Jennifer M. Martin

A diagnostic assessment for introductory molecular and cell biology.

We have developed and validated a tool for assessing understanding of a selection of fundamental concepts and basic knowledge in undergraduate introductory molecular and cell biology, focusing on areas in which students often have misconceptions. This multiple-choice Introductory Molecular and Cell Biology Assessment (IMCA) instrument is designed for use as a pre- and posttest to measure student learning gains. To develop the assessment, we first worked with faculty to create a set of learning goals that targeted important concepts in the field and seemed likely to be emphasized by most instructors teaching these subjects. We interviewed students using open-ended questions to identify commonly held misconceptions, formulated multiple-choice questions that included these ideas as distracters, and reinterviewed students to establish validity of the instrument. The assessment was then evaluated by 25 biology experts and modified based on their suggestions. The complete revised assessment was administered to more than 1300 students at three institutions. Analysis of statistical parameters including item difficulty, item discrimination, and reliability provides evidence that the IMCA is a valid and reliable instrument with several potential uses in gauging student learning of key concepts in molecular and cell biology.

Transmembrane Domains 1 and 2 of the Latent Membrane Protein 1 of Epstein-Barr Virus Contain a Lipid Raft Targeting Signal and Play a Critical Role in Cytostasis

ABSTRACT The latent membrane protein 1 (LMP-1) oncoprotein of Epstein-Barr virus (EBV) is a constitutively active, CD40-like cell surface signaling protein essential for EBV-mediated human B-cell immortalization. Like ligand-activated CD40, LMP-1 activates NF-κB and Jun kinase signaling pathways via binding, as a constitutive oligomer, to tumor necrosis factor receptor-associated factors (TRAFs). LMP-1s lipid raft association and oligomerization have been linked to its activation of cell signaling pathways. Both oligomerization and lipid raft association require the function of LMP-1s polytopic multispanning transmembrane domain, a domain that is indispensable for LMP-1s growth-regulatory signaling activities. We have begun to address the sequence requirements of the polytopic hydrophobic transmembrane domain for LMP-1s signaling and biochemical activities. Here we report that transmembrane domains 1 and 2 are sufficient for LMP-1s lipid raft association and cytostatic activity. Transmembrane domains 1 and 2 support NF-κB activation, albeit less potently than does the entire polytopic transmembrane domain. Interestingly, LMP-1s first two transmembrane domains are not sufficient for oligomerization or TRAF binding. These results suggest that lipid raft association and oligomerization are mediated by distinct and separable activities of LMP-1s polytopic transmembrane domain. Additionally, lipid raft association, mediated by transmembrane domains 1 and 2, plays a significant role in LMP-1 activation, and LMP-1 can activate NF-κB via an oligomerization/TRAF binding-independent mechanism. To our knowledge, this is the first demonstration of an activitys being linked to individual membrane-spanning domains within LMP-1s polytopic transmembrane domain.

EBNA2 and Activated Notch Induce Expression of BATF

ABSTRACT The immortalization of human B lymphocytes by Epstein-Barr virus (EBV) requires the virus-encoded transactivator EBNA2 and the products of both viral and cellular genes which serve as EBNA2 targets. In this study, we identified BATF as a cellular gene that is up-regulated dramatically within 24 h following the infection of established and primary human B cells with EBV. The transactivation of BATF is mediated by EBNA2 in a B-cell-specific manner and is duplicated in non-EBV-infected B cells by the expression of mammalian Notch proteins. In contrast to other target genes activated by EBNA2, the BATF gene encodes a member of the AP-1 family of transcription factors that functions as a negative regulator of AP-1 activity and as an antagonist of cell growth. A potential role for BATF in promoting EBV latency is supported by studies in which BATF was shown to negatively impact the expression of a BZLF1 reporter gene and to reduce the frequency of lytic replication in latently infected cells. The identification of BATF as a cellular target of EBV provides important new information on how programs of viral and cellular gene expression may be coordinated to promote viral latency and control lytic-cycle entry.

The cytoplasmic amino-terminus of the Latent Membrane Protein-1 of Epstein-Barr Virus: relationship between transmembrane orientation and effector functions of the carboxy-terminus and transmembrane domain.

