SARS coronavirus 2-reactive antibodies in bovine colostrum.

Objective: To determine if bovine colostrum and sera have antibodies that react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Animals: Dairy and beef cattle from North America and Europe, sampled before and after the SARS-CoV-2 pandemic. Procedures: Indirect ELISAs using whole bovine coronavirus (BCoV) and SARS-CoV-2; whole SARS-CoV-2 Spike 1, Spike 2, and nucleocapsid proteins; and SARS-CoV-2-specific nucleocapsid peptide as antigens. Virus neutralization assay for BCoV. Surrogate virus neutralization assay for SARS-CoV-2. Results: Antibodies reactive to BCoV were highly prevalent in samples collected from cattle before and after the SARS-CoV-2 pandemic. Antibodies reactive with SARS-CoV-2 were present in the same samples, and apparently increased in prevalence after the SARS-CoV-2 pandemic. These antibodies had variable reactivity with the spike and nucleocapsid proteins of SARS-CoV-2 but were apparently not specific for SARS-CoV-2. Conclusions: Bovine coronavirus continues to be endemic in cattle populations, as indicated by the high prevalence of antibodies to the virus in colostrum and serum samples. Also, the prevalent antibodies to SARS-CoV-2 in bovine samples, before and after the pandemic, are likely the result of responses to epitopes on the spike and nucleocapsid proteins that are shared between the 2 betacoronaviruses. Cross-reactive antibodies in bovine colostrum could be examined for prophylactic or therapeutic effects on SARS-CoV-2 infections in humans.

Anticorps réactifs au coronavirus du SRAS 2 dans le colostrum bovin. Objectif: Déterminer si le colostrum et des échantillons de sérum bovins contiennent des anticorps qui réagissent avec le coronavirus 2 du syndrome respiratoire aigu sévère (SRAS-CoV-2). Animaux: Bovins laitiers et bovins de boucherie d'Amérique du Nord et d'Europe, échantillonnés avant et après la pandémie de SARS-CoV-2. Procédures: Épreuves ELISA indirectes utilisant le coronavirus bovin entier (BCoV) et le SARS-CoV-2; ensemble des protéines SARS-CoV-2 Spicule 1, Spicule 2 et nucléocapside; et le peptide de nucléocapside spécifique du SARS-CoV-2 comme antigènes. Test de neutralisation du virus pour le BCoV. Virus de substitution pour le test de neutralisation du SRAS-CoV-2. Résultats: Les anticorps réactifs au BCoV étaient très répandus dans les échantillons prélevés sur les bovins avant et après la pandémie de SRAS-CoV-2. Des anticorps réactifs au SRAS-CoV-2 étaient présents dans les mêmes échantillons et leur prévalence a apparemment augmenté après la pandémie de SRAS-CoV-2. Ces anticorps avaient une réactivité variable avec les protéines de spicule et de nucléocapside du SARS-CoV-2 mais n'étaient apparemment pas spécifiques du SARS-CoV-2. Conclusion: Le coronavirus bovin continue d'être endémique dans les populations bovines, comme l'indique la forte prévalence d'anticorps dirigés contre le virus dans les échantillons de colostrum et de sérum. De plus, les anticorps prévalents contre le SRAS-CoV-2 dans les échantillons de bovins, avant et après la pandémie, sont probablement le résultat de réponses à des épitopes sur les protéines de spicule et de nucléocapside qui sont partagées entre les 2 bêtacoronavirus. Les anticorps à réaction croisée dans le colostrum bovin pourraient être examinés pour leurs effets prophylactiques ou thérapeutiques sur les infections par le SRAS-CoV-2 chez l'humain.(Traduit par Dr Serge Messier).

DDX41 is required for cGAS-STING activation against DNA virus infection.

