Neurons are particularly vulnerable to perturbations in endo-lysosomal transport, as several neurological disorders are caused by a primary deficit in this pathway. In this report, we used positional cloning to show that the spontaneously occurring neurological mutation teetering (tn) is a single nucleotide substitution in hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). The tn mice exhibit hypokenesis, muscle weakness, reduced muscle size and early perinatal lethality by 5-weeks of age. Although HGS has been suggested to be essential for the sorting of ubiquitinated membrane proteins to the lysosome, there were no alterations in receptor tyrosine kinase levels in the central nervous system, and only a modest decrease in tropomyosin receptor kinase B (TrkB) in the sciatic nerves of the tn mice. Instead, loss of HGS resulted in structural alterations at the neuromuscular junction (NMJ), including swellings and ultra-terminal sprouting at motor axon terminals and an increase in the number of endosomes and multivesicular bodies. These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ. These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction. In addition to the deficits in neuronal function, mutation of Hgs resulted in both hypermyelinated and dysmyelinated axons in the tn mice, which supports a growing body of evidence that ESCRTs are required for proper myelination of peripheral nerves. Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.
Endocytic trafficking involves the internalization, endosomal sorting and lysosomal degradation of cell surface cargo. Many factors involved in endosomal sorting in mammalian cells have been identified, and mutations in these components are associated with a variety of neurological disorders. While the function of endosomal sorting components has been intensely studied in immortalized cell lines, it is not known what role these factors play in endosomal sorting in the nervous system. In this study, we show that the teetering (tn) gene encodes the hepatocyte
growth factor regulated tyrosine kinase
substrate (Hgs), a core component of the endosomal sorting pathway. The tn mice exhibit several signs of motor neuron disease, including reduced muscle mass, muscle weakness and motor abnormalities. Although HGS is predicted to be required for the lysosomal degradation of receptor tyrosine kinases, there was no change in the levels of receptor tyrosine kinases in the spinal cords of the tn mice. Instead, we found that HGS is required for synaptic transmission at the neuromuscular junction and for the proper myelination of the peripheral nervous system.
Pancreatic ductal adenocarcinomas (PDACs) overexpress pro-angiogenic factors but are not viewed as vascular. Using data from The Cancer Genome Atlas we demonstrate that a subset of PDACs exhibits a strong pro-angiogenic signature that includes 37 genes, such as HDAC9, that are overexpressed in PDAC arising in KRC mice, which express mutated Kras and lack RB. Moreover, patient-derived orthotopic xenografts can exhibit tumor angiogenesis, whereas conditioned media (CM) from KRC-derived pancreatic cancer cells (PCCs) enhance endothelial cell (EC) growth and migration, and activate canonical TGF-β signaling and STAT3. Inhibition of the type I TGF-β receptor with SB505124 does not alter endothelial activation in vitro, but decreases pro-angiogenic gene expression and suppresses angiogenesis in vivo. Conversely, STAT3 silencing or JAK1–2 inhibition with ruxolitinib blocks CM-enhanced EC proliferation. STAT3 disruption also suppresses endothelial HDAC9 and blocks CM-induced HDAC9 expression, whereas HDAC9 re-expression restores CM-enhanced endothelial proliferation. Moreover, ruxolitinib blocks mitogenic EC/PCC cross-talk, and suppresses endothelial p-STAT3 and HDAC9, and PDAC progression and angiogenesis in vivo, while markedly prolonging survival of KRC mice. Thus, targeting JAK1–2 with ruxolitinib blocks a final pathway that is common to multiple pro-angiogenic factors, suppresses EC-mediated PCC proliferation, and may be useful in PDACs with a strong pro-angiogenic signature.
Pancreatic cancer; Angiogenesis; TGF-β; STAT3; mouse model
Ubiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14’s catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14’s catalytic activity is required for neuromuscular junction (NMJ) structure and function.
Mice expressing catalytically inactive USP14 in the nervous system exhibited motor deficits, altered NMJ structure, and synaptic transmission deficits that were similar to what is observed in the USP14-deficient ataxia mice. Acute pharmacological inhibition of USP14 in wild type mice also reduced NMJ synaptic transmission. However, there was no evidence of altered proteasome activity when USP14 was inhibited either genetically or pharmacologically. Instead, these manipulations increased the levels of non-proteasome targeting ubiquitin conjugates. Specifically, we observed enhanced proteasome-independent ubiquitination of mixed lineage kinase 3 (MLK3). Consistent with the direct activation of MLK3 by ubiquitination, we also observed increased activation of its downstrea targets MAP kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK). In vivo inhibition of JNK improved motor function and synapse structure in the USP14 catalytic mutant mice.