The Latent Membrane Protein 1 (LMP-1) protein of Epstein-Barr virus (EBV) is localized in the plasma membrane of the infected cell. LMP-1 possesses a hydrophobic membrane spanning domain, and charged, intracellular amino- and carboxy-termini. Two models have been proposed for the contribution of the amino-terminus to LMP-1s function: (i) as an effector domain, interacting with cellular proteins, or (ii) as a structural domain dictating the correct orientation of transmembrane domains and thereby positioning LMP-1s critical effector domains (i.e. the carboxy-terminus). However, no studies to date have addressed directly the structural contributions of LMP-1s cytoplasmic amino-terminus to function. This study was designed to determine if LMP-1s cytoplasmic amino-terminus (N-terminus) encodes information required solely for maintenance of proper topological orientation. We have constructed LMP-1 chimeras in which the cytoplasmic N-terminus of LMP-1 is replaced with an unrelated domain of similar size and charge, but of different primary sequence. Retention of the charged amino-terminal (N-terminal) cytoplasmic domain and first predicted transmembrane domain was required for correct transmembrane topology. The absolute primary sequence of the cytoplasmic N-terminus was not critical for LMP-1s cytoskeletal association, turnover, plasma membrane patching, oligomerization, Tumor Necrosis Factor Receptor-associated factor (TRAF) binding, NF-κB activation, rodent cell transformation and cytostatic activity. Furthermore, our results point to the hydrophobic transmembrane domain, independent of the cytoplasmic domains, as the primary LMP-1 domain mediating oligomerization, patching and cytoskeletal association. The cytoplasmic amino-terminus provides the structural information whereby proper transmembrane orientation is achieved.

The Late Lytic LMP-1 Protein of Epstein-Barr Virus Can Negatively Regulate LMP-1 Signaling

ABSTRACT The BNLF-1 open reading frame of Epstein-Barr virus (EBV) encodes two related proteins, latent membrane protein-1 (LMP-1) and lytic LMP-1 (lyLMP-1). LMP-1 is a latent protein required for immortalization of human B cells by EBV, whereas lyLMP-1 is expressed during the lytic cycle and is found in the EBV virion. We show here that, in contrast to LMP-1, lyLMP-1 is stable, with a half-life of >20 h in tetradecanoyl phorbol acetate- and butyrate-treated B95-8 cells. Although lyLMP-1 itself has negligible effects on NF-κB activity, it inhibits NF-κB activation by LMP-1 in a dose-dependent manner. The lyLMP-1 protein does not oligomerize with LMP-1, and the negative effect of lyLMP-1 on NF-κB activation by LMP-1 does not result from lyLMP-1-mediated disruption of LMP-1 oligomers. Modulation of LMP-1-activated signaling pathways is the first identified biological activity associated with lyLMP-1, and this activity may contribute to the progression of EBVs lytic cycle.

The Epstein-Barr Virus-Encoded LMP-1 Oncoprotein Negatively Affects Tyk2 Phosphorylation and Interferon Signaling in Human B Cells

ABSTRACT Epstein-Barr virus (EBV) establishes a persistent infection in the human host and is associated with a variety of human cancers. Persistent infection results from a balance between the host immune response and viral immune evasion mechanisms. EBV infection is controlled initially by the innate immune response and later by T-cell-mediated adaptive immunity. EBV has evolved mechanisms to evade the host immune response so that it can persist for the lifetime of the host. Latent membrane protein 1 (LMP-1) is the EBV oncoprotein essential for B-cell immortalization by EBV. We show here that LMP-1 interacts with Tyk2, a signaling intermediate in the alpha interferon (IFN-α) signaling pathway, via a previously uncharacterized LMP-1 signaling domain. LMP-1 prevents Tyk2 phosphorylation and inhibits IFN-α-stimulated STAT2 nuclear translocation and interferon-stimulated response element transcriptional activity. Long-term culture of EBV+ lymphoblastoid cells in IFN-α is associated with outgrowth of a population expressing elevated LMP-1 protein levels, suggesting that cells expressing higher levels of LMP-1 survive the antiproliferative selective pressure imposed by IFN-α. These results show that LMP-1 can protect EBV+ cells from the IFN-α-stimulated antiviral/antiproliferative response and suggest that chronic IFN-α treatment may encourage the outgrowth of cells expressing elevated, and therefore potentially oncogenic, LMP-1 levels in EBV+ individuals.

Latent membrane protein 1 associated signaling pathways are important in tumor cells of Epstein-Barr virus negative Hodgkin's disease.