Upon binding double-stranded DNA (dsDNA), cyclic GMP-AMP synthase (cGAS) is activated and initiates the cGAS-stimulator of IFN genes (STING)-type I interferon pathway. DEAD-box helicase 41 (DDX41) is a DEAD-box helicase, and mutations in DDX41 cause myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). Here, we show that DDX41-knockout (KO) cells have reduced type I interferon production after DNA virus infection. Unexpectedly, activations of cGAS and STING are affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibits ATP-dependent DNA-unwinding activity and ATP-independent strand-annealing activity. The MDS/AML-derived mutant R525H has reduced unwinding activity but retains normal strand-annealing activity and stimulates greater cGAS dinucleotide-synthesis activity than wild-type DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells results in higher type I interferon production. Our results have led to the hypothesis that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and single-stranded DNA (ssDNA), which, in turn, regulates cGAS-STING activation.

Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus.

Coronaviruses that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are speculated to have originated in bats. The mechanisms by which these viruses are maintained in individuals or populations of reservoir bats remain an enigma. Mathematical models have predicted long-term persistent infection with low levels of periodic shedding as a likely route for virus maintenance and spillover from bats. In this study, we tested the hypothesis that bat cells and MERS coronavirus (CoV) can co-exist in vitro. To test our hypothesis, we established a long-term coronavirus infection model of bat cells that are persistently infected with MERS-CoV. We infected cells from Eptesicus fuscus with MERS-CoV and maintained them in culture for at least 126 days. We characterized the persistently infected cells by detecting virus particles, protein and transcripts. Basal levels of type I interferon in the long-term infected bat cells were higher, relative to uninfected cells, and disrupting the interferon response in persistently infected bat cells increased virus replication. By sequencing the whole genome of MERS-CoV from persistently infected bat cells, we identified that bat cells repeatedly selected for viral variants that contained mutations in the viral open reading frame 5 (ORF5) protein. Furthermore, bat cells that were persistently infected with ΔORF5 MERS-CoV were resistant to superinfection by wildtype virus, likely due to reduced levels of the virus receptor, dipeptidyl peptidase 4 (DPP4) and higher basal levels of interferon in these cells. In summary, our study provides evidence for a model of coronavirus persistence in bats, along with the establishment of a unique persistently infected cell culture model to study MERS-CoV-bat interactions.

Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover.

Bats harbor a myriad of viruses and some of these viruses may have spilled over to other species including humans. Spillover events are rare and several factors must align to create the "perfect storm" that would ultimately lead to a spillover. One of these factors is the increased shedding of virus by bats. Several studies have indicated that bats have unique defense mechanisms that allow them to be persistently or latently infected with viruses. Factors leading to an increase in the viral load of persistently infected bats would facilitate shedding of virus. This article reviews the unique nature of bat immune defenses that regulate virus replication and the various molecular mechanisms that play a role in altering the balanced bat⁻virus relationship.

Interferon Regulatory Factor 3-Mediated Signaling Limits Middle-East Respiratory Syndrome (MERS) Coronavirus Propagation in Cells from an Insectivorous Bat.

Insectivorous bats are speculated to be ancestral hosts of Middle-East respiratory syndrome (MERS) coronavirus (CoV). MERS-CoV causes disease in humans with thirty-five percent fatality, and has evolved proteins that counteract human antiviral responses. Since bats experimentally infected with MERS-CoV do not develop signs of disease, we tested the hypothesis that MERS-CoV would replicate less efficiently in bat cells than in human cells because of its inability to subvert antiviral responses in bat cells. We infected human and bat (Eptesicus fuscus) cells with MERS-CoV and observed that the virus grew to higher titers in human cells. MERS-CoV also effectively suppressed the antiviral interferon beta (IFNß) response in human cells, unlike in bat cells. To determine if IRF3, a critical mediator of the interferon response, also regulated the response in bats, we examined the response of IRF3 to poly(I:C), a synthetic analogue of viral double-stranded RNA. We observed that bat IRF3 responded to poly(I:C) by nuclear translocation and post-translational modifications, hallmarks of IRF3 activation. Suppression of IRF3 by small-interfering RNA (siRNA) demonstrated that IRF3 was critical for poly(I:C) and MERS-CoV induced induction of IFNß in bat cells. Our study demonstrates that innate antiviral signaling in E. fuscus bat cells is resistant to MERS-CoV-mediated subversion.

Arousal from hibernation and reactivation of Eptesicus fuscus gammaherpesvirus (EfHV) in big brown bats.