USP14’s catalytic activity is required for nervous system structure and function and has an ongoing role in NMJ synaptic transmission. By regulating the ubiquitination status of protein kinases, USP14 can coordinate the activity of intracellular signaling pathways that control the development and activity of the NMJ.
Electronic supplementary material
The online version of this article (doi:10.1186/1750-1326-10-3) contains supplementary material, which is available to authorized users.
USP14; JNK; Ubiquitin; Neuromuscular junction; MLK3; Synapse; Ubiquitin proteasome system; K63-linked ubiquitin; Motor neuron; Motor endplate disease
Motivation: The identification of suitable conditions for crystallization is a rate-limiting step in protein structure determination. The pH of an experiment is an important parameter and has the potential to be used in data-mining studies to help reduce the number of crystallization trials required. However, the pH is usually recorded as that of the buffer solution, which can be highly inaccurate.
Results: Here, we show that a better estimate of the true pH can be predicted by considering not only the buffer pH but also any other chemicals in the crystallization solution. We use these more accurate pH values to investigate the disputed relationship between the pI of a protein and the pH at which it crystallizes.
Availability and implementation: Data used to generate models are available as Supplementary Material.
Supplementary data are available at Bioinformatics online.
Ubiquitin-specific protease 14 (USP14) is a major deubiquitinating enzyme and a key determinant of neuromuscular junction (NMJ) structure and function. We have previously reported dramatic ubiquitin depletion in the nervous systems of the USP14-deficient ataxia (axJ) mice and demonstrated that transgenic ubiquitin overexpression partially rescues the axJ neuromuscular phenotype. However, later work has shown that ubiquitin overexpression does not correct the axJ deficits in hippocampal short term plasticity, and that transgenic expression of a catalytically inactive form of USP14 in the nervous system mimics the neuromuscular phenotype observed in the axJ mice, but causes a only a modest reduction of free ubiquitin. Instead, increased ubiquitin conjugates and aberrant activation of pJNK are observed in the nervous systems of the USP14 catalytic mutant mice. In this report, we demonstrate that restoring free ubiquitin levels in the USP14 catalytic mutant mice improved NMJ structure and reduced pJNK accumulation in motor neuron terminals, but had a negative impact on measures of NMJ function, such as motor performance and muscle development. Transgenic expression of ubiquitin had a dose-dependent effect on NMJ function in wild type mice: moderate levels of overexpression improved NMJ function while more robust ubiquitin overexpression reduced muscle development and motor coordination. Combined, these results suggest that maintenance of free ubiquitin levels by USP14 contributes to NMJ structure, but that USP14 regulates NMJ function through a separate pathway.
neuromuscular junction; ubiquitin; USP14; pJNK; proteasomes; motor neuron and deubiquitinating enzyme
Shell beads appear to have been one of the earliest examples of personal adornments. Marine shells identified far from the shore evidence long-distance transport and imply networks of exchange and negotiation. However, worked beads lose taxonomic clues to identification, and this may be compounded by taphonomic alteration. Consequently, the significance of this key early artefact may be underestimated. We report the use of bulk amino acid composition of the stable intra-crystalline proteins preserved in shell biominerals and the application of pattern recognition methods to a large dataset (777 samples) to demonstrate that taxonomic identification can be achieved at genus level. Amino acid analyses are fast (<2 hours per sample) and micro-destructive (sample size <2 mg). Their integration with non-destructive techniques provides a valuable and affordable tool, which can be used by archaeologists and museum curators to gain insight into early exploitation of natural resources by humans. Here we combine amino acid analyses, macro- and microstructural observations (by light microscopy and scanning electron microscopy) and Raman spectroscopy to try to identify the raw material used for beads discovered at the Early Bronze Age site of Great Cornard (UK). Our results show that at least two shell taxa were used and we hypothesise that these were sourced locally.
Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by excessive mitogenic pathway activation and marked chemoresistance to a broad spectrum of chemotherapeutic drugs. Dual specificity protein phosphatase 1 (DUSP1) is a key negative regulator of mitogen activated protein kinases (MAPKs). Yet, DUSP1 is overexpressed in pancreatic cancer cells (PCCs) in PDAC where it paradoxically enhances colony formation in soft agar and promotes in vivo tumorigenicity. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human PCCs, we show that gemcitabine activates c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Gemcitabine-induced JNK and p38 MAPK activation mediates increased apoptosis, but also transcriptionally upregulates DUSP1, as evidenced by increased DUSP1 mRNA levels and RNA polymerase II loading at DUSP1 gene body. Conversely, shRNA-mediated inhibition of DUSP1 enhances JNK and p38 MAPK activation and gemcitabine chemosensitivity. Using doxycycline-inducible knockdown of DUSP1 in established orthotopic pancreatic tumors, we found that combining gemcitabine with DUSP1 inhibition improves animal survival, attenuates angiogenesis, and enhances apoptotic cell death, as compared with gemcitabine alone. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the advantage of enhancing gemcitabine chemosensitivity while suppressing angiogenesis.
In this study, we identified and characterized an N-ethyl-N-nitrosourea (ENU) induced mutation in Usp14 (nmf375) that leads to adult-onset neurological disease. The nmf375 mutation causes aberrant splicing of Usp14 mRNA, resulting in a 95% reduction in USP14. We previously showed that loss of USP14 in ataxia (axJ) mice results in reduced ubiquitin levels, motor endplate disease, Purkinje cell axonal dystrophy and decreased hippocampal paired pulse facilitation (PPF) during the first 4-6 weeks of life, and early postnatal lethality by two months of age. Although the loss of USP14 is comparable between the nmf375 and axJ mice, the nmf375 mice did not exhibit these axJ developmental abnormalities. However, by 12 weeks of age the nmf375 mutants present with ubiquitin depletion and motor endplate disease, indicating a continual role for USP14-mediated regulation of ubiquitin pools and neuromuscular junction (NMJ) structure in adult mice. The observation that motor endplate disease was only seen after ubiquitin depletion suggests that the preservation of NMJ structure requires the stable maintenance of synaptic ubiquitin pools. Differences in genetic background were shown to affect ubiquitin expression and dramatically alter the phenotypes caused by USP14 deficiency.
The purpose of this study was to investigate the effects of 1st-grade number knowledge tutoring with contrasting forms of practice. Tutoring occurred 3 times per week for 16 weeks. In each 30-min session, the major emphasis (25 min) was number knowledge; the other 5 min provided practice in 1 of 2 forms. Nonspeeded practice reinforced relations and principles addressed in number knowledge tutoring. Speeded practice promoted quick responding and use of efficient counting procedures to generate many correct responses. At-risk students were randomly assigned to number knowledge tutoring with speeded practice (n = 195), number knowledge tutoring with nonspeeded practice (n = 190), and control (no tutoring, n = 206). Each tutoring condition produced stronger learning than control on all 4 mathematics outcomes. Speeded practice produced stronger learning than nonspeeded practice on arithmetic and 2-digit calculations, but effects were comparable on number knowledge and word problems. Effects of both practice conditions on arithmetic were partially mediated by increased reliance on retrieval, but only speeded practice helped at-risk children compensate for weak reasoning ability.
mathematics; practice; fluency; arithmetic; word problems
Aboriginal people living in Canada have a high prevalence of obesity, type 2 diabetes, and cardiovascular disease (CVD). To better understand the pre and postnatal influences on the development of adiposity and related cardio-metabolic factors in adult Aboriginal people, we will recruit and follow prospectively Aboriginal pregnant mothers and their children – the Aboriginal Birth Cohort (ABC) study.
We aim to recruit 300 Aboriginal pregnant mothers and their newborns from the Six Nations Reserve, and follow them prospectively to age 3 years. Key details of environment and health including maternal nutrition, glucose tolerance, physical activity, and weight gain will be collected. At birth, cord blood and placenta samples will be collected, as well as newborn anthropometric measurements. Mothers and offspring will be followed annually with serial measurements of diet and physical activity, growth trajectory, and adiposity.
There is an urgent need to understand maternal and child factors that underlie the early development of adiposity and type 2 diabetes in Aboriginal people. The information generated from this cohort will assist the Six Nations community in developing interventions to prevent early adiposity in Aboriginal children.
