An integral feature of gammaherpesvirus infections is the ability to establish lifelong latency in B cells. During latency, the viral genome is maintained as an extrachomosomal episome, with stable maintenance in dividing cells mediated by the viral proteins Epstein-Barr nuclear antigen 1 (EBNA-1) for Epstein-Barr virus and latencyassociated nuclear antigen (LANA) for Kaposi's sarcoma-associated herpesvirus. It is believed that the expression of episome maintenance proteins is turned off in the predominant long-term latency reservoir of resting memory B cells, suggesting that chronic gammaherpesvirus infection is primarily dormant. However, the kinetics of LANA/ EBNA-1 expression in individual B-cell subsets throughout a course of infection has not been examined. The infection of mice with murine gammaherpesvirus 68 (MHV68, ␥HV68) provides a model to determine the specific cellular and molecular events that occur in vivo during lifelong gammaherpesvirus latency. In work described here, we make use of a heterologously expressed enzymatic marker to define the types of B cells that express the LANA homolog (mLANA) during chronic MHV68 infection. Our data demonstrate that mLANA is expressed in a stable fraction of B cells throughout chronic infection, with a prominent peak at 28 days. The expression of mLANA was detected in naïve follicular B cells, germinal-center B cells, and memory B cells throughout infection, with germinalcenter and memory B cells accounting for more than 80% of the mLANA-expressing cells during the maintenance phase of latency. These findings suggest that the maintenance phase of latency is an active process that involves the ongoing proliferation or reseeding of latently infected memory B cells.Gammaherpesviruses such as Epstein-Barr virus (EBV), Kaposi's sarcoma-associated virus (KSHV, HHV-8), and murine gammaherpesvirus 68 (MHV68, ␥HV68) are associated with lymphoproliferative diseases and a variety of malignancies of both epithelial and lymphoid origin. The strict species specificity exhibited by gammaherpesviruses has limited research on the human viruses primarily to in vitro studies. MHV68 is genetically colinear to the human gammaherpesviruses and exhibits many similar pathogenic features (54, 62). MHV68 is a natural pathogen of rodents (6, 9, 44), making the inoculation of mice with MHV68 a useful small-animal model to study gammaherpesvirus infection in vivo.A hallmark of gammaherpesvirus infections is the establishment of lifelong latency in B cells. During latency, the viral genome is maintained as an extrachromosomal episome, viral gene expression is highly restricted, and no new progeny virions are generated. The stable maintenance of the episome in dividing cells requires regulated plasmid DNA replication and the efficient partitioning of replicated genomes to daughter cells. These processes are mediated by critical episome maintenance protein Epstein-Barr nuclear antigen 1 (EBNA-1) for EBV and latency-associated nuclear antigen (LANA) for KSHV. EBNA-1 and LANA facilitate the re...
Gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesis in vivo is unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis of in vitro and in vivo MHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virus in vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infected in vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replication in vitro and in vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease.
Epstein-Barr virus (EBV) is a well-established B-cell-tropic virus associated with various lymphoproliferative diseases of both B-cell and non-B-cell origin.EBV is associated with a number of T-cell lymphomas; however, in vitro studies utilizing prototypical EBV type 1 (EBV-1) laboratory strains have generally failed to readily infect mature T cells in culture. The difficulties in performing in vitro T-cell experiments have left questions regarding the role of EBV in the pathogenesis of EBV-positive T-cell lymphoproliferative diseases largely unresolved. We report here that the EBV type 2 (EBV-2) strain displays a unique cell tropism for T cells. In remarkable contrast to EBV-1, EBV-2 readily infects primary T cells in vitro, demonstrating a propensity for CD8 ؉ T cells. EBV-2 infection of purified T cells results in expression of latency genes and ultimately leads to T-cell activation, substantial proliferation, and profound alteration of cytokine expression. The pattern of cytokine production is strikingly skewed toward chemokines with roles in lymphocyte migration, demonstrating that EBV-2 has the ability to modulate normal T-cell processes. Collectively, these novel findings identify a previously unknown cell population potentially utilized by EBV-2 to establish latency and lay the foundation for further studies to elucidate the role of EBV in the pathogenesis of T-cell lymphoproliferative diseases. IMPORTANCE The ability of EBV to infect T cells is made apparent by its association with a variety of T-cell lymphoproliferative disorders.However, studies to elucidate the pathogenic role of EBV in these diseases have been limited by the inability to conduct in vitro T-cell infection experiments. Here, we report that EBV-2 isolates, compromised in the capacity to immortalize B cells, infect CD3 ؉ T cells ex vivo and propose a working model of EBV-2 persistence where alteration of T-cell functions resulting from EBV-2 infection enhances the establishment of latency in B cells. If indeed EBV-2 utilizes T cells to establish a persistent infection, this could provide one mechanism for the association of EBV with T-cell lymphomas. The novel finding that EBV-2 infects T cells in culture will provide a model to understand the role EBV plays in the development of T-cell lymphomas. While Epstein-Barr virus (EBV) establishes lifelong latency in B cells and is associated with B-cell malignancies, it is also associated with malignancies and diseases that originate from T cells, including NK/T-cell lymphomas (1), hemophagocytic lymphohistiocytosis (2), hydroa vacciniforme (HV) (3), and chronic active EBV (CAEBV) (4, 5). In these diseases, EBV can be detected in CD4 ϩ T cells, CD8 ϩ T cells, or ␥␦ T cells (6, 7), with the virus predominantly existing as a latent infection (8, 9). The etiology of these T-cell diseases, and in particular whether EBV infection of T cells is an aberrancy in a virus known for its B-cell tropism in vitro and in vivo, remains unknown.Based on genetic differences in the Epstein-Barr nuclear ...
The human gammaherpesviruses Epstein-Barr virus (EBV)and Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 ) are ubiquitous human pathogens that are associated with the development of numerous types of malignancies, including B cell lymphomas. Murine gammaherpesvirus 68 (MHV68) is genetically related to EBV and KSHV and causes lymphoma and lymphoproliferative disease in mice, providing a useful small-animal model for mechanistic in vivo studies of the virus-host relationship. Both the human and murine viruses subvert the antiviral immune response to establish lifelong latent infections in mature B cells. However, it is not clearly understood how these viruses gain access to specific mature B cell subsets or whether latent infection of these subsets is actively maintained over time. One intriguing possibility is that gammaherpesviruses gain entry to the circulating mature B cell compartment via infection of B cell progenitors. Mature B cells arise via a highly regulated, multistep developmental process that results in the daily generation of thousands of new cells. Thus, any developing B cell subsets could provide a potential access point for recurrent entry of the virus into the long-lived mature B cell reservoir.
Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies.
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