Many viruses that cause serious and often fatal disease in humans have spilled over from bats. Recent evidence suggests that stress may enhance virus shedding by bats increasing the possibility of transmission to other species. To understand the reasons for spillover is therefore important to determine the molecular pathways that link stress to virus reactivation and shedding in bats. We recently isolated and characterized a gammaherpesvirus (Eptesicus fuscus herpesvirus, EfHV) autochthonous to North American big brown bats. Since herpesviruses are known to reactivate from latent infections in response to a wide variety of stressors, EfHV presents us with an opportunity to study how physiological, behavioural or environmental changes may influence the big brown bats' relationship with EfHV. To understand the biology of the virus and how the extended periods of torpor experienced by these bats during hibernation along with the stress of arousal might influence the virus-host relationship, we attempted to detect the virus in the blood of wild-caught non-hibernating bats as well as captive bats arising from hibernation. We compared the prevalence of EfHV in the blood (using PCR) and EfHV-specific antibodies (using ELISA) between captive hibernating bats and wild-caught non-hibernating bats. We detected EfHV only in the blood of captive hibernating bats (27.8% = 10/36) and not in wild-caught non-hibernating bats (0.0% = 0/43). In contrast, the EfHV-specific antibody titres were higher in the non-hibernating bats compared to the hibernating bats. Our study suggests that: (a) viral DNA in blood indicates reactivation from latency, (b) long periods of hibernation lead to suppression of immunity, (c) stress of arousal from hibernation reactivates the virus in bats with lower levels of anti-viral immunity (indicated by humoral immune response), and (d) levels of anti-viral immunity increase in non-hibernating bats following reactivation.

White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats.

Spillover of viruses from bats to other animals may be associated with increased contact between them, as well as increased shedding of viruses by bats. Here, we tested the prediction that little brown bats (Myotis lucifugus) co-infected with the M. lucifugus coronavirus (Myl-CoV) and with Pseudogymnoascus destructans (Pd), the fungus that causes bat white-nose syndrome (WNS), exhibit different disease severity, viral shedding and molecular responses than bats infected with only Myl-CoV or only P. destructans. We took advantage of the natural persistence of Myl-CoV in bats that were experimentally inoculated with P. destructans in a previous study. Here, we show that the intestines of virus-infected bats that were also infected with fungus contained on average 60-fold more viral RNA than bats with virus alone. Increased viral RNA in the intestines correlated with the severity of fungus-related pathology. Additionally, the intestines of bats infected with fungus exhibited different expression of mitogen-activated protein kinase pathway and cytokine related transcripts, irrespective of viral presence. Levels of coronavirus antibodies were also higher in fungal-infected bats. Our results suggest that the systemic effects of WNS may down-regulate anti-viral responses in bats persistently infected with M. lucifugus coronavirus and increase the potential of virus shedding.

Isolation, characterization and prevalence of a novel Gammaherpesvirus in Eptesicus fuscus, the North American big brown bat.

Little is known about the relationship of Gammaherpesviruses with their bat hosts. Gammaherpesviruses are of interest because of their long-term infection of lymphoid cells and their potential to cause cancer. Here, we report the characterization of a novel bat herpesvirus isolated from a big brown bat (Eptesicus fuscus) in Canada. The genome of the virus, tentatively named Eptesicus fuscus herpesvirus (EfHV), is 166,748 base pairs. Phylogenetically EfHV is a member of Gammaherpesvirinae, in which it belongs to the Genus Rhadinovirus and is closely related to other bat Gammaherpesviruses. In contrast to other known Gammaherpesviruses, the EfHV genome contains coding sequences similar to those of class I and II host major histocompatibility antigens. The virus is capable of infecting and replicating in human, monkey, cat and pig cell lines. Although we detected EfHV in 20 of 28 big brown bats tested, these bats lacked neutralizing antibodies against the virus.

A persistently infecting coronavirus in hibernating Myotis lucifugus, the North American little brown bat.

