Ex Vivo Stimulation of B Cells Latently Infected with Gammaherpesvirus 68 Triggers Reactivation from Latency

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Gammaherpesvirus 68 (␥HV68, or MHV68) is a naturally occurring rodent pathogen that replicates to high titer in cell culture and is amenable to in vivo experimental evaluation of viral and host determinants of gammaherpesvirus disease. However, the inability of MHV68 to transform primary murine B cells in culture, the absence of a robust cell culture latency system, and the paucity of MHV68-positive tumor cell lines have limited an understanding of the molecular mechanisms by which MHV68 modulates the host cell during latency and reactivation. To facilitate a more complete understanding of viral and host determinants that regulate MHV68 latency and reactivation in B cells, we generated a recombinant MHV68 virus that encodes a hygromycin resistance protein fused to enhanced green fluorescent protein as a means to select cells in culture that harbor latent virus. We utilized this virus to infect the A20 murine mature B-cell line and evaluate reactivation competence following treatment with diverse stimuli to reveal viral gene expression, DNA replication, and production of progeny virions. Comparative analyses of parental and infected A20 cells indicated a correlation between infection and alterations in DNA damage signaling following etoposide treatment. The data described in this study highlight the potential utility of this new cell culture-based system to dissect molecular mechanisms that regulate MHV68 latency and reactivation, as well as having the potential of illuminating biochemical alterations that contribute to gammaherpesvirus pathogenesis. In addition, such cell lines may be of value in evaluating targeted therapies to gammaherpesvirus-related tumors.Gammaherpesvirus 68 (␥HV68 or MHV68) is a rodent rhadinovirus found naturally in European bank voles and wood mice (6, 7). As a rodent pathogen, MHV68 infection of laboratory mice offers an experimental system to investigate aspects of gammaherpesvirus pathogenesis. Following experimental inoculation of laboratory mice, MHV68 undergoes productive replication at the site of inoculation, which facilitates dissemination to distal organs and is followed by quiescence or latency, a form of infection where viral gene expression is restricted and progeny virions are not produced (41,51,71). In the case of MHV68, latent infection is established in B lymphocytes, as well as epithelial cells, macrophages, and dendritic cells (19,56,58,74). Latency is maintained for the lifetime of the host, and latently infected cells retain the capacity to reinitiate the productive replication cycle, a process termed reactivation (45, 51, 52).The genetic malleability of MHV68 enables the identification of viral genes involved in specific aspects of viral pathogenesis by targeted mutagenesis (1). For example, such approaches have demonstrated the importance of the unique MHV68 M2 gene product in latency establishment, reactivation from latency, and promoting B-cell differentiation (24, 26), as well as defining the roles of conserved genes, such as orf73 (LANA homolog) in latency mai...

mentioning

confidence: 71%

Section: Murine B-cell Lines Do Not Support Lytic Mhv68 Replicationmentioning

confidence: 99%

Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade

Forrest

Speck

2008

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“…TPA and ionomycin induce protein kinase C-and Ca 2ϩ -mediated signaling, thus promoting B cell proliferation and mimicking the signals induced by B cell receptor (BCR) cross-linking (44). Of note, CD40 and Ig crosslinking has been shown to induce MHV68 reactivation from latency (45). Since XBP-1s reflects a stress signal in a terminally differentiating cell, it is evident that the virus has evolved mechanisms to reactivate from latency when the host cell is at risk of undergoing cell death.…”

