Search

Epstein-Barr virus (EBV) is associated with a number of T-cell diseases, including some peripheral T-cell lymphomas, hemophagocytic lymphohistiocytosis, and chronic active EBV disease. The tropism of EBV for B cells and epithelial cell infection has been well characterized, but infection of T cells has been minimally explored. We have recently shown that the EBV type 2 (EBV-2) strain has the unique ability to infect mature T cells. Utilizing an ex vivo infection model, we sought to understand the viral glycoprotein and cellular receptor required for EBV-2 infection of T cells. Here, using a neutralizing-antibody assay, we found that viral gp350 and complement receptor 2 (CD21) are required for CD3 + T-cell infection. Using the HB5 anti-CD21 antibody clone but not the Bly-4 anti-CD21 antibody clone, we detected expression of CD21 on both CD4 + and CD8 + T cells, with the highest expression on naive CD4 and CD8 + T-cell subsets. Using CRISPR to knock out CD21, we demonstrated that CD21 is necessary for EBV entry into the Jurkat T-cell line. Together, these results indicate that EBV uses the same viral glycoprotein and cellular receptor for both T- and B-cell infection. IMPORTANCE Epstein-Barr virus (EBV) has a well-described tropism for B cells and epithelial cells. Recently, we described the ability of a second strain of EBV, EBV type 2, to infect mature peripheral T cells. Using a neutralizing antibody assay, we determined that EBV uses the viral glycoprotein gp350 and the cellular protein CD21 to gain entry into mature peripheral T cells. CRISPR-Cas9 deletion of CD21 on the Jurkat T-cell line confirmed that CD21 is required for EBV infection. This study has broad implications, as we have defined a function for CD21 on mature peripheral T cells, i.e., as a receptor for EBV. In addition, the requirement for gp350 for T-cell entry has implications for EBV vaccine studies currently targeting the gp350 glycoprotein to prevent EBV-associated diseases., (Copyright © 2020 American Society for Microbiology.)

Exercise capacity is a strong predictor of all-cause mortality. Skeletal muscle mitochondrial respiratory capacity, its biggest contributor, adapts robustly to changes in energy demands induced by contractile activity. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are regulated. Here, we show that exercise training or muscle disuse alters mitochondrial membrane phospholipids including phosphatidylethanolamine (PE). Addition of PE promoted, whereas removal of PE diminished, mitochondrial respiratory capacity. Unexpectedly, skeletal muscle-specific inhibition of mitochondria-autonomous synthesis of PE caused respiratory failure because of metabolic insults in the diaphragm muscle. While mitochondrial PE deficiency coincided with increased oxidative stress, neutralization of the latter did not rescue lethality. These findings highlight the previously underappreciated role of mitochondrial membrane phospholipids in dynamically controlling skeletal muscle energetics and function.

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level., Competing Interests: The following authors, L.M.O., A.K.K., R.E.K., A.N.K., C.B.C, R.R., L.v.D. and E.T.C. have declared that no competing interests exist. I have read the journal's policy and the following authors of this manuscript have the following competing interests: T.C. and B.A. are employees of EMD Millipore and Luminex respectively and have a potential financial conflict of interest.

Many lymphoproliferative disorders (LPDs) are considered "EBV associated" based on detection of the virus in tumor tissue. EBV drives proliferation of LPDs via expression of the viral latent genes and many pre-clinical and clinical studies have shown EBV-associated LPDs can be treated by exploiting the viral life cycle. After a brief review of EBV virology and the natural life cycle within a host we will discuss the importance of the viral gene programs expressed during specific viral phases, as well as within immunocompetent vs. immunocompromised hosts and corresponding EBV-associated LPDs. We will then review established and emerging treatment approaches for EBV-associated LPDs based on EBV gene expression programs. Patients with EBV-associated LPDs can have a poor performance status, multiple comorbidities, and/or are immunocompromised from organ transplantation, autoimmune disease, or other congenital or acquired immunodeficiency making them poor candidates to receive intensive cytotoxic chemotherapy. With the emergence of EBV-directed therapy there is hope that we can devise more effective therapies that confer milder toxicity.

