Antibody-mediated control of persistent gamma-herpesvirus infection

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine gamma-herpesvirus-68 has biological and structural similarities to the human gamma-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28(-/-) mice were used to address the role of Abs in control of persistent murine gamma-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28(-/-) mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28(-/-) mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of gamma-herpesvirus latency, and have significant implications for clinical intervention.     

Early establishment of gamma-herpesvirus latency: implications for immune control

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, infect >90% of the population worldwide, and latent infection is associated with numerous malignancies. Rational vaccination and therapeutic strategies require an understanding of virus-host interactions during the initial asymptomatic infection. Primary EBV infection is associated with virus replication at epithelial sites and entry into the circulating B lymphocyte pool. The virus exploits the life cycle of the B cell and latency is maintained long term in resting memory B cells. In this study, using a murine gamma-herpesvirus model, we demonstrate an early dominance of latent virus at the site of infection, with lung B cells harboring virus almost immediately after infection. These data reinforce the central role of the B cell not only in the later phase of infection, but early in the initial infection. Early inhibition of lytic replication does not impact the progression of the latent infection, and latency is established in lymphoid tissues following infection with a replication-deficient mutant virus. These data demonstrate that lytic viral replication is not a requirement for gamma-herpesvirus latency in vivo and suggest that viral latency can be disseminated by cellular proliferation. These observations emphasize that prophylactic vaccination strategies must target latent gamma-herpesvirus at the site of infection.     

Reduced prevalence of Epstein-Barr virus-related lymphocryptovirus infection in sera from a new world primate

The recent discovery of an Epstein-Barr virus (EBV)-related lymphocryptovirus (LCV) naturally infecting common marmosets demonstrated that gamma-1 herpesviruses are not limited to human and Old World nonhuman primate hosts. We developed serologic assays to detect serum antibodies against lytic- and latent-infection marmoset LCV antigens in order to perform the first seroepidemiologic study of LCV infection in New World primates. In three different domestic colonies and in animals recently captured from the wild, we found that the seroprevalence of marmoset LCV infection was not as ubiquitous as with EBV or Old World LCV. These biologic differences in LCV infection of New World versus human and Old World primate hosts correlate with the evolution of the LCV viral gene repertoire.     

Efficacy of methylenecyclopropane analogs of nucleosides against herpesvirus replication in vitro

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8). Ten of the analogs had activity against at least one of the viruses tested. One compound had moderate activity against HSV-1 and six had activity against VZV. All but one compound was active against HCMV with a mean EC50 of 2.1 +/- 0.6 microM, compared with a mean EC50 of 3.9 +/- 0.8 microM for ganciclovir. Of special interest was the fact that eight of the ten compounds were active against both HHV-6A and HHV-6B with mean EC50 values of 6.0 +/- 5.2 mciroM and <2.4 +/- 1.5 microM, respectively. Only two compounds had activity against EBV, whereas all but one compound was active against HHV-8 with a mean EC50 of 3.1 +/- 1.7 microM. These results indicate that members of this series of methylenecyclopropane analogs are highly active against HCMV, HHV-6, and HHV-8 but are less active against HSV, VZV, and EBV.     

Survey of Nonhuman Primates for Antibodies Reactive With Epstein-Barr Virus (EBV) Antigens and Susceptibility of Their Lymphocytes for Immortalization With EBV

The susceptibility to transformation with Epstein-Barr virus (EBV) and the prevalence of antibodies reactive to EBV were examined in 43 primate species. In vitro EBV infection was revealed in lymphocytes from Old World monkeys, including patas monkeys and the colobines, as well as in lymphocytes from the apes. Antibodies reactive to EBV-early antigen/viral capsid antigen (EA/VCA) were detected in all the species of Old World monkeys and apes examined and in two out of seven species of New World monkeys.     

