentionally induce EBV lytic replication in combination with nucleotide analogues. Recent advances in EBV lytic‐based strategies are beginning to show promise in the treatment and/or prevention of EBV‐related tumors.     

HIV, EBV and KSHV: viral cooperation in the pathogenesis of human malignancies

Malignancies associated with Epstein-Barr virus (EBV) and/or Kaposi's sarcoma human herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is frequently found in patients infected with HIV. Both these human gammaherpesviruses are known for their oncogenic properties, for the viral products that mimic or interfere with the functions of critical cellular proteins, and the ability to escape the immune responses. The introduction of the highly active anti-retroviral therapy (HAART) has significantly decreased the frequency of Kaposi's sarcoma (KS), non-Hodgkin's lymphoma (NHL), and primary central nervous system lymphoma (PCNSL); conversely, for some lymphomas the incidence diminished only slightly, as in Burkitt's lymphoma (BL), or had no significant variations, as Hodgkin's lymphoma (HL). These observations may indicate that HAART might have a direct impact on KSHV and EBV biology, that there is a reconstitution of the immune system in HIV-infected patients under HAART, or even that HAART perhaps has a detrimental impact in the pathogenic interactions between HIV, EBV and KSHV. The present review aim to evaluate and to discuss the data available for these hypotheses, in order to shed more light on the mechanisms for the cooperation among HIV-1, EBV and KSHV that may culminate in cell transformation and cancer development in humans.     

Virological and molecular characterisation of a new B lymphoid cell line, established from an AIDS patient with primary effusion lymphoma, harbouring both KSHV/HHV8 and EBV viruses

We report here a new case of primary effusion lymphoma (PEL), occurring in a French homosexual HIV-1 infected male with a pericardial, pleural and mesenteric tumour dissemination, and the establishment from his pleural effusion of a new cell line, Cra-BCBL, dually infected by EBV and KSHV/HHV8. Cra-BCBL cells are of B-cell origin as judged by their clonal immunoglobulin heavy chain (IgH) gene rearrangement, identical to that of the parental tumour. Both the cell line and the lymphoma cells expressed CD38 and CD45 antigens but no classical B-cell or T-cell lineage-restricted antigens. Cra-BCBL harbours a type I EBV virus, expressing a latency type II. Expression of KSHV/HHV8 ORF72 and ORF75 was detected by RT/PCR. In addition, KSHV lytic replication could be induced by treatment by n-butyrate. An equivalent and high copy number of KSHV genomes (20 to 200 copies by cell) was detected both in the primary tumour cells and in the cell line. Southern blot (SB) analysis of EBV terminal repeats (TR) displayed the same unique band in the cell line DNA and in the original tumour cells, consistent with a monoclonal infection of EBV. Furthermore, SB analysis of KSHV/HHV8 TR revealed the same hybridisation pattern between Cra-BCBL and the effusion cells, with a common band at around 30-40 kb corresponding to the fused termini of the viral episomes and a 5 Kb rearranged fragment. The new cell line characterised here could be a useful model to study interactions between two human herpes viruses and their contribution to lymphomagenesis.     

EB病毒对伯基特淋巴瘤细胞系宿主基因表达的影响

背景与目的：Epstein—Barr病毒（Epstein—Barrvirus,EBV）以潜伏状态存在于Burkitt’s淋巴瘤（Burkitt’s lymphoma,BL）细胞中,其基因表达呈高度限制模式,只有少数肿瘤细胞存在病毒裂解性复制。BL细胞株可被诱导进入病毒裂解周期,通过刺激表面免疫球蛋白分子启动病毒复制过程。此过程中,有许多EBV基因表达,这些基因可能影响宿主基因的表达。本实验目的在于鉴定BL细胞中由EBV所调节的宿主基因及由表面IgG连接所调节的宿主基因。方法：利用微阵列检测EBV阳性Akata细胞和EBV阴性AK31细胞中差异性表达的基因。结果：Akata和AK31细胞中共有91个人类基因呈差异性表达,细胞受刺激进入裂解性复制后,检测到198个差异性表达基因。其中myd88基因的差异性表达与TLR9信号通路受损有关。结论：EBV下调了在裂解周期激活早期阶段由表面Ig交联作用所调节的大部分基因。这些基因与细胞存活、信号转导、转录控制以及可能调节B淋巴细胞和其他细胞（如可能影响病毒复制的HDAC4）EBV转化的免疫应答过程有关。     

