Browsing by Subject "Herpesvirus 4, Human"

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    Analysis of Epstein-Barr virus-regulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1-mediated NF-κB activation.
    (Journal of virology, 2012-10) Price, Alexander M; Tourigny, Jason P; Forte, Eleonora; Salinas, Raul E; Dave, Sandeep S; Luftig, Micah A
    Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-κB activity. It was previously thought that the major viral protein responsible for NF-κB activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-κB activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-κB activation during the first week after infection increased the transformation efficiency, while early NF-κB inhibition had no effect on transformation. Rather, inhibition of NF-κB was not toxic to EBV-infected cells until LMP1 levels and NF-κB activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.
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    Epstein-Barr virus induces global changes in cellular mRNA isoform usage that are important for the maintenance of latency.
    (Journal of virology, 2013-11) Homa, Nicholas J; Salinas, Raul; Forte, Eleonora; Robinson, Timothy J; Garcia-Blanco, Mariano A; Luftig, Micah A
    Oncogenic viruses promote cell proliferation through the dramatic reorganization of host transcriptomes. In addition to regulating mRNA abundance, changes in mRNA isoform usage can have a profound impact on the protein output of the transcriptome. Using Epstein-Barr virus (EBV) transformation of primary B cells, we have studied the ability of an oncogenic virus to alter the mRNA isoform profile of its host. Using the algorithm called SplicerEX with two complementary Affymetrix microarray platforms, we uncovered 433 mRNA isoform changes regulated by EBV during B-cell transformation. These changes were largely orthogonal with the 2,163 mRNA abundance changes observed during transformation, such that less than one-third of mRNAs changing at the level of isoform also changed in overall abundance. While we observed no preference for a mechanistic class of mRNA isoform change, we detected a significant shortening of 3' untranslated regions and exclusion of cassette exons in EBV-transformed cells relative to uninfected B cells. Gene ontology analysis of the mRNA isoform changes revealed significant enrichment in nucleic acid binding proteins. We validated several of these isoform changes and were intrigued by those in two mRNAs encoding the proteins XBP1 and TCF4, which have both been shown to bind and activate the promoter of the major EBV lytic trans-activator BZLF1. Our studies indicate that EBV latent infection promotes the usage of mRNA isoforms of XBP1 and TCF4 that restrict BZLF1 activation. Therefore, characterization of global changes in mRNA isoform usage during EBV infection identifies a new mechanism for the maintenance of latent infection.
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    Human Immunodeficiency Virus Type 1 Persistence Following Systemic Chemotherapy for Malignancy.
    (The Journal of infectious diseases, 2017-07) Henrich, Timothy J; Hobbs, Kristen S; Hanhauser, Emily; Scully, Eileen; Hogan, Louise E; Robles, Yvonne P; Leadabrand, Kaitlyn S; Marty, Francisco M; Palmer, Christine D; Jost, Stephanie; Körner, Christian; Li, Jonathan Z; Gandhi, Rajesh T; Hamdan, Ayad; Abramson, Jeremy; LaCasce, Ann S; Kuritzkes, Daniel R

    Background

    Systemic chemotherapies for various malignancies have been shown to significantly, yet transiently, decrease numbers of CD4+ T lymphocytes, a major reservoir for human immunodeficiency virus type 1 (HIV-1) infection. However, little is known about the impact of cytoreductive chemotherapy on HIV-1 reservoir dynamics, persistence, and immune responses.

    Methods

    We investigated the changes in peripheral CD4+ T-cell-associated HIV-1 DNA and RNA levels, lymphocyte activation, viral population structure, and virus-specific immune responses in a longitudinal cohort of 15 HIV-1-infected individuals receiving systemic chemotherapy or subsequent autologous stem cell transplantation for treatment of hematological malignancies and solid tumors.

    Results

    Despite a transient reduction in CD4+ T cells capable of harboring HIV-1, a 1.7- and 3.3-fold increase in mean CD4+ T-cell-associated HIV-1 RNA and DNA, respectively, were observed months following completion of chemotherapy in individuals on antiretroviral therapy. We also observed changes in CD4+ T-cell population diversity and clonal viral sequence expansion during CD4+ T-cell reconstitution following chemotherapy cessation. Finally, HIV-1 DNA was preferentially, and in some cases exclusively, detected in cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-responsive CD4+ T cells following chemotherapy.

    Conclusions

    Expansion of HIV-infected CMV/EBV-specific CD4 + T cells may contribute to maintenance of the HIV DNA reservoir following chemotherapy.
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    SplicerEX: a tool for the automated detection and classification of mRNA changes from conventional and splice-sensitive microarray expression data.
    (RNA (New York, N.Y.), 2012-08) Robinson, Timothy J; Forte, Eleonora; Salinas, Raul E; Puri, Shaan; Marengo, Matthew; Garcia-Blanco, Mariano A; Luftig, Micah A
    The key postulate that one gene encodes one protein has been overhauled with the discovery that one gene can generate multiple RNA transcripts through alternative mRNA processing. In this study, we describe SplicerEX, a novel and uniquely motivated algorithm designed for experimental biologists that (1) detects widespread changes in mRNA isoforms from both conventional and splice sensitive microarray data, (2) automatically categorizes mechanistic changes in mRNA processing, and (3) mitigates known technological artifacts of exon array-based detection of alternative splicing resulting from 5' and 3' signal attenuation, background detection limits, and saturation of probe set signal intensity. In this study, we used SplicerEX to compare conventional and exon-based Affymetrix microarray data in a model of EBV transformation of primary human B cells. We demonstrated superior detection of 3'-located changes in mRNA processing by the Affymetrix U133 GeneChip relative to the Human Exon Array. SplicerEX-identified exon-level changes in the EBV infection model were confirmed by RT-PCR and revealed a novel set of EBV-regulated mRNA isoform changes in caspases 6, 7, and 8. Finally, SplicerEX as compared with MiDAS analysis of publicly available microarray data provided more efficiently categorized mRNA isoform changes with a significantly higher proportion of hits supported by previously annotated alternative processing events. Therefore, SplicerEX provides an important tool for the biologist interested in studying changes in mRNA isoform usage from conventional or splice-sensitive microarray platforms, especially considering the expansive amount of archival microarray data generated over the past decade. SplicerEX is freely available upon request.
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    The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells.
    (Journal of virology, 2012-06) Forte, Eleonora; Salinas, Raul E; Chang, Christina; Zhou, Ting; Linnstaedt, Sarah D; Gottwein, Eva; Jacobs, Cassandra; Jima, Dereje; Li, Qi-Jing; Dave, Sandeep S; Luftig, Micah A
    Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.