Primary lymphocyte infection models for KSHV and its putative tumorigenesis mechanisms in B cell lymphomas

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the latest addition to the human herpesvirus family. Unlike alpha- and beta-herpesvirus subfamily members, gamma-herpesviruses, including Epstein-Barr virus (EBV) and KSHV, cause various tumors in humans. KSHV primarily infects endothelial and B cells in vivo, and is associated with at least three malignancies: Kaposi’s sarcoma and two B cell lymphomas, respectively. Although KSHV readily infects endothelial cells in vitro and thus its pathogenic mechanisms have been extensively studied, B cells had been refractory to KSHV infection. As such, functions of KSHV genes have mostly been elucidated in endothelial cells in the context of viral infection but not in B cells. Whether KSHV oncogenes, defined in endothelial cells, play the same roles in the tumorigenesis of B cells remains an open question. Only recently, through a few ground-breaking studies, B cell infection models have been established. In this review, those models will be compared and contrasted and putative mechanisms of KSHV-induced B cell transformation will be discussed.     

Novel simian homologues of Epstein-Barr virus

Thirty different lymphocryptoviruses (LCV), 26 of them novel, were detected in primates by a panherpesvirus PCR assay. Nineteen LCV from chimpanzees, bonobos, gorillas, and other Old World primates were closely related to Epstein-Barr virus (EBV), the type species of the genus lymphocryptovirus. Seven LCV originating from New World primates were related to callitrichine herpesvirus 3 (CalHV-3), the first recognized New World LCV. Importantly, a second LCV from gorillas and three LCV from orangutans and gibbons were only distantly related to EBV and CalHV-3. They were tentatively assigned to a novel genogroup of Old World primate LCV. The work described in the paper may also help identify an as yet unknown human LCV.     

Expansion of effector memory TCR Vbeta4+ CD8+ T cells is associated with latent infection-mediated resistance to transplantation tolerance

Therapies that control largely T cell-dependent allograft rejection in humans also possess the undesirable effect of impairing T cell function, leaving transplant recipients susceptible to opportunistic viruses. Prime among these opportunists are the ubiquitous herpesviruses. To date, studies are lacking that address the effect of viruses that establish a true latent state on allograft tolerance or the effect of tolerance protocols on the immune control of latent viruses. By using a mixed chimerism-based tolerance-induction protocol, we found that mice undergoing latent infection with gammaHV68, a murine gamma-herpesvirus closely related to human gamma-herpesviruses such as EBV and Kaposi's sarcoma-associated herpesvirus, significantly resist tolerance to allografts. Limiting the degree of virus reactivation or innate immune response did not reconstitute chimerism in latently infected mice. However, gammaHV68-infected mice showed increased frequency of CD8+ T cell alloreactivity and, interestingly, expansion of virus-induced, alloreactive, "effector/effector memory" TCR Vbeta4+CD8+ T cells driven by the gammaHV68-M1 gene was associated with resistance to tolerance induction in studies using gammaHV68-M1 mutant virus. These results define the viral gene and immune cell types involved in latent infection-mediated resistance to allograft tolerance and underscore the influence of latent herpesviruses on allograft survival.     

Comparative analysis identifies conserved tumor necrosis factor receptor-associated factor 3 binding sites in the human and simian Epstein-Barr virus oncogene LMP1.

Nonhuman primates are naturally infected with a B-lymphotropic herpesvirus closely related to Epstein-Barr virus (EBV). These simian EBV share considerable genetic, biologic, and epidemiologic features with human EBV, including virus-induced tumorigenesis. However, latent, transformation-associated viral genes demonstrate marked sequence divergence among species despite the conserved functions. We have cloned the latent membrane protein 1 (LMP1) homologs from the simian EBV naturally infecting baboons (cercopithicine herpesvirus 12, herpesvirus papio) and rhesus monkeys (cercopithicine herpesvirus 15) for a comparative study with the human EBV oncogene. The transmembrane domains are well conserved, but there is striking sequence divergence of the carboxy-terminal cytoplasmic domain essential for B-cell immortalization and interaction with the tumor necrosis factor receptor signaling pathway. Nevertheless, the simian EBV LMP1s retain most functions in common with EBV LMP1, including the ability to induce NF-(kappa)B activity in human cells, to bind the tumor necrosis factor-associated factor 3 (TRAF3) in vitro, and to induce expression of tumor necrosis factor-responsive genes, such as ICAM1, in human B lymphocytes. Multiple TRAF3 binding sites containing a PXQXT/S core sequence can be identified in the simian EBV LMP1s by an in vitro binding assay. A PXQXT/S-containing sequence is also present in the cytoplasmic domain of the Hodgkin's disease marker, CD30, and binds TRAF3 in vitro. The last 13 amino acids containing a PXQXT/S sequence are highly conserved in human and simian EBV LMP1 but do not bind TRAF3, suggesting a distinct role for this conserved region of LMP1. The conserved TRAF3 binding sites in LMP1 and the CD30 Hodgkin's disease marker provides further evidence that a TRAF3-mediated signal transduction pathway may be important in malignant transformation.     