Aboriginal; Birth cohort; Early origins; Adiposity
Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (Usp14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that Usp14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using Usp14-deficient axJ mice, we investigated if loss of Usp14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of Usp14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the Usp14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the Usp14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of Usp14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.
The ubiquitin-proteasome system (UPS) controls protein abundance and is essential for many aspects of neuronal function. In ataxia (axJ) mice, profound neurological and synaptic defects result from a loss-of-function mutation in the proteasome-associated deubiquitinating enzyme Usp14, which is required for recycling ubiquitin from proteasomal substrates. Here, we show that transgenic complementation of axJ mice with neuronally-expressed ubiquitin prevents early postnatal lethality, restores muscle mass, and corrects developmental and functional deficits resulting from the loss of Usp14, demonstrating that ubiquitin deficiency is a major cause of the neurological defects observed in the axJ mice. We also show that proteasome components are normally induced during the first two weeks of postnatal development, which coincides with dramatic alterations in polyubiquitin chain formation. These data demonstrate a critical role for ubiquitin homeostasis in synaptic development and function, and show that ubiquitin deficiency may contribute to diseases characterized by synaptic dysfunction.
Metabotropic glutamate receptors (mGluRs), which are coupled to second messenger pathways via G proteins, modulate glutamatergic and GABAergic neurotransmission. Because of their role in modulating neurotransmission, mGluRs are attractive therapeutic targets for anxiety disorders. Previously we showed that mGluR8−/− male mice showed higher measures of anxiety in the open field and elevated plus maze than age-matched wild-type mice. In this study, we assessed the potential effects of acute pharmacological modulation of mGluR8 on measures of avoidable and unavoidable anxiety. In addition to wild-type mice, we also tested apolipoprotein E-deficient (Apoe−/−) mice, as these mice show increased levels of anxiety-like behaviors and therefore might show an altered sensitivity to mGluR8 stimulation. mGluR8 stimulation with the specific agonist DCPG, or modulation with AZ12216052, a new, positive allosteric modulator of mGluR8 reduced measures of anxiety in both wild-type mice. The effects of mGluR8 positive allosteric modulators, which only affect neurotransmission in the presence of extracellular glutamate, seem particularly promising for patients with anxiety disorders showing benzodiazepine insensitivity.
metabotropic; group-III mGluR; allosteric modulator; behavior
Dysfunction of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many neurological diseases, including Alzheimer's, spinocerebellar ataxia, and several motor neuron diseases. Recent research indicates that changes in synaptic transmission may play a critical role in the progression of neurological disease; however, the mechanisms by which the UPS regulates synaptic structure and function have not been well characterized. In this report, we show that Usp14 is indispensable for synaptic development and function at neuromuscular junctions (NMJs). Usp14-deficient axJ mice display a resting tremor, a reduction in muscle mass, and notable hind-limb rigidity without any detectable loss of motor neurons. Instead, loss of Usp14 causes developmental defects at motor neuron endplates. Presynaptic defects include phosphorylated neurofilament accumulations, nerve terminal sprouting and poor arborization of the motor nerve terminals, while postsynaptic acetylcholine receptors display immature plaque-like morphology. These structural changes in the NMJ correlated with ubiquitin loss in the spinal cord and sciatic nerve. Further studies demonstrated that the greatest loss of ubiquitin was found in synaptosomal fractions, suggesting that the endplate swellings may be caused by decreased protein turnover at the synapse. Transgenic restoration of Usp14 in the nervous system corrected the levels of monomeric ubiquitin in the motor neuron circuit and the defects that were observed in the motor endplates and muscles of the axJ mice. These data define a critical role for Usp14 at mammalian synapses and suggest a requirement for local ubiquitin recycling by the proteasome to control the development and function of NMJs.