Bats are important reservoir hosts for emerging viruses, including coronaviruses that cause diseases in people. Although there have been several studies on the pathogenesis of coronaviruses in humans and surrogate animals, there is little information on the interactions of these viruses with their natural bat hosts. We detected a coronavirus in the intestines of 53/174 hibernating little brown bats (Myotis lucifugus), as well as in the lungs of some of these individuals. Interestingly, the presence of the virus was not accompanied by overt inflammation. Viral RNA amplified from little brown bats in this study appeared to be from two distinct clades. The sequences in clade 1 were very similar to the archived sequence derived from little brown bats and the sequences from clade 2 were more closely related to the archived sequence from big brown bats. This suggests that two closely related coronaviruses may circulate in little brown bats. Sequence variation among coronavirus detected from individual bats suggested that infection occurred prior to hibernation, and that the virus persisted for up to 4 months of hibernation in the laboratory. Based on the sequence of its genome, the coronavirus was placed in the Alphacoronavirus genus, along with some human coronaviruses, bat viruses and the porcine epidemic diarrhoea virus. The detection and identification of an apparently persistent coronavirus in a local bat species creates opportunities to understand the dynamics of coronavirus circulation in bat populations.

Lack of inflammatory gene expression in bats: a unique role for a transcription repressor.

In recent years viruses similar to those that appear to cause no overt disease in bats have spilled-over to humans and other species causing serious disease. Since pathology in such diseases is often attributed to an over-active inflammatory response, we tested the hypothesis that bat cells respond to stimulation of their receptors for viral ligands with a strong antiviral response, but unlike in human cells, the inflammatory response is not overtly activated. We compared the response of human and bat cells to poly(I:C), a viral double-stranded RNA surrogate. We measured transcripts for several inflammatory, interferon and interferon stimulated genes using quantitative real-time PCR and observed that human and bat cells both, when stimulated with poly(I:C), contained higher levels of transcripts for interferon beta than unstimulated cells. In contrast, only human cells expressed robust amount of RNA for TNFα, a cell signaling protein involved in systemic inflammation. We examined the bat TNFα promoter and found a potential repressor (c-Rel) binding motif. We demonstrated that c-Rel binds to the putative c-Rel motif in the promoter and knocking down c-Rel transcripts significantly increased basal levels of TNFα transcripts. Our results suggest bats may have a unique mechanism to suppress inflammatory pathology.

Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen.

It is speculated that bats are important reservoir hosts for numerous viruses, with 27 viral families reportedly detected in bats. Majority of these viruses have not been isolated and there is little information regarding their biology in bats. Establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. Currently, few bat cell lines have been developed and only Tb1-Lu, derived from Tadarida brasiliensis is commercially available. Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). To our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. The cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture.

Activation of innate immune-response genes in little brown bats (Myotis lucifugus) infected with the fungus Pseudogymnoascus destructans.

Recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern Americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. However, despite these catastrophic events little Information is available on bat defences or how they interact with their pathogens. Even less is known about the response of bats to infection during torpor or long-term hibernation. Using tissue samples collected at the termination of an experiment to explore the pathogenesis of White Nose Syndrome in Little Brown Bats, we determined if hibernating bats infected with the fungus Pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. Lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. However, we were unable to obtain sufficient amounts of RNA from these sites. We therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. We found that bats responded to infection with a significant increase in lungs of transcripts for Cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. In conclusion, hibernating bats can respond to experimental P. destructans infection by activating expression of innate immune response genes.

Zhangfei/CREB-ZF - a potential regulator of the unfolded protein response.

Cells respond to perturbations in the microenvironment of the endoplasmic reticulum (ER), and to the overloading of its capacity to process secretory and membrane-associate proteins, by activating the Unfolded Protein Response (UPR). Genes that mediate the UPR are regulated by three basic leucine-zipper (bLZip) motif-containing transcription factors - Xbp1s, ATF4 and ATF6. A failure of the UPR to achieve homeostasis and its continued stimulation leads to apoptosis. Mechanisms must therefore exist to turn off the UPR if it successfully restores normalcy. The bLZip protein Zhangfei/CREBZF/SMILE is known to suppress the ability of several, seemingly structurally unrelated, transcription factors. These targets include Luman/CREB3 and CREBH, ER-resident bLZip proteins known to activate the UPR in some cell types. Here we show that Zhangfei had a suppressive effect on most UPR genes activated by the calcium ionophore thapsigargin. This effect was at least partially due to the interaction of Zhangfei with Xbp1s. The leucine zipper of Zhangfei was required for this interaction, which led to the subsequent proteasomal degradation of Xbp1s. Zhangfei suppressed the ability of Xbp1s to activate transcription from a promoter containing unfolded protein response elements and significantly reduced the ability to Xbp1s to activate the UPR as measured by RNA and protein levels of UPR-related genes. Finally, specific suppression of endogenous Zhangfei in thapsigargin-treated primary rat sensory neurons with siRNA directed to Zhangfei transcripts, led to a significant increase in transcripts and proteins of UPR genes, suggesting a potential role for Zhangfei in modulating the UPR.