The latent membrane protein 1 (LMP1) oncogene of Epstein-Barr virus (EBV) is selectively expressed in the Reed-Sternberg (RS) cells of EBV-associated Hodgkins disease (HD). However, no differences in clinical presentation and course are found between EBV positive and EBV negative forms of HD suggesting a common pathogenetic mechanism. We have studied the LMP1 associated signaling pathways and their dominant negative inhibition in the myelomonocytic HD-MyZ and the B-lymphoid L-428 HD cell lines. In both EBV negative cell lines expression of LMP1 is associated with the formation of multinuclear RS cells. Dominant negative inhibition of NF-κB mediated signaling at the step of IκB-α phosphorylation results in increased cell death with only a few typical RS cells resistant to overexpression of the dominant negative inhibitor IκB-α-NΔ54. However, dominant negative inhibition of NF-κB mediated signaling at the early step of TRAF2 interaction results in the formation of multinuclear cells in both cell lines and, in addition, in clusters of small mononuclear cells in the HD-MyZ cell line. In HD-MyZ cells overexpression of the powerful JBD-inhibitor of the JNK signal transduction pathway is restricted to small cells and never observed in RS cells. These small cells undergo apoptosis as shown by the TUNEL technique. Apoptosis of small cells is still observed after co-transfection of JBD and LMP1 but in addition a few apoptotic HD-MyZ cells with large fused nuclear masses are identified suggesting that specific inhibition of JNK leads also to apoptosis of LMP1 induced RS cells. Thus, activation of the JNK signaling pathway is also important in the formation of Reed-Sternberg cells. Our findings are consistent with a model where all three LMP1 associated functions, i.e. NF-κB mediated transcription, TRAF2 dependent signaling, and c-Jun activation act as a common pathogenetic denominator of both EBV negative and EBV positive HD.

Transmembrane peptides used to investigate the homo‐oligomeric interface and binding hotspot of latent membrane protein 1

Epstein-Barr virus (EBV), a human γ-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. Although normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein latent membrane protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV. The constitutive signaling activity of LMP-1 is dependent on homo-oligomerization of its six-spanning hydrophobic transmembrane domain (TMD). However, the mechanism driving LMP-1 intermolecular interaction is poorly understood. Here, we show that the fifth transmembrane helix (TM5) of LMP-1 strongly self-associates, forming a homotrimeric complex mediated by a polar residue embedded in the membrane, D150. Replacement of this aspartic acid residue with alanine disrupts TM5 self-association in detergent micelles and bacterial cell membranes. A full-length LMP-1 variant harboring the D150A substitution is deficient in NFκB activation, supporting the key role of the fifth transmembrane helix in constitutive activation of signaling by this oncoprotein.

Unexpected Absence of the Epstein-Barr Virus (EBV) lyLMP-1 Open Reading Frame in Tumor Virus Isolates: Lack of Correlation between Met129 Status and EBV Strain Identity

ABSTRACT The lytic cycle-associated lytic latent membrane protein-1 (lyLMP-1) of Epstein-Barr virus (EBV) is an amino-terminally truncated form of the oncogenic LMP-1. Although lyLMP-1 shares none of LMP-1s transforming and signal transducing activities, we recently reported that lyLMP-1 can negatively regulate LMP-1-stimulated NF-κB activation. The lyLMP-1 protein encoded by the B95-8 strain of EBV initiates from methionine 129 (Met129) of the LMP-1 open reading frame (ORF). The recent report that Met129 in the B95-8 LMP-1 ORF is not conserved in the Akata strain of EBV prompted us to screen a panel of EBV-positive cell lines for conservation of Met129 and lyLMP-1 expression. We found that 15 out of 16 tumor-associated virus isolates sequenced encoded an ATT or ACC codon in place of ATG in the LMP-1 ORF at position 129, and tumor cell lines harboring isolates lacking an ATG at codon 129 did not express the lyLMP-1 protein. In contrast, we found that EBV DNA from 22 out of 37 healthy seropositive donors retained the Met129 codon. Finally, the lyLMP-1 initiator occurs variably within distinct EBV strains and its presence cannot be predicted by EBV strain identity. Thus, Met129 is not peculiar to the B95-8 strain of EBV, but rather can be found in the background of several evolutionarily distinct EBV strains. Its absence from EBV isolates from tumors raises the possibility of selective pressure on Met129 in EBV-dependent tumors.

Using Pre-Assessment and In-Class Questions to Change Student Understanding of Molecular Movements.

Understanding how different types of molecules move through cell membranes is a fundamental part of cell biology. To identify and address student misconceptions surrounding molecular movement through cell membranes, we surveyed student understanding on this topic using pre-class questions, in-class clicker questions, and subsequent exam questions in a large introductory biology course. Common misconceptions identified in student responses to the pre-class assessment questions were used to generate distractors for clicker questions. Two-tier diagnostic clicker questions were used to probe incoming common student misconceptions (first tier) and their reasoning (second tier). Two subsequent lectures with assessment clicker questions were used to help students construct a new framework to understand molecular movement through cell membranes. Comparison of pre-assessment and post-assessment (exam) performance showed dramatic improvement in students’ understanding of molecular movement: student answers to exam questions were 74.6% correct with correct reasoning while only 1.3% of the student answers were correct with correct reasoning on the pre-class assessment. Our results show that students’ conceptual understanding of molecular movement through cell membranes progressively increases through discussions of a series of clicker questions and suggest that this clicker-based teaching strategy was highly effective in correcting common student misconceptions on this topic.