proteasome; ubiquitin; neuromuscular junction; Usp14; synapse; Motoneuron
Hematopoietic differentiation of embryonic stem (ES) cells can be enhanced by co-culture with stromal cells derived from hematopoietic tissues and by overexpression of the transcription factor HOXB4. In this study, we compare the hematopoietic inductive effects of stromal cell lines derived from different subregions of the embryonic aorta-gonad-mesonephros tissue with the commonly used OP9 stromal cell line and with HOXB4 activation. We show that stromal cell lines derived from the aorta and surrounding mesenchyme (AM) act at an earlier stage of the differentiation process compared with the commonly used OP9 stromal cells. AM stromal cells were able to promote the further differentiation of isolated brachyury-GFP+ mesodermal cells into hematopoietic progenitors, whereas the OP9 stromal cells could not support the differentiation of these cells. Co-culture and analyses of individual embryoid bodies support the hypothesis that the AM stromal cell lines could enhance the de novo production of hematopoietic progenitors, lending support to the idea that AM stromal cells might act on prehematopoietic mesoderm. The induction level observed for AM stromal cells was comparable to HOXB4 activation, but no additive effect was observed when these 2 inductive strategies were combined. Addition of a γ-secretase inhibitor reduced the inductive effects of both the stromal cell line and HOXB4, providing clues to possible shared molecular mechanisms.
Homozygous ataxia (axJ) mice have reduced expression of ubiquitin-specific protease 14 (Usp14), resulting in severe neuromuscular defects and death by 2 months of age. Transgenic expression of Usp14 exclusively in the nervous system of axJ mice (axJ-Tg) prevents early lethality and restores motor system function to the axJ mice, enabling an analysis of the reproductive capabilities of Usp14-deficient mice. Although female axJ-Tg mice had a 75% reduction of Usp14 in the ovaries, they were able to produce normal litters. Ovary transfer experiments also demonstrated that the ovaries of axJ mice were capable of producing viable pups. In contrast, male axJ and axJ-Tg mice displayed a 50% reduction in testicular Usp14 levels and were infertile, indicating that Usp14 is required for development and function of the male reproductive system. Immunohistochemistry experiments showed that Usp14 is found in the redundant nuclear envelope and cytoplasmic droplet of epididymal spermatozoa. Analysis of axJ testes demonstrated a 50% reduction in testis weight, a 100-fold reduction in sperm number and the presence of abnormal spermatozoa in the epididymis. Histological examination of the Usp14-deficient testes revealed abnormal spermatogenesis and the presence of degenerating germ cells, indicating that Usp14 and the ubiquitin proteasome system are required for spermatid differentiation during spermiogenesis.
ubiquitin; proteasome; testis; Usp14; fertility
Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8+ T-cell responses to six Ebola virus proteins were examined. CD8+ T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8+ cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8+ cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.
Cytotoxic T lymphocytes (CTLs) are proposed to be critical for protection from intracellular pathogens such as Ebola virus. However, there have been no demonstrations that protection against Ebola virus is mediated by Ebola virus-specific CTLs. Here, we report that C57BL/6 mice vaccinated with Venezuelan equine encephalitis virus replicons encoding the Ebola virus nucleoprotein (NP) survived lethal challenge with Ebola virus. Vaccination induced both antibodies to the NP and a major histocompatibility complex class I-restricted CTL response to an 11-amino-acid sequence in the amino-terminal portion of the Ebola virus NP. Passive transfer of polyclonal NP-specific antiserum did not protect recipient mice. In contrast, adoptive transfer of CTLs specific for the Ebola virus NP protected unvaccinated mice from lethal Ebola virus challenge. The protective CTLs were CD8+, restricted to the Db class I molecule, and recognized an epitope within amino acids 43 to 53 (VYQVNNLEEIC) in the Ebola virus NP. The demonstration that CTLs can prevent lethal Ebola virus infection affects vaccine development in that protective cellular immune responses may be required for optimal protection from Ebola virus.
Retinoblastoma protein (Rb) is a key regulator of cellular proliferation, controlling entry into G1/S in the cell cycle, largely through its action in binding the cellular transcription factor E2F, which activates genes important in DNA synthesis. Small DNA tumor viruses encode gene products which can functionally inactivate Rb, promoting cellular proliferation and viral DNA synthesis. In this study, the Epstein-Barr virus (EBV) immediate-early lytic gene product, BRLF1 (R), is shown to bind Rb in vivo, shortly after induction of the viral lytic cycle in EBV-infected Akata cells. Furthermore, the temporal kinetics of R-Rb interaction correlate with displacement of E2F1 from Rb. Mapping of the domains required for the interaction of R and Rb proteins reveals that R binds specifically to the N terminus of Rb, outside the Rb pocket, and that the first 200 amino acids of R are required for this interaction. The interaction of R and Rb may initiate cell cycle progression and facilitate viral DNA synthesis during lytic replication.