pUL36 Deubiquitinase Activity Augments Both the Initiation and the Progression of Lytic Herpes Simplex Virus Infection in IFN-Primed Cells.

The evolutionarily conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal deubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo, as it acts as a potent viral immune evasin. Interferon (IFN) induces the expression of hundreds of antiviral factors, including many ubiquitin modulators, which HSV-1 needs to neutralize to efficiently initiate a productive infection. Herein, we discover two functions of the conserved pUL36 DUB during lytic replication in cell culture in an understudied but equally important scenario of HSV-1 infection in IFN-treated cells. Our data indicate that the pUL36 DUB contributes to overcoming the IFN-mediated suppression of productive infection in both the early and late phases of HSV-1 infection. We show that incoming tegument-derived pUL36 DUB activity contributes to the IFN resistance of HSV-1 in IFN-primed cells to efficiently initiate lytic virus replication. Subsequently, the de novo expressed DUB augmented the efficiency of virus replication and increased the output of infectious virus. Notably, the DUB defect was only apparent when IFN was applied prior to infection. Our data indicate that IFN-induced defense mechanisms exist and that they work to both neutralize infectivity early on and slow the progression of HSV-1 replication in the late stages of infection. Also, our data indicate that pUL36 DUB activity contributes to the disarming of these host responses. IMPORTANCE HSV-1 is a ubiquitous human pathogen that is responsible for common cold sores and may also cause life-threatening disease. pUL36 is an essential, conserved herpesvirus protein with N-terminal deubiquitinating (DUB) activity. The DUB is dispensable for HSV-1 replication in cell culture but represents an important viral immune evasin in vivo. IFN plays a pivotal role in HSV-1 infection and suppresses viral replication both in vitro and in vivo. Here, we show that DUB activity contributes to overcoming IFN-induced cellular resistance in order to more efficiently initiate lytic replication and produce infectious virions. As such, DUB activity in the incoming virions increases their infectivity, while the de novo synthesized DUB augments productive infection. Thus, the HSV-1 DUB antagonizes the activity of IFN-inducible effector proteins to facilitate productive infection at multiple levels. Our findings underscore the importance of using more challenging cell culture systems to fully understand virus protein functions.

Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry.

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.

A bimodal switch in global protein translation coupled to eIF4H relocalisation during advancing cell-cell transmission of herpes simplex virus.

We used the bioorthogonal protein precursor, homopropargylglycine (HPG) and chemical ligation to fluorescent capture agents, to define spatiotemporal regulation of global translation during herpes simplex virus (HSV) cell-to-cell spread at single cell resolution. Translational activity was spatially stratified during advancing infection, with distal uninfected cells showing normal levels of translation, surrounding zones at the earliest stages of infection with profound global shutoff. These cells further surround previously infected cells with restored translation close to levels in uninfected cells, reflecting a very early biphasic switch in translational control. While this process was dependent on the virion host shutoff (vhs) function, in certain cell types we also observed temporally altered efficiency of shutoff whereby during early transmission, naïve cells initially exhibited resistance to shutoff but as infection advanced, naïve target cells succumbed to more extensive translational suppression. This may reflect spatiotemporal variation in the balance of oscillating suppression-recovery phases. Our results also strongly indicate that a single particle of HSV-2, can promote pronounced global shutoff. We also demonstrate that the vhs interacting factor, eIF4H, an RNA helicase accessory factor, switches from cytoplasmic to nuclear localisation precisely correlating with the initial shutdown of translation. However translational recovery occurs despite sustained eIF4H nuclear accumulation, indicating a qualitative change in the translational apparatus before and after suppression. Modelling simulations of high multiplicity infection reveal limitations in assessing translational activity due to sampling frequency in population studies and how analysis at the single cell level overcomes such limitations. The work reveals new insight and a revised model of translational manipulation during advancing infection which has important implications both mechanistically and with regards to the physiological role of translational control during virus propagation. The work also demonstrates the potential of bioorthogonal chemistry for single cell analysis of cellular metabolic processes during advancing infections in other virus systems.

Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy.

