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Search Results: 1 - 10 of 155534 matches for " Samuel H. Speck "
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Tracking Murine Gammaherpesvirus 68 Infection of Germinal Center B Cells In Vivo
Christopher M. Collins, Samuel H. Speck
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0033230
Abstract: Infection of mice with murine gammaherpesvirus 68 (MHV68) provides a tractable small animal model to study various aspects of persistent gammaherpesvirus infection. We have previously utilized a transgenic MHV68 that expresses enhanced yellow fluorescent protein (EYFP) to identify infected cells. While this recombinant MHV68 has been useful for identifying infected cell populations by flow cytometry, it has been suboptimal for identification of infected cells in tissue sections due to the high solubility of EYFP. Efficient detection of EYFP expressed from the MHV68 genome in tissue sections requires fixation of whole organs prior to sectioning, which frequently leads to over-fixation of some cellular antigens precluding their detection. To circumvent this issue, we describe the generation and characterization of a transgenic MHV68 harboring a fusion gene composed of the EYFP coding sequence fused to the histone H2B open reading frame. Because the H2bYFP fusion protein is tightly bound in nucleosomes in the nucleus it does not freely diffuse out of unfixed tissue sections, and thus eliminates the need for tissue fixation. We have used the MHV68-H2bYFP recombinant virus to assess the location and distribution of virus infected B cells in germinal centers during the peak of MHV68 latency in vivo. These analyses show that the physical location of distinct populations of infected germinal center B cells correlates well with their surface phenotype. Furthermore, analysis of the distribution of virus infection within germinal center B cell populations revealed that ca. 70% of MHV68 infected GC B cells are rapidly dividing centroblasts, while ca. 20% have a clear centrocyte phenotype. Finally, we have shown that marking of infected cells with MHV68-H2bYFP is extended long after the onset of latency – which should facilitate studies to track MHV68 latently infected cells at late times post-infection.
A Tissue Culture Model of Murine Gammaherpesvirus Replication Reveals Roles for the Viral Cyclin in Both Virus Replication and Egress from Infected Cells
Francine M. Scott, Samuel H. Speck
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0093871
Abstract: Passage through the eukaryotic cell cycle is regulated by the activity of cyclins and their cyclin-dependent kinase partners. Rhadinoviruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), encode a viral homologue of mammalian D-type cyclins. In MHV68, the interaction of the viral cyclin with its CDK partners is important for acute replication in the lungs following low dose inoculation. Attempts to further study this requirement in vitro have been limited by the lack of available tissue culture models that mimic the growth defect observed in vivo. It is hypothesized that analysis of virus replication in a cell line that displays properties of primary airway epithelium, such as the ability to polarize, might provide a suitable environment to characterize the role of the v-cyclin in virus replication. We report here MHV68 replication in the rat lung cell line RL-65, a non-transformed polarizable epithelial cell line. These analyses reveal a role for the v-cyclin in both virus replication, as well as virus egress from infected cells. As observed for acute replication in vivo, efficient replication in RL-65 cells requires CDK binding. However, we show that the KSHV v-cyclin (K-cyclin), which utilizes different CDK partners (CDK4 and CDK6) than the MHV68 v-cyclin (CDK2 and CDC2), can partially rescue the replication defect observed with a v-cyclin null mutant – both in vitro and in vivo. Finally, we show that MHV68 is shed from both the apical and basolateral surfaces of polarized RL-65 cells. In summary, the RL-65 cell line provides an attractive in vitro model that mimics critical aspects of MHV68 replication in the lungs.
Expansion of Murine Gammaherpesvirus Latently Infected B Cells Requires T Follicular Help
Christopher M. Collins,Samuel H. Speck
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1004106
Abstract: X linked lymphoproliferative disease (XLP) is an inherited immunodeficiency resulting from mutations in the gene encoding the slam associated protein (SAP). One of the defining characteristics of XLP is extreme susceptibility to infection with Epstein-Barr virus (EBV), a gammaherpesvirus belonging to the genus Lymphocryptovirus, often resulting in fatal infectious mononucleosis (FIM). However, infection of SAP deficient mice with the related Murine gammaherpesvirus 68 (MHV68), a gammaherpesvirus in the genus Rhadinovirus, does not recapitulate XLP. Here we show that MHV68 inefficiently establishes latency in B cells in SAP deficient mice due to insufficient CD4 T cell help during the germinal center response. Although MHV68 infected B cells can be found in SAP-deficient mice, significantly fewer of these cells had a germinal center phenotype compared to SAP-sufficient mice. Furthermore, we show that infected germinal center B cells in SAP-deficient mice fail to proliferate. This failure to proliferate resulted in significantly lower viral loads, and likely accounts for the inability of MHV68 to induce a FIM-like syndrome. Finally, inhibiting differentiation of T follicular helper (TFH) cells in SAP-sufficient C57Bl/6 mice resulted in decreased B cell latency, and the magnitude of the TFH response directly correlated with the level of infection in B cells. This requirement for CD4 T cell help during the germinal center reaction by MHV68 is in contrast with EBV, which is thought to be capable of bypassing this requirement by expressing viral proteins that mimic signals provided by TFH cells. In conclusion, the outcome of MHV68 infection in mice in the setting of loss of SAP function is distinct from that observed in SAP-deficient patients infected with EBV, and may identify a fundamental difference between the strategies employed by the rhadinoviruses and lymphocryptoviruses to expand B cell latency during the early phase of infection.