Modulation of the autophagy pathway by human tumor viruses

Autophagy is a highly conserved and regulated process in eukaryotic cells by which components of the cytoplasm, such as damaged organelles and foreign pathogens, become enveloped into double-membrane autophagosome vesicles that fuse with the lysosome for degradation. Viruses are adept at subverting host cellular pathways for their replication and survival. The human tumor viruses, Epstein-Barr virus (EBV), Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Hepatitis B virus (HBV), and Hepatitis C virus (HCV), have evolved novel ways of modulating autophagy during productive and latent stages of the virus life cycle. This review will discuss how the autophagy pathway becomes activated upon viral infection and the role of viral proteins in regulating the autophagy pathway. Specifically, we will examine how virus-encoded homologs of autophagy proteins evade autophagy-mediated degradation by blocking the induction, elongation, or maturation steps in the autophagy pathway. We will also discuss how certain viruses enhance autophagy induction or usurp autophagic machinery for their own replication. A comprehensive understanding of the autophagic response to tumor viruses may enable the discovery of novel antiviral and/or anticancer drug therapies.     

Immune evasion in Kaposi's sarcoma-associated herpes virus associated oncogenesis

A hallmark of herpesviruses is a lifelong persistent infection, which often leads to diseases upon immune suppression of infected host. Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is etiologically linked to the development of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and Multicentric Castleman's disease (MCD). In order to establish a persistent infection, KSHV dedicates a large portion of its genomic information to sabotage almost every aspect of host immune system. Thus, understanding the interplay between KSHV and the host immune system is important in not only unraveling the complexities of viral persistence and pathogenesis, but also discovering novel therapeutic targets. This review summarizes current knowledge of host immune evasion strategies of KSHV and their contributions to KSHV-associated diseases.     

The role of metabolic reprogramming in γ‐herpesvirus‐associated oncogenesis

The γ‐herpesviruses, EBV and KSHV, are closely associated with a number of human cancers. While the signal transduction pathways exploited by γ‐herpesviruses to promote cell growth, survival and transformation have been reported, recent studies have uncovered the impact of γ‐herpesvirus infection on host cell metabolism. Here, we review the mechanisms used by γ‐herpesviruses to induce metabolic reprogramming in host cells, focusing on their ability to modulate the activity of metabolic regulators and manipulate metabolic pathways. While γ‐herpesviruses alter metabolic phenotypes as a means to support viral infection and long‐term persistence, this modulation can inadvertently contribute to cancer development. Strategies that target deregulated metabolic phenotypes induced by γ‐herpesviruses provide new opportunities for therapeutic intervention.     

Efficient lytic induction of kaposi's sarcoma-associated herpesvirus (KSHV) by the anthracyclines

Lytic induction of latent Kaposi''s sarcoma-associated herpesvirus (KSHV) has been considered as a therapeutic option for efficient treatment of several KSHV-associated malignancies. Here, we developed a robust high-throughput screening system that allows an easy and quantitative measurement of lytic induction of latent KSHV and discovered three anthracyclines as potent inducers from screen of FDA-approved drugs. Lytic induction of latent KSHV by three compounds was verified by the significant induction of lytic genes and subsequent production of infectious KSHV. Importantly, lytic induction by three compounds was much more efficient than that by sodium butyrate, a well-characterized inducer of KSHV lytic cycle. Mechanistically, the anthracyclines caused lytic induction of KSHV through apoptosis induced by their DNA intercalation rather than topoisomerase II inhibition. Consequently, our results clearly demonstrated a role of anthracyclines as effective lytic inducers of KSHV and also provided a molecular basis of their use for efficient treatment of diseases associated with KSHV infection.     

The ubiquitin/proteasome system in Epstein-Barr virus latency and associated malignancies

Epstein-Barr virus (EBV) is a human herpesvirus associated with lymphoid and epithelial malignancies. Three viral proteins, the EBV nuclear antigen 1 and the latent membrane proteins-1 and -2A, regulate viral latency by manipulating ubiquitin-dependent proteolysis. The activation of cellular oncogenes is instrumental for the progression of EBV infected cells to full malignancy. Constitutively activation of c-myc correlates with decreased proteasomal activity and upregulation of compensatory proteolytic pathways in Burkitt's lymphomas. Knowledge of these multiple strategies of interference with regulated proteolysis may provide new clues for the treatment of EBV-associated malignancies.     