We investigated the spatiotemporal dynamics of HSV genome transport during the initiation of infection using viruses containing bioorthogonal traceable precursors incorporated into their genomes (HSVEdC). In vitro assays revealed a structural alteration in the capsid induced upon HSVEdC binding to solid supports that allowed coupling to external capture agents and demonstrated that the vast majority of individual virions contained bioorthogonally-tagged genomes. Using HSVEdC in vivo we reveal novel aspects of the kinetics, localisation, mechanistic entry requirements and morphological transitions of infecting genomes. Uncoating and nuclear import was observed within 30 min, with genomes in a defined compaction state (ca. 3-fold volume increase from capsids). Free cytosolic uncoated genomes were infrequent (7-10% of the total uncoated genomes), likely a consequence of subpopulations of cells receiving high particle numbers. Uncoated nuclear genomes underwent temporal transitions in condensation state and while ICP4 efficiently associated with condensed foci of initial infecting genomes, this relationship switched away from residual longer lived condensed foci to increasingly decondensed genomes as infection progressed. Inhibition of transcription had no effect on nuclear entry but in the absence of transcription, genomes persisted as tightly condensed foci. Ongoing transcription, in the absence of protein synthesis, revealed a distinct spatial clustering of genomes, which we have termed genome congregation, not seen with non-transcribing genomes. Genomes expanded to more decondensed forms in the absence of DNA replication indicating additional transitional steps. During full progression of infection, genomes decondensed further, with a diffuse low intensity signal dissipated within replication compartments, but frequently with tight foci remaining peripherally, representing unreplicated genomes or condensed parental strands of replicated DNA. Uncoating and nuclear entry was independent of proteasome function and resistant to inhibitors of nuclear export. Together with additional data our results reveal new insight into the spatiotemporal dynamics of HSV genome uncoating, transport and organisation.

Spatial and Temporal Resolution of Global Protein Synthesis during HSV Infection Using Bioorthogonal Precursors and Click Chemistry.

We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection. A striking feature was the rapid accumulation of newly synthesised proteins not only in a general nuclear pattern but additionally in newly forming sub-compartments represented by small discrete foci. These newly synthesised protein domains (NPDs) were similar in size and morphology to PML domains but were more numerous, and whereas PML domains were progressively disrupted, NPDs were progressively induced and persisted. Immediate-early proteins ICP4 and ICP0 were excluded from NPDs, but using an ICP0 mutant defective in PML disruption, we show a clear spatial relationship between NPDs and PML domains with NPDs frequently forming immediately adjacent and co-joining persisting PML domains. Further analysis of location of the chaperone Hsc70 demonstrated that while NPDs formed early in infection without overt Hsc70 recruitment, later in infection Hsc70 showed pronounced recruitment frequently in a coat-like fashion around NPDs. Moreover, while ICP4 and ICP0 were excluded from NPDs, ICP22 showed selective recruitment. Our data indicate that NPDs represent early recruitment of host and viral de novo translated protein to distinct structural entities which are precursors to the previously described VICE domains involved in protein quality control in the nucleus, and reveal new features from which we propose spatially linked platforms of newly synthesised protein processing after nuclear import.

Evasion of innate cytosolic DNA sensing by a gammaherpesvirus facilitates establishment of latent infection.

Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to α- and ß-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections.

Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission.

UNLABELLED: Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cycle, HSV suppresses host DNA synthesis and promotes viral DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover, using pulse-chase analysis, we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis is then shut off and replaced with virus DNA synthesis. Using nonpropagating viruses or conditioned medium, we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways in which virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as of mechanisms governing host DNA synthesis. IMPORTANCE: We show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus-induced paracrine effector. The field has had no conception that this process occurs, and the work changes our interpretation of virus-host interaction during advancing infection and has implications for understanding controls of host DNA synthesis. Our findings demonstrate the utility of chemical biology techniques in analysis of infection processes, reveal distinct processes when infection is examined in multiround transmission versus single-step growth curves, and reveal a hitherto-unknown process in virus infection, likely relevant for other viruses (and other infectious agents) and for remote signaling of other processes, including transcription and protein synthesis.

