Amy L. Adamson

Epstein-Barr Virus Immediate-Early Protein BZLF1 Is SUMO-1 Modified and Disrupts Promyelocytic Leukemia Bodies

ABSTRACT Although the immediate-early proteins of both herpes simplex virus (HSV) and cytomegalovirus (CMV) are known to modify promyelocytic leukemia (PML) (ND10) bodies in the nucleus of the host cell, it has been unclear whether lytic infection with gamma herpesviruses induces a similar effect. The PML protein is induced by interferon, involved in major histocompatibility complex class I presentation, and necessary for certain types of apoptosis. Therefore, it is likely that PML bodies function in an antiviral capacity. SUMO-1 modification of PML is known to be required for the formation of PML bodies. To examine whether Epstein-Barr virus (EBV) lytic replication interferes with PML bodies, we expressed the EBV immediate-early genes BZLF1 (Z) and BRLF1 (R) in EBV-positive cell lines and examined PML localization. Both Z and R expression resulted in PML dispersion in EBV-positive cells. Z but not R expression is sufficient to disrupt PML bodies in EBV-negative cell lines. We show that dispersion of PML bodies by Z requires a portion of the transcriptional activation domain of Z but not the DNA-binding function. As was previously reported for the HSV-1 ICP0 and CMV IE1 proteins, Z reduces the amount of SUMO-1-modified PML. We also found that Z itself is SUMO-1 modified (through amino acid 12) and that Z competes with PML for limiting amounts of SUMO-1. These results suggest that disruption of PML bodies is important for efficient lytic replication of EBV. Furthermore, Z may potentially alter the function of a variety of cellular proteins by inhibiting SUMO-1 modification.

Epstein-Barr Virus Immediate-Early Proteins BZLF1 and BRLF1 Activate the ATF2 Transcription Factor by Increasing the Levels of Phosphorylated p38 and c-Jun N-Terminal Kinases

ABSTRACT Expression of either Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) or BRLF1 (R) is sufficient to convert EBV infection from the latent to lytic form. Disruption of viral latency requires transcriptional activation of the Z and R promoters. The Z and R proteins are transcriptional activators, and each immediate-early protein activates expression of the other immediate-early protein. Z activates the R promoter through a direct binding mechanism. However, R does not bind directly to the Z promoter. In this study, we demonstrate that the ZII element (a cyclic AMP response element site) in the Z promoter is required for efficient activation by R. The ZII element has been shown to be important for induction of lytic EBV infection by tetradecanoyl phorbol acetate and surface immunoglobulin cross-linking and is activated by Z through an indirect mechanism. We demonstrate that both R and Z activate the cellular stress mitogen-activated protein (MAP) kinases, p38 and JNK, resulting in phosphorylation (and activation) of the cellular transcription factor ATF2. Furthermore, we show that the ability of R to induce lytic EBV infection in latently infected cells is significantly reduced by inhibition of either the p38 kinase or JNK pathways. In contrast, inhibition of stress MAP kinase pathways does not impair the ability of Z expression vectors to disrupt viral latency, presumably because expression of Z under the control of a strong heterologous promoter bypasses the need to activate Z transcription. Thus, both R and Z can activate the Z promoter indirectly by inducing ATF2 phosphorylation, and this activity appears to be important for R-induced disruption of viral latency.

Glycolytic control of vacuolar-type ATPase activity: a mechanism to regulate influenza viral infection.

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection.

Modeling early Epstein-Barr virus infection in Drosophila melanogaster : The BZLF1 protein

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several forms of cancer, including lymphomas and nasopharyngeal carcinoma. The EBV immediate-early protein BZLF1 functions as a transcriptional activator of EBV early gene expression and is essential for the viral transition between latent and lytic replication. In addition to its role in the EBV life cycle, BZLF1 (Z) also has profound effects upon the host cellular environment, including disruption of cell cycle regulation, signal transduction pathways, and transcription. In an effort to understand the nature of Z interactions with the host cellular environment, we have developed a Drosophila model of early EBV infection, where we have expressed Z in the Drosophila eye. Using this system, we have identified a highly conserved interaction between the Epstein-Barr virus Z protein and shaven, a Drosophila homolog of the human Pax2/5/8 family of genes. Pax5 is a well-characterized human gene involved with B-cell development. The B-cell-specific Pax5 also promotes the transcription of EBV latent genes from the EBV Wp promoter. Our work clearly demonstrates that the Drosophila system is an appropriate and powerful tool for identifying the underlying genetic networks involved in human infectious disease.