Epstein-Barr virus (EBV) has been classified into two strains, EBV type 1 (EBV-1) and EBV type 2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B cells in culture while EBV-2 is poorly transforming. The differing abilities to immortalize B cells in vitro suggest that in vivo these viruses likely use alternative approaches to establish latency. Indeed, we recently reported that EBV-2 has a unique cell tropism for T cells, infecting T cells in culture and in healthy Kenyan infants, strongly suggesting that EBV-2 infection of T cells is a natural part of the EBV-2 life cycle. However, limitations of human studies hamper further investigation into how EBV-2 utilizes T cells. Therefore, BALB/c Rag2 null IL2rγ null SIRPα humanized mice were utilized to develop an EBV-2 in vivo model. Infection of humanized mice with EBV-2 led to infection of both T and B cells, unlike infection with EBV-1, in which only B cells were infected. Gene expression analysis demonstrated that EBV-2 established a latency III infection with evidence of ongoing viral reactivation in both B and T cells. Importantly, EBV-2-infected mice developed tumors resembling diffuse large B cell lymphoma (DLBCL). These lymphomas had morphological features comparable to those of EBV-1-induced DLBCLs, developed at similar rates with equivalent frequencies, and expressed a latency III gene profile. Thus, despite the impaired ability of EBV-2 to immortalize B cells in vitro , EBV-2 efficiently induces lymphomagenesis in humanized mice. Further research utilizing this model will enhance our understanding of EBV-2 biology, the consequence of EBV infection of T cells, and the capacity of EBV-2 to drive lymphomagenesis. IMPORTANCE EBV is a well-established B cell-tropic virus. However, we have recently shown that the EBV type 2 (EBV-2) strain also infects primary T cells in culture and in healthy Kenyan children. This finding suggests that EBV-2, unlike the well-studied EBV-1 strain, utilizes the T cell compartment to persist. As EBV is human specific, studies to understand the role of T cells in EBV-2 persistence require an in vivo model. Thus, we developed an EBV-2 humanized mouse model, utilizing immunodeficient mice engrafted with human cord blood CD34 + stem cells. Characterization of the EBV-2-infected humanized mice established that both T cells and B cells are infected by EBV-2 and that the majority of infected mice develop a B cell lymphoma resembling diffuse large B cell lymphoma. This new in vivo model can be utilized for studies to enhance our understanding of how EBV-2 infection of T cells contributes to persistence and lymphomagenesis., (Copyright © 2018 American Society for Microbiology.)

Barth Syndrome (BTHS) is an X-linked recessive disorder characterized by cardiomyopathy and muscle weakness. The underlying cause of BTHS is a mutation in the tafazzin (TAZ) gene, a key enzyme of cardiolipin biosynthesis. The lack of CL arising from loss of TAZ function results in destabilization of the electron transport system, promoting oxidative stress that is thought to contribute to development of cardioskeletal myopathy. Indeed, in vitro studies demonstrate that mitochondria-targeted antioxidants improve contractile capacity in TAZ-deficient cardiomyocytes. The purpose of the present study was to determine if resolving mitochondrial oxidative stress would be sufficient to prevent cardiomyopathy and skeletal myopathy in vivo using a mouse model of BTHS. To this end we crossed mice that overexpress catalase in the mitochondria (MCAT mice) with TAZ-deficient mice (TAZKD) to produce TAZKD mice that selectively overexpress catalase in the mitochondria (TAZKD+MCAT mice). TAZKD+MCAT mice exhibited decreased mitochondrial H 2 O 2 emission and lipid peroxidation compared to TAZKD littermates, indicating decreased oxidative stress. Despite the improvements in oxidative stress, TAZKD+MCAT mice developed cardiomyopathy and mild muscle weakness similar to TAZKD littermates. These findings indicate that resolving oxidative stress is not sufficient to suppress cardioskeletal myopathy associated with BTHS., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Background: Palatine tonsils are principally B cell organs that are the initial line of defense against many oral pathogens, as well as the site of infection for others. While the size of palatine tonsils changes greatly in the first five years of life, the cellular changes during this period are not well studied. Epstein Barr virus (EBV) is a common orally transmitted virus that infects tonsillar B cells. Naïve B cells are thought to be the target of primary infection with EBV in vivo, suggesting that they are targeted by the virus. EBV enters B cells through CD21, but studies of older children and adults have not shown differences in surface CD21 between naïve B cells and other tonsil B cell populations., Methods: In this study, we used an 11-color flow cytometry panel to detail the changes in B cell subpopulations in human tonsils over the first five years of life from 33 healthy US children., Results: We provide reference ranges for tonsil B cell subpopulations over this age range. We show that the frequency of naïve tonsil B cells decreases over the early years of life, and that naïve B cells expressed higher surface levels of CD21 relative to other tonsil B cell populations., Conclusions: We show that young children have a higher frequency of naïve tonsil B cells, and importantly that these cells express increased surface EBV receptor, suggesting that young children have a larger pool of cells that can be infected by the virus. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: The 2 strains of Epstein-Barr virus (EBV), EBV type 1 (EBV-1) and EBV-2, differ in latency genes, suggesting that they use distinct mechanisms to establish latency. We previously reported that EBV-2 infects T cells in vitro. In this study, we tested the possibility that EBV-2 infects T cells in vivo., Methods: Purified T-cell fractions isolated from children positive for EBV-1 or EBV-2 and their mothers were examined for the presence of EBV and for EBV type., Results: We detected EBV-2 in all T-cell samples obtained from EBV-2-infected children at 12 months of age, with some children retaining EBV-2-positive T cells through 24 months of age, suggesting that EBV-2 persists in T cells. We were unable to detect EBV-2 in T-cell samples from mothers but could detect EBV-2 in samples of their breast milk and saliva., Conclusions: These data suggest that EBV-2 uses T cells as an additional latency reservoir but that, over time, the frequency of infected T cells may drop below detectable levels. Alternatively, EBV-2 may establish a prolonged transient infection in the T-cell compartment. Collectively, these novel findings demonstrate that EBV-2 infects T cells in vivo and suggest EBV-2 may use the T-cell compartment to establish latency., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: We previously reported that infants in Kenya were infected with Epstein-Barr virus (EBV) at <6 months of age, suggesting that mothers were the likely source of transmissible virus to the infant. In this study, we investigated whether breast milk contained infectious EBV and the role of malaria in EBV shedding in breast milk., Methods: Breast milk samples were obtained from Kenyan mothers at postpartum weeks 6, 10, 14, and 18 and analyzed for presence of infectious EBV., Results: We found that the prevalence of EBV DNA and the mean EBV load were significantly higher at 6 weeks and decreased through postpartum week 18 (P < .0001). High EBV load in breast milk correlated with mothers who had Plasmodium falciparum malaria at delivery. To determine whether viral DNA was encapsidated, breast milk samples were treated with DNAse before DNA extraction. Sixty percent of samples were DNAse resistant, suggesting that the viral DNA in breast milk was encapsidated. Next, we exposed peripheral blood mononuclear cells to breast milk supernatant, which resulted in the generation of EBV-positive lymphoblastoid cell lines, indicating that the virus in breast milk was infectious., Conclusions: Our data suggest that breast milk contains infectious EBV and is a potential source of viral transmission to infants living in malaria-endemic regions., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: 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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: 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., Importance: Gammaherpesviruses such as EBV and KSHV are widespread pathogens that establish lifelong infections and are associated with the development of numerous types of diseases, including cancer. Gammaherpesviruses encode many small noncoding RNAs called microRNAs (miRNAs). It is predicted that gammaherpesvirus miRNAs facilitate infection and disease by suppressing host target transcripts involved in a wide range of key cellular pathways; however, the precise contribution that these regulatory RNAs make to in vivo virus infection and pathogenesis is unknown. Here, we generated a mutated form of murine gammaherpesvirus (MHV68) to dissect the function of gammaherpesvirus miRNAs in vivo. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. Moreover, the MHV68 miRNAs were essential for the development of virus-associated pneumonia, implicating them as a critical component of gammaherpesvirus-associated disease., (Copyright © 2014 Feldman et al.)

Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the recurrent infection and vBcl-2-mediated survival of developing B cells.

Diabetes is a major risk factor for cardiovascular disease and is associated with increased intimal thickening and accelerated vascular smooth muscle cell (VSMC) proliferation. We measured the expression of two microRNAs that promote intimal thickening, miR-221/222, and mRNA encoding a downstream target, p27(Kip1), in internal mammary artery (IMA) segments collected from 37 subjects undergoing coronary artery bypass grafting. The segments were stratified into three groups: non-diabetic subjects (ND), diabetic subjects not on metformin (DMMet-), and diabetic subjects on metformin (DMMet+). The DMMet- group exhibited a significant increase in miR-221/222 and decrease in p27(Kip1) mRNA compared to both the ND and DMMet+ groups. miR-221/222 levels inversely correlated with metformin dose. VSMCs isolated from the IMAs of the DMMet- group proliferate at a faster rate than those of the ND and DMMet+ groups. Further studies into the importance of miR-221/222 in the increased intimal thickening observed in diabetic subjects is warranted., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68; also known as gammaherpesvirus 68 [γHV68] or murine herpesvirus 4 [MuHV-4]), establish lifelong latency in the resting memory B cell compartment. However, little is known about how this reservoir of infected mature B cells is maintained for the life of the host. In the context of a normal immune system, the mature B cell pool is naturally maintained by the renewable populations of developing B cells that arise from hematopoiesis. Thus, recurrent infection of these developing B cell populations could allow the virus continual access to the B cell lineage and, subsequent to differentiation, the memory B cell compartment. To begin to address this hypothesis, we examined whether MHV68 establishes latency in developing B cells during a normal course of infection. In work described here, we demonstrate the presence of viral genome in bone marrow pro-pre-B cells and immature B cells during early latency and immature B cells during long-term latency. Further, we show that transitional B cells in the spleen are latently infected and express the latency-associated nuclear antigen (LANA) throughout chronic infection. Because developing B cells normally exhibit a short life span and a high rate of turnover, these findings suggest a model in which gammaherpesviruses may gain access to the mature B cell compartment by recurrent seeding of developing B cells.