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8)

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8, HHV-8) are members of gamma-herpes virus family. Both viruses infect to B cells and cause malignancies such as lymphoma. Since EBV and HHV-8 are so-called 'oncovirus', their oncogenecities have been focused in the researches on EBV and KSHV for a long time. EBV was discovered in 1964, whereas KSHV was identified in 1994. However, KSHV was analyzed rapidly in these fifteen years. One of the recent progresses in the research on EBV and KSHV is that virus-encoded small RNAs were identified in their genomes and characterized. EBV is the first human virus in whose genome microRNA was identified. The oncogenecity of EBV and KSHV remains unclear. Here, I discuss the pathogenesis by EBV and KSHV with special reference to recent progress in this field.     

Simian homologues of human herpesvirus 8

Gamma-herpesviruses can be found in most primates including Old World an New World monkeys. The gamma-herpesvirinae are grouped into two classes: lymphocryptoviruses (gamma1) and rhadinoviruses (gamma2). The lymphocryptoviruses include Epstein-Barr virus, lymphocryptovirus of rhesus monkeys, and Herpesvirus papio of baboons. Rhadinoviruses that infect New World monkeys include Herpesvirus saimiri, whose natural host is the squirrel monkey, and Herpesvirus ateles, which infects spider monkeys. Rhadinoviruses that infect hominoids and Old World monkeys include Kaposi's sarcoma-associated herpesvirus, also known as HHV-8, and rhesus monkey rhadinovirus.     

Animal in vivo models of EBV-associated lymphoproliferative diseases: special references to rabbit models

Animal models of human EBV-associated diseases are essential to elucidate the pathogenesis of EBV-associated diseases. Here we review those previous models using EBV or EBV-like herpesviruses and describe the details on our two newly-developed rabbit models of lymphoproliferative diseases (LPD) induced by simian EBV-like viruses. The first is Cynomolgus-EBV-induced T-cell lymphomas in rabbits inoculated intravenously (77-90%) and orally (82-89%) during 2-5 months. EBV-DNA was detected in peripheral blood by PCR from 2 days after oral inoculation, while anti-EBV-VCA IgG was raised 3 weeks later. Rabbit lymphomas and their cell lines contained EBV-DNA and expressed EBV-encoded RNA-1 (EBER-1). Rabbit lymphoma cell lines, most of which have specific chromosomal abnormality, showed tumorigenicity in nude mice. The second is the first animal model for EBV-infected T-cell LPD with virus-associated hemophagocytic syndrome (VAHS), using rabbits infected with an EBV-like herpesvirus, Herpesvirus papio (HVP). Rabbits inoculated intravenously with HVP-producing cells showed increased anti-EBV-VCA-IgG titers, and most (85%) subsequently died of fatal LPD and VAHS, with bleeding and hepatosplenomegaly, during 22-105 days. Peroral spray of cell-free HVP induced viral infection with seroconversion in 3 out of 5 rabbits, with 2 of the 3 infected rabbits dying of LPD with VAHS. Atypical T lymphocytes containing HVP-DNA and expressing EBER-1 were observed in many organs. Hemophagocytic histiocytosis was observed in the lymph nodes, spleen, bone marrow, and thymus. These rabbit models are also useful and inexpensive alternative experimental model systems for studying the biology and pathogenesis of EBV, and prophylactic and therapeutic regimens.     

Efficacy of Methylenecyclopropane Analogs of Nucleosides Against Herpesvirus Replication In Vitro

Abstract

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8). Ten of the analogs had activity against at least one of the viruses tested. One compound had moderate activity against HSV-1 and six had activity against VZV. All but one compound was active against HCMV with a mean EC₅₀ of 2.1 ± 0.6 µM, compared with a mean EC₅₀ of 3.9 ± 0.8 µM for ganciclovir. Of special interest was the fact that eight of the ten compounds were active against both HHV-6A and HHV-6B with mean EC₅₀ values of 6.0 ± 5.2 µM and <2.4 ± 1.5 µM, respectively. Only two compounds had activity against EBV, whereas all but one compound was active against HHV-8 with a mean EC₅₀ of 3.1 ± 1.7 µM. These results indicate that members of this series of methylenecyclopropane analogs are highly active against HCMV, HHV-6, and HHV-8 but are less active against HSV, VZV, and EBV.     