Mechanism for the induction of cell death in ONS-76 medulloblastoma cells by Zhangfei/CREB-ZF.

Cells from medulloblastoma lines do not contain detectable amounts of the basic leucine-zipper protein Zhangfei. However, we have previously shown that expression of this protein in cells of the ONS-76 and UW228 medulloblastoma lines causes the cells to stop growing and develop processes that resemble neurites. Our objective was to determine the molecular mechanisms by which Zhangfei influences ONS-76 cells. We infected ONS-76 cells with adenovirus vectors expressing either Zhangfei or the control protein LacZ and then compared the following parameters in Zhangfei and LacZ-expressing cells: (a) markers of apoptosis, autophagy and macropinocytosis, (b) transcripts for genes involved in neurogenesis and apoptosis, (c) phosphorylation of peptide targets of selected cellular protein kinases, and (d) activation of transcription factors. Zhangfei-expressing cells appeared to succumb to apoptosis. Increased staining for autophagic vesicles and upregulated expression of autophagy response genes in these cells indicated that they were undergoing autophagy, possibly associated with apoptosis. Within our analysis, patterns of gene expression and phosphorylation-mediated signal transduction activity in Zhangfei-expressing cells indicated that the mitogen-activated protein kinase (MAPK) pathway was active. In addition, we found that the transcription factor Brn3a as well as factors implicated in differentiation were also active in Zhangfei-expressing cells. We tested the hypothesis that Zhangfei enhances the expression of Brn3a, a known inducer of TrkA, the high-affinity receptor for nerve growth factor (NGF). TrkA then engages NGF in an autocrine manner triggering the MAPK pathway and leading to differentiation of ONS-76 cells into neuron and glia-like cells-a process that eventually brings about cell death. We showed that: (a) Zhangfei could enhance transcription from the isolated Brn3a promoter, (b) ONS-76 cells produced NGF and (c) antibodies against NGF and inhibitors of TrkA and selected components of the MAPK pathway could partially restore the growth of Zhangfei-expressing ONS-76 cells.

Zhangfei induces the expression of the nerve growth factor receptor, trkA, in medulloblastoma cells and causes their differentiation or apoptosis.

Interactions between nerve growth factor (NGF) and its receptor-the tropomyosin related kinase A (trkA)-regulate many neuronal functions including the correct development of sensory neurons during embryogenesis, the survival of sensory neurons and the differentiation and apoptosis of neuronal tumours. Zhangfei is a transcriptional factor that is expressed in differentiated neurons. Since we could detect Zhangfei in mature neurons but not in neuronal tumour cells, we hypothesised that ectopic expression of the protein in medulloblastoma cells may induce the differentiation of these cells. We show that in ONS-76 medulloblastoma cells, resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei, trkA and Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stopped growing soon after treatment with resveratrol. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked some of the effects of resveratrol. Ectopically expressed Zhangfei in ONS-76 cells led to the increased expression of trkA and Egr1, phosphorylation of extracellular signal-regulated kinase (Erk1), and caused ONS-76 cells to display markers of apoptosis. UW228, another medulloblastoma cell-line, was also susceptible to the suppressive effects of resveratrol and Zhangfei. In contrast, while resveratrol suppressed the growth of human diploid fibroblasts (MRC5), Zhangfei had relatively little effect on these cells.

Zhangfei, a novel regulator of the human nerve growth factor receptor, trkA.