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 latency-associated 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, gammaHV68) 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 germinal-center 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.

This study identifies transcriptional regulation of stress response element (STRE) genes in space in the model eukaryotic organism, Saccharomyces cerevisiae. To determine transcription-factor dependence, gene expression changes in space were examined in strains bearing green fluorescent protein-tagged (GFP-tagged) reporters for YIL052C (Sfp1 dependent with stress), YST-2 (Sfp1/Rap1 dependent with stress), or SSA4 (Msn4 dependent with stress), along with strains of SSA4-GFP and YIL052C-GFP with individual deletions of the Msn4 or Sfp1. When compared to parallel ground controls, spaceflight induces significant gene expression changes in SSA4 (35% decrease) and YIL052C (45% decrease), while expression of YST-2 (0.08% decrease) did not change. In space, deletion of Sfp1 reversed the SSA4 gene expression effect (0.00% change), but Msn4 deletion yielded a similar decrease in SSA4 expression (34% change), which indicates that SSA4 gene expression is dependent on the Sfp1 transcription factor in space, unlike other stresses. For YIL052C, deletion of Sfp1 reversed the effect (0.01% change), and the Msn4 deletion maintained the decrease in expression (30% change), which indicates that expression of YIL052C is also dependent on Sfp1 in space. Spaceflight has selective and specific effects on SSA4 and YIL052C gene expression, indicated by novel dependence on Sfp1.

To understand the cellular effects of magnetic traps requires independent analysis of the effects of magnetic field, gravity, and buoyancy. In the current study, buoyancy is manipulated by addition of Ficoll, a viscous substance that can create gradients of buoyancy without significantly affecting osmolality. Specifically, we investigated whether Ficoll induces concentration dependent changes in cell growth, cell cycle, and gene expression in Saccharomyces cerevisiae, with special attention paid to the neutrally buoyant concentration of 35% Ficoll. Cell growth and cell cycle analysis were examined in three strains: wild-type (WT) yeast and strains with deletions in transcription factors Msn4 (Msn4Delta) or Sfp1 (Sfp1Delta). Changes in growth were observed in all three strains with WT and Msn4Delta strains showing strong concentration dependence. In addition, these changes in growth were supported by changes in the cell cycle of all three strains. Gene expression changes were observed in seven GFP-reporter strains including: SSA4, YIL052C, YST2, Msn4DeltaSSA4, Sfp1DeltaSSA4, Msn4DeltaYIL052C, and Sfp1DeltaYIL052C. Buoyancy forces had selective concentration dependent effects on gene expression of SSA4 and YIL052C with transcription factor dependence on Msn4. Additionally, SSA4 expression was dependent on Sfp1. YST2 gene expression was not dependent on changes in buoyancy force. This study shows that buoyancy has selective and concentration dependent effects on growth, cell cycle and gene expression, some of which are Msn4 and Sfp1 dependent. For the first time, SSA4 gene expression is shown to be dependent on Sfp1 and YIL052C gene expression is dependent on Msn4.

Inhomogeneous magnetic fields are used in magnetic traps to levitate biological specimens by exploiting the natural diamagnetism of virtually all materials. Using Saccharomyces cerevisiae, this report investigates whether magnetic field (B) induces changes in growth, cell cycle, and gene expression. Comparison to the effects of gravity and temperature allowed determination of whether the responses are general pathways or stimulus specific. Growth and cell cycle analysis were examined in wild-type (WT) yeast and strains with deletions in transcription factors Msn4 or Sfp1. Msn4, Sfp1, and Rap1 have been implicated in responses to physical forces, but only Msn4 and Sfp1 deletions are viable. Gene expression changes were examined in strains bearing GFP-tagged reporters for YIL052C (Sfp1-dependent), YST-2 (Sfp1/Rap1-dependent), or SSA4 (Msn4-dependent). The cell growth and gene expression responses were highly stimulus specific. B increased growth only following Msn4 or Sfp1 deletion, associated with decreased G1 and G2/M and increased S phase of the cell cycle. In addition, B suppressed expression of both YIL052C and YST2. Gravity decreased growth in an Sfp1 but not Msn4-dependent manner, in association with decreased G2/M and increased S phase of the cell cycle. Additionally, gravity decreased expression of SSA4 and YIL052C genes. Temperature increased cell growth in an Msn4- and Sfp1-dependent manner in association with increased G1 and G2/M with decreased S phase of the cell cycle. In addition, temperature increased YIL052C gene expression. This study shows that B has selective effects on cell growth, cell cycle, and gene expression that are stimulus specific., ((c) 2007 Wiley Periodicals, Inc.)