Murine Gammaherpesvirus M2 Protein Induction of IRF4 via the NFAT Pathway Leads to IL-10 Expression in B Cells
Udaya S. Rangaswamy,Samuel H. Speck
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1003858
Abstract: Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner – leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4). Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA) resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4 – a key player in plasma cell differentiation – which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation.
Interleukin 21 Signaling in B Cells Is Required for Efficient Establishment of Murine Gammaherpesvirus Latency
Christopher M. Collins?,Samuel H. Speck
PLOS Pathogens , 2015, DOI: 10.1371/journal.ppat.1004831
Abstract: The human gammaherpesviruses take advantage of normal B cell differentiation pathways to establish life-long infection in memory B cells. Murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice also leads to life-long infection in memory B cells. To gain access to the memory B cell population, MHV68 infected B cells pass through the germinal center reaction during the onset of latency and require signals from T follicular helper (TFH) cells for proliferation. Interleukin 21 (IL-21), one of the secreted factors produced by TFH cells, plays an important role in both the maintenance of the germinal center response as well as in the generation of long-lived plasma cells. Using IL-21R deficient mice, we show that IL-21 signaling is required for efficient establishment of MHV68 infection. In the absence of IL-21 signaling, fewer infected splenocytes are able to gain access to either the germinal center B cell population or the plasma cell population – the latter being a major site of MHV68 reactivation. Furthermore, the germinal center B cell population in IL-21R-/- mice is skewed towards the non-proliferating centrocyte phenotype, resulting in reduced expansion of infected B cells. Additionally, the reduced frequency of infected plasma cells results in a significant reduction in the frequency of splenocytes capable of reactivating virus. This defect in establishment of MHV68 infection is intrinsic to B cells, as MHV68 preferentially establishes infection in IL-21R sufficient B cells in mixed bone marrow chimeric mice. Taken together, these data indicate that IL-21 signaling plays multiple roles during establishment of MHV68 infection, and identify IL-21 as a critical TFH cell-derived factor for efficient establishment of gammaherpesvirus B cell latency.
The MHV68 M2 Protein Drives IL-10 Dependent B Cell Proliferation and Differentiation
Andrea M. Siegel,Jeremy H. Herskowitz,Samuel H. Speck
PLOS Pathogens , 2008, DOI: 10.1371/journal.ppat.1000039
Abstract: Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1α. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10?/? B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells—perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis—identifying a strategy that appears to be conserved between at least EBV and MHV68.
Characterization of Omental Immune Aggregates during Establishment of a Latent Gammaherpesvirus Infection
Kathleen S. Gray, Christopher M. Collins, Samuel H. Speck
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043196
Abstract: Herpesviruses are characterized by their ability to establish lifelong latent infection. The gammaherpesvirus subfamily is distinguished by lymphotropism, establishing and maintaining latent infection predominantly in B lymphocytes. Consequently, gammaherpesvirus pathogenesis is closely linked to normal B cell physiology. Murine gammaherpesvirus 68 (MHV68) pathogenesis in laboratory mice has been extensively studied as a model system to gain insights into the nature of gammaherpesvirus infection in B cells and their associated lymphoid compartments. In addition to B cells, MHV68 infection of macrophages contributes significantly to the frequency of viral genome-positive cells in the peritoneal cavity throughout latency. The omentum, a sheet of richly-vascularized adipose tissue, resides in the peritoneal cavity and contains clusters of immune cell aggregates termed milky spots. Although the value of the omentum in surgical wound-healing has long been appreciated, the unique properties of this tissue and its contribution to both innate and adaptive immunity have only recently been recognized. To determine whether the omentum plays a role in gammaherpesvirus pathogenesis we examined this site during early MHV68 infection and long-term latency. Following intraperitoneal infection, immune aggregates within the omentum expanded in size and number and contained virus-infected cells. Notably, a germinal-center B cell population appeared in the omentum of infected animals with earlier kinetics and greater magnitude than that observed in the spleen. Furthermore, the omentum harbored a stable frequency of viral genome-positive cells through early and into long-term latency, while removal of the omentum prior to infection resulted in a slight decrease in the establishment of splenic latency following intraperitoneal infection. These data provide the first evidence that the omentum is a site of chronic MHV68 infection that may contribute to the maintenance of chronic infection.
A Persistent Interest in Viruses
Samuel Speck
PLOS Pathogens , 2016, DOI: 10.1371/journal.ppat.1005327
Unbiased Mutagenesis of MHV68 LANA Reveals a DNA-Binding Domain Required for LANA Function In Vitro and In Vivo
Clinton R. Paden,J. Craig Forrest,Scott A. Tibbetts,Samuel H. Speck
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002906
Abstract: The Latency-Associated Nuclear Antigen (LANA), encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses). The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-herpesvirus, murine gammaherpesvirus 68 (MHV68), the LANA homolog (mLANA) is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s), knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR) of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i) replication fitness; (ii) efficiency of latency establishment; and (iii) reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function.
Murine Gamma-herpesvirus Immortalization of Fetal Liver-Derived B Cells Requires both the Viral Cyclin D Homolog and Latency-Associated Nuclear Antigen
Xiaozhen Liang,Clinton R. Paden,Francine M. Morales,Ryan P. Powers,Joshy Jacob,Samuel H. Speck
PLOS Pathogens , 2011, DOI: 10.1371/journal.ppat.1002220
Abstract: Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities.
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