Kaposi sarcoma-associated herpesvirus (KSHV): Molecular biology and oncogenesis

Kaposi sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA herpesvirus belonging to the γ-herpesvirinae subfamily. KSHV has been associated with the development of three neoplastic diseases: Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). In this review, we discuss the three KSHV-associated malignancies, KSHV genome, latent and lytic aspects of the viral lifecycle, putative viral oncogenes, as well as therapeutic regimens used for the treatment of KS, PEL, and MCD.     

Targeting human herpesvirus-8 for treatment of Kaposi's sarcoma and primary effusion lymphoma

PURPOSE OF REVIEW: Human herpesvirus-8, also called the Kaposi's sarcoma herpesvirus, is present in all cases of Kaposi's sarcoma and primary effusion lymphoma and in some cases of multicentric Castleman's disease. This review discusses mechanisms by which human herpesvirus-8 contributes to tumorigenesis and how this knowledge can be used to target the virus for the treatment of these tumors. RECENT FINDINGS: Most primary effusion lymphomas and Kaposi's sarcoma tumor cells are latently infected with human herpesvirus-8 and hence resistant to antiherpesvirus drugs that are dependent on lytic replication. In contrast, many of the cells infected with human herpesvirus-8 in multicentric Castleman's disease support lytic replication, so that clinical improvement frequently occurs in response to treatment with antiherpesvirus drugs. The resistance of latently-infected tumor cells to antiherpesvirus drugs can be overcome by inducing human herpesvirus-8 to reenter the lytic cascade in the presence of antiherpesvirus drugs. This leads to apoptosis of virally infected cells without increasing production of infectious virus. Alternatively, the replication and maintenance of the human herpesvirus-8 episome during latency can be disrupted by glycyrrhizic acid or hydroxyurea so that the virus no longer contributes to tumorigenesis. Both the innate and acquired immune systems can also be augmented to help prevent or treat human herpesvirus-8-associated tumors. SUMMARY: Novel strategies targeting human herpesvirus-8, which is present in all cases of Kaposi's sarcoma and primary effusion lymphoma, provide opportunities for selectively killing tumor cells.     

Mono/oligoclonal pattern of Kaposi Sarcoma-associated herpesvirus (KSHV/HHV-8) episomes in primary effusion lymphoma cells

Primary effusion lymphoma (PEL) is a rare lymphoma of B-cell origin, developed in serous cavities. PEL tumor cells are latently infected with Kaposi sarcoma-associated herpesvirus (KSHV) and in most cases co-infected with Epstein-Barr virus (EBV). In 15 primary PEL tumors including 10 EBV-positive cases, we analyzed the fused terminal repeat (TR) regions of KSHV episomes using pulsed-field gel electrophoresis and Southern blot. On the same genomic DNA samples, the cellular clonality was assessed by Southern blot and PCR detection of monoclonal immunoglobulin heavy chain (IGH) VDJ gene rearrangements, associated in the EBV-infected cases, with Southern blot analysis of the fused termini of EBV episomes. Monoclonal IGH gene rearrangements were detected in 13 tumors using Southern blot, in 11 cases using PCR, and in all cases considering both methods. EBV infection was monoclonal in all EBV-positive cases. However, only 5 PEL tumors were found to be monoclonally infected with KSHV. In the 10 other cases, we found a biclonal (2 bands; n = 4) or an oligoclonal pattern (3-6 bands; n = 6) of KSHV episomes. We hypothesized that the apparent discrepancy between viral and cellular clonalities in PEL might be due to several phenomena including complex mechanisms of genomic recircularization, insertion of duplicated sequences into the TR region and simultaneous infection of tumor cells with defective KSHV variants. KSHV infection of contaminating nontumoral cells, superinfection from lytically infected cells or viral integration events might also explain the oligoclonal pattern of KSHV infection. Several of these mechanisms, not mutually exclusive, might coexist in a single tumor.