Inhibition of mTORC1 inhibits lytic replication of Epstein-Barr virus in a cell-type specific manner

BackgroundEpstein-Barr virus is a human herpesvirus that infects a majority of the human population. Primary infection of Epstein-Barr virus (EBV) causes the syndrome infectious mononucleosis. This virus is also associated with several cancers, including Burkitt’s lymphoma, post-transplant lymphoproliferative disorder and nasopharyngeal carcinoma. As all herpesvirus family members, EBV initially replicates lytically to produce abundant virus particles, then enters a latent state to remain within the host indefinitely.MethodsThrough a genetic screen in Drosophila, we determined that reduction of Drosophila Tor activity altered EBV immediate-early protein function. To further investigate this finding, we inhibited mTOR in EBV-positive cells and investigated subsequent changes to lytic replication via Western blotting, flow cytometry, and quantitative PCR. The student T-test was used to evaluate significance.ResultsmTOR, the human homolog of Drosophila Tor, is an important protein at the center of a major signaling pathway that controls many aspects of cell biology. As the EBV immediate-early genes are responsible for EBV lytic replication, we examined the effect of inhibition of mTORC1 on EBV lytic replication in human EBV-positive cell lines. We determined that treatment of cells with rapamycin, which is an inhibitor of mTORC1 activity, led to a reduction in the ability of B cell lines to undergo lytic replication. In contrast, EBV-positive epithelial cell lines underwent higher levels of lytic replication when treated with rapamycin.ConclusionsOverall, the responses of EBV-positive cell lines vary when treated with mTOR inhibitors, and this may be important when considering such inhibitors as anti-cancer therapeutic agents.

A Drosophila Model for Genetic Analysis of Influenza Viral/Host Interactions

Influenza viruses impose a constant threat to vertebrates susceptible to this family of viruses. We have developed a new tool to study virus–host interactions that play key roles in viral replication and to help identify novel anti-influenza drug targets. Via the UAS/Gal4 system we ectopically expressed the influenza virus M2 gene in Drosophila melanogaster and generated dose-sensitive phenotypes in the eye and wing. We have confirmed that the M2 proton channel is properly targeted to cell membranes in Drosophila tissues and functions as a proton channel by altering intracellular pH. As part of the efficacy for potential anti-influenza drug screens, we have also demonstrated that the anti-influenza drug amantadine, which targets the M2 proton channel, suppressed the UAS-M2 mutant phenotype when fed to larvae. In a candidate gene screen we identified mutations in components of the vacuolar V1V0 ATPase that modify the UAS-M2 phenotype. Importantly, in this study we demonstrate that Drosophila genetic interactions translate directly to physiological requirements of the influenza A virus for these components in mammalian cells. Overexpressing specific V1 subunits altered the replication capacity of influenza virus in cell culture and suggests that drugs targeting the enzyme complex via these subunits may be useful in anti-influenza drug therapies. Moreover, this study adds credence to the idea of using the M2 “flu fly” to identify new and previously unconsidered cellular genes as potential drug targets and to provide insight into basic mechanisms of influenza virus biology.

Epstein-Barr Virus BZLF1 Protein Binds to Mitotic Chromosomes

ABSTRACT Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and is associated with several types of cancers, including nasopharyngeal carcinoma and Burkitts lymphoma. An EBV protein that plays an integral role during lytic replication is the immediate-early protein BZLF1. Our laboratory has found that BZLF1 (Z) localizes to host chromosomes during mitosis. Two Z-interacting proteins are also found localized to mitotic chromosomes in the presence of Z. The association between Z and mitotic chromosomes may lead to the sequestering of Z-interacting proteins within the cell and potentially cause an alteration of chromosome compaction or chromatin structure.

A Study of Epstein-Barr Virus BRLF1 Activity in a Drosophila Model System

Epstein-Barr virus, a member of the herpesvirus family, infects a large majority of the human population and is associated with several diseases, including cancer. We have created Drosophila model systems to study the interactions between host cellular proteins and the Epstein-Barr virus (EBV) immediate-early genes BRLF1 and BZLF1. BRLF1 and BZLF1 function as transcription factors for viral transcription and are also potent modifiers of host cell activity. Here we have used our model systems to identify host cell genes whose proteins modulate BRLF1 and BZLF1 functions. Via our GMR-R model system, we have found that BRLF1 expression results in overproliferation of fly tissue, unlike BZLF1, and does so through the interaction with known tumor suppressor genes. Through an additional genetic screen, we have identified several Drosophila genes, with human homologs, that may offer further insights into the pathways that BRLF1 interacts with in order to promote EBV replication.