The replication of herpes simplex virus (HSV) in epithelial cells, and during reactivation from latency in sensory neurons, depends on a ubiquitous cellular protein called host cell factor (HCF). The HSV transactivator, VP16, which initiates the viral replicative cycle, binds HCF as do some other cellular proteins. Of these, the neuronal transcription factor Zhangfei suppresses the ability of VP16 to initiate the replicative cycle. It also suppresses Luman, another cellular transcription factor that binds HCF. Interactions of nerve growth factor (NGF) and its receptor tropomyosin-related kinase (trkA) appear to be critical for maintaining HSV latency. Because the neuronal transcription factor Brn3a, which regulates trkA expression, has a motif for binding HCF, we investigated if Zhangfei had an effect on its activity. We found that Brn3a required HCF for activating the trkA promoter and Zhangfei suppressed its activity in non-neuronal cells. However, in neuron-like NGF-differentiated PC12 cells, both Brn3a and Zhangfei activated the trkA promoter and induced the expression of endogenous trkA. In addition, capsaicin, a stressor, which activates HSV in in vitro models of latency, decreased levels of Zhangfei and trkA transcripts in NGF-differentiated PC12 cells.

Zhangfei is a potent and specific inhibitor of the host cell factor-binding transcription factor Luman.

Host cell factor (HCF) was initially discovered as a cellular co-factor required for the activation of herpes simplex virus immediate early gene expression by the virion associated transactivator VP16. HCF also participates in a variety of cellular processes, although the mechanism of its action is not known. VP16 binds to HCF through a 4-amino acid motif (EHAY), which closely resembles the HCF binding domain of two cellular basic leucine-zipper proteins, Luman and Zhangfei. Luman is a powerful transcription factor that, in transient expression assays, activates promoters containing cAMP or unfolded protein response elements (UPRE). In contrast, Zhangfei neither binds consensus recognition elements for basic leucine-zipper proteins nor does it activate promoters containing them. Here we show that Zhangfei suppresses the ability of Luman to activate transcription. HCF appeared to be required for efficient suppression. A mutant of Zhangfei, which was unable to bind HCF, was impaired in its ability to suppress Luman. Zhangfei did not suppress ATF6, a transcription factor closely related to Luman but that does not bind HCF, unless the HCF binding motif of Luman was grafted onto it. Zhangfei inhibited the HCF-dependent activation of a UPRE-containing promoter by a Gal4-Luman fusion protein but was unable to inhibit the HCF-independent activation by Gal4-Luman of a promoter that contained Gal4 binding motifs. Binding of HCF by Zhangfei was required for the co-localization of Luman and Zhangfei to nuclear domains, suggesting that HCF might target the proteins to a common location.

Luman, the cellular counterpart of herpes simplex virus VP16, is processed by regulated intramembrane proteolysis.

Luman is a human basic leucine zipper transcription factor that, like the herpes simplex virus transcription factor VP16, requires the host cell factor, HCF, for activity. Although both HCF and Luman have been implicated in cell growth, their biological roles have not been clearly defined. Luman conforms to a type II membrane-associated glycoprotein with its carboxyl terminus embedded in cellular membranes and its amino terminus, which contains all its identified functional domains, in the cytoplasm. Here we show that Luman is processed by regulated intramembrane proteolysis (RIP). The site 1 protease (S1P), a Golgi apparatus-resident enzyme responsible for catalyzing the first step in the RIP pathway of the sterol regulatory element binding proteins (SREBPs) and ATF6, may also be involved in the processing of Luman. Thus, processing of Luman was highly stimulated by brefeldin A, a compound that causes the reflux of Golgi apparatus enzymes to the endoplasmic reticulum (ER). In addition, coexpression of Luman with S1P containing a KDEL ER retrieval signal resulted in virtually quantitative cleavage of Luman in the absence of any treatment. Finally, Luman contains a sequence, RQLR, immediately downstream from the transmembrane domain which bears similarity to the consensus S1P cleavage site identified by others. Substitution of arginine residues within this motif abolished S1P cleavage, providing robust evidence that S1P is involved in Luman processing. We observed that following S1P cleavage, the majority of the cleaved Luman was retained in cytoplasmic membranes, indicating that an additional step or enzymes yet to be identified are involved in complete cleavage and release to yield the product which ultimately enters the nuclei of cells.