Simian homologues of Epstein-Barr virus

Gamma-herpesviruses closely related to the Epstein-Barr virus (EBV) are known to naturally infect Old World non-human primates and are classified in the same lymphocryptovirus (LCV) genera. LCV infecting humans and Old World primates share similar biology, and recent studies have demonstrated that these viruses share a similar repertoire of viral genes. Surprisingly, the latent infection genes associated with cell growth transformation demonstrate the most striking sequence divergence, but the functional mechanisms for these genes are generally well conserved. The recent discovery of LCVs naturally infecting New World primates has rewritten the old paradigm of LCV host range restriction to humans and Old World non-human primates, so that these viruses are more widespread than previously believed. However, the New World LCV genome has significant and interesting differences from EBV and other Old World LCVs despite similar biological properties. Thus, the simian homologues of EBV can provide an important animal model for studying LCV pathogenesis, and the similarities and differences that have evolved among these related viruses can provide a unique perspective towards a better understanding of EBV.     

A virus-like particle-based Epstein-Barr virus vaccine

Epstein-Barr Virus (EBV) is an ubiquitous human herpesvirus which can lead to infectious mononucleosis and different cancers. In immunocompromised individuals, this virus is a major cause for morbidity and mortality. Transplant patients who did not encounter EBV prior to immunosuppression frequently develop EBV-associated malignancies, but a prophylactic EBV vaccination might reduce this risk considerably. Virus-like particles (VLPs) mimic the structure of the parental virus but lack the viral genome. Therefore, VLPs are considered safe and efficient vaccine candidates. We engineered a dedicated producer cell line for EBV-derived VLPs. This cell line contains a genetically modified EBV genome which is devoid of all potential viral oncogenes but provides viral proteins essential for the assembly and release of VLPs via the endosomal sorting complex required for transport (ESCRT). Human B cells readily take up EBV-based VLPs and present viral epitopes in association with HLA molecules to T cells. Consequently, EBV-based VLPs are highly immunogenic and elicit humoral and strong CD8+ and CD4+ T cell responses in vitro and in a preclinical murine model in vivo. Our findings suggest that VLP formulations might be attractive candidates to develop a safe and effective polyvalent vaccine against EBV.     

Retrovirus restriction by TRIM5alpha variants from Old World and New World primates

The TRIM5alpha proteins of humans and some Old World monkeys have been shown to block infection of particular retroviruses following virus entry into the host cell. Infection of most New World monkey cells by the simian immunodeficiency virus of macaques (SIVmac) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5alpha orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5alpha proteins functionally resembled human TRIM5alpha, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5alpha proteins from several New World monkey species restricted infection by SIVmac and the SIV of African green monkeys, SIVagm. Spider monkey TRIM5alpha, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIVmac, SIVagm, HIV-1, and N-MLV. Tandem duplications in the TRIM5alpha B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5alpha proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5alpha proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.     

Concluding overview: looking back, looking forward

The gamma-herpesviruses are a group of related agents which share the same broad strategy for infection of and persistence within the lymphoid tissues of their hosts. Yet in evolutionary terms these agents are sufficiently diverse to display multiple different molecular mechanisms whereby that strategy can be achieved. Attempts are made to relate the different in vitro growth transforming capacities of the gamma1-herpesviruses, the T-lymphotropic gamma2-herpesviruses and the B-lymphotropic gamma2-herpesviruses to what is known about the biology of these virus infections in their natural or in experimental hosts. The review then summarizes the evidence linking gamma-herpesviruses with oncogenesis and proposes that the diverse spectrum of Epstein-Barr virus and human herpesvirus 8-associated human tumours falls into three pathogenetically distinct categories. Many questions remain unanswered in the areas of gamma-herpesvirus biology and disease pathogenesis: resolving these questions will require a broadening of our experimental approaches and a willingness to relinquish 'single-model' panaceas.     

Tyrosine 129 of the Murine Gammaherpesvirus M2 Protein Is Critical for M2 Function In Vivo

A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes – predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.