Section: Resultsmentioning

confidence: 99%

Murine Gammaherpesvirus 68 Reactivation from B Cells Requires IRF4 but Not XBP-1

Matar

Rangaswamy

Wakeman

et al. 2014

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Gammaherpesviruses display tropism for B cells and, like all known herpesviruses, exhibit distinct lytic and latent life cycles. One well-established observation among members of the gammaherpesvirus family is the link between viral reactivation from latently infected B cells and plasma cell differentiation. Importantly, a number of studies have identified a potential role for a CREB/ATF family member, X-box binding protein 1 (XBP-1), in trans-activating the immediate early BZLF-1 or BRLF1/gene 50 promoters of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), respectively. XBP-1 is required for the unfolded protein response and has been identified as a critical transcription factor in plasma cells. Here, we demonstrate that XBP-1 is capable of trans-activating the murine gammaherpesvirus 68 (MHV68) RTA promoter in vitro, consistent with previous observations for EBV and KSHV. However, we show that in vivo there does not appear to be a requirement for XBP-1 expression in B cells for virus reactivation. The MHV68 M2 gene product under some experimental conditions plays an important role in virus reactivation from B cells. M2 has been shown to drive B cell differentiation to plasma cells, as well as interleukin-10 (IL-10) production, both of which are dependent on M2 induction of interferon regulatory factor 4 (IRF4) expression. IRF4 is required for plasma cell differentiation, and consistent with a role for plasma cells in MHV68 reactivation from B cells, we show that IRF4 expression in B cells is required for efficient reactivation of MHV68 from splenocytes. Thus, the latter analyses are consistent with previous studies linking plasma cell differentiation to MHV68 reactivation from B cells. The apparent independence of MHV68 reactivation from XBP-1 expression in plasma cells may reflect redundancy among CREB/ATF family members or the involvement of other plasma cell-specific transcription factors. Regardless, these findings underscore the importance of in vivo studies in assessing the relevance of observations made in tissue culture models. IMPORTANCEAll known herpesviruses establish a chronic infection of their respective host, persisting for the life of the individual. A critical feature of these viruses is their ability to reactivate from a quiescent form of infection (latency) and generate progeny virus. In the case of gammaherpesviruses, which are associated with the development of lymphoproliferative disorders, including lymphomas, reactivation from latently infected B lymphocytes occurs upon terminal differentiation of these cells to plasma cellsthe cell type that produces antibodies. A number of studies have linked a plasma cell transcription factor, XBP-1, to the induction of gammaherpesvirus reactivation, and we show here that indeed in tissue culture models this cellular transcription factor can trigger expression of the murine gammaherpesvirus gene involved in driving virus reactivation. However, surprisingly, when we examined the role of XBP-1 in the setting of infe...

“…Thus, transferring latently infected B cells into naïve recipient mice (which lack specific antiviral immunity) allows virus reactivation in the donor B cells and leads to de novo infection of host B cells, confirming clinical observations of virus-seronegative patients becoming seropositive after organ transplant (7). Stimulation of latently infected B cells by treatment with antiIg/anti-CD40 antibodies or various Toll-like receptor ligands has been demonstrated to induce ␥HV68 reactivation from latency (5,14,18), suggesting that inadvertent B cell stimulation during the transfer process could facilitate virus reactivation. However, the kinetics of reactivation after transfer argued against the likelihood that lytic virus or reactivating B cells were transferred, as we were unable to detect lytic virus in the spleens and lungs of recipient mice until 7 days after transfer (Fig.…”

Section: Robust Infection Of Donor-derived B Cells Following Reactivamentioning

confidence: 75%

De Novo Infection of B Cells during Murine Gammaherpesvirus 68 Latency

Freeman

Burkum

Yager

et al. 2011

The mechanisms by which gammaherpesviruses maintain latency are unclear. Here we used a murine gammaherpesvirus model to show that previously uninfected B cells in immunocompetent mice can acquire virus during latency. In vivo depletion of T cells allowed viral reactivation, as measured by increased viral loads, but not enhanced transfer of virus to new cells. In the absence of both immune T cells and antibody following the transfer of latently infected cells into naïve animals, there was robust infection of new B cells. These data confirm that both T cells and antibody contribute to the control of gammaherpesvirus latency, reactivation, and spread.The hallmark of herpesvirus (HV) infections is the establishment of latency, a lifelong condition characterized by persistent viral genome maintenance and restricted, low-level viral gene expression. The ability to reactivate from latency, reenter the lytic life cycle, and generate infectious virions is responsible for many of the secondary pathological consequences of HV infections, including autoimmunity, cancer, immunoproliferative disorders, recrudescent skin lesions, and transplant rejection (15,19). An unresolved issue in the field of herpesvirology is how latent infections are maintained in the host over time. In the case of alphaherpesvirus (␣HV) latency, the viral genomes reside in nonreplicating cells such as neurons, and so latency is likely propagated by the mere survival of latently infected cells, with periodic de novo infections following reactivation events. ␤HV and ␥HV infections are different, as they establish latency in immune system cells, which, unlike neurons, not only can proliferate but also can express high levels of major histocompatibility complex (MHC) class I and are targets for T cell immunity. Since long-term latency persists even in the presence of functional virus-specific immunity, it is crucial to understand how viral latency is propagated in an immunocompetent host in order to develop rational strategies to control ␥HV infections.Mechanisms for ␥HV latency persistence include viral immune evasion strategies; proliferation of latently infected B cells, in which latent viral episomes are replicated and transferred to the daughter cells; direct cell-to-cell transfer of infectious virus to naïve cells, in the absence of cell-free virus; and asymptomatic shedding of lytic virus, leading to the infection of new B cells. There is evidence in the literature for each of these mechanisms. For example, Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) protein is associated with driving the proliferation of infected B cells (8). Furthermore, it has been shown that efficient EBV infection of epithelial cell lines requires cell-to-cell contact with virally infected cells in cocultures (3, 10), a process that is refractory to inhibition by blocking antibodies (Abs), suggesting that cell-free virus is not required for infection (22). In one study, long-term valacyclovir treatment did not affect the number of EBV DNA copies per B cell but did red...