An orchestrated program regulating secretory pathway genes and cargos by the transmembrane transcription factor CREB-H.

CREB3 proteins comprise a set of ER-localized bZip transcription factors defined by the presence of a transmembrane domain. They are regulated by inter-compartmental transport, Golgi cleavage and nuclear transport where they promote appropriate transcriptional responses. Although CREB3 proteins play key roles in differentiation, inflammation and metabolism, a general framework relating their defining features to these diverse activities is lacking. We identify unique features of CREB3 organization including the ATB domain, which we show it is essential for transcriptional activity. This domain is absent in all other human bZip factors, but conserved in Drosophila CREBA, which controls secretory pathway genes (SPGs). Furthermore, each of the five human CREB3 factors was capable of activating SPGs in Drosophila, dependent upon the ATB domain. Expression of the CREB3 protein, CREB-H, in 293 cells, upregulated genes involved in secretory capacity, extracellular matrix formation and lipid metabolism and increased secretion of specific cargos. In liver cells, which normally express CREB-H, the active form specifically induced secretion of apolipoproteins, including ApoA-IV, ApoAI, consistent with data implicating CREB-H in metabolic homeostasis. Based on these data and other recent studies, we propose a general role for the CREB3 family in regulating secretory capacity, with particular relevance to specialized cargos.

Polarized cell migration during cell-to-cell transmission of herpes simplex virus in human skin keratinocytes.

In addition to transmission involving extracellular free particles, a generally accepted model of virus propagation is one wherein virus replicates in one cell, producing infectious particles that transmit to the next cell via cell junctions or induced polarized contacts. This mechanism of spread is especially important in the presence of neutralizing antibody, and the concept underpins analysis of virus spread, plaque size, viral and host functions, and general mechanisms of virus propagation. Here, we demonstrate a novel process involved in cell-to-cell transmission of herpes simplex virus (HSV) in human skin cells that has not previously been appreciated. Using time-lapse microscopy of fluorescent viruses, we show that HSV infection induces the polarized migration of skin cells into the site of infection. In the presence of neutralizing antibody, uninfected skin cells migrate to the initial site of infection and spread over infected cells to become infected in a spatially confined cluster containing hundreds of cells. The cells in this cluster do not undergo cytocidal cell lysis but harbor abundant enveloped particles within cells and cell-free virus within interstitial regions below the cluster surface. Cells at the base and outside the cluster were generally negative for virus immediate-early expression. We further show, using spatially separated monolayer assays, that at least one component of this induced migration is the paracrine stimulation of a cytotactic response from infected cells to uninfected cells. The existence of this process changes our concept of virus transmission and the potential functions, virus, and host factors involved.

Interstitial cystitis patients' use and rating of complementary and alternative medicine therapies.

INTRODUCTION AND HYPOTHESIS: The purpose of this study was to describe the use of complementary and alternative medicine (CAM) therapies among interstitial cystitis (IC) patients, patients' perception of CAM therapies' effectiveness, and the association of time since diagnosis with perceived effectiveness of these therapies. METHODS: In April 2009, the Interstitial Cystitis Association (ICA) initiated an Internet-based survey on CAM. Respondents indicated whether they received an IC diagnosis and how long ago, whether they tried CAM, and who recommended it. On a 5-point scale, respondents rated 49 therapies. For respondents confirming a diagnosis, we used a chi-square goodness-of-fit test to assess which therapies were rated positively or negatively by a majority of patients who tried them. Using separate one-way analyses of variance, we assessed differences in mean perceived effectiveness among groups based on time since diagnosis and conducted post hoc tests, if necessary. Using chi-square tests, we explored the association of time since diagnosis with the use of CAM and the number of therapies tried. RESULTS: A total of 2,101 subjects responded to the survey; 1,982 confirmed an IC diagnosis. Most (84.2 %) had tried CAM, and 55 % said physicians had recommended CAM. Of those trying CAM, 82.8 % had tried diet or physical therapy and 69.2 % other therapies. Of the therapies, 22 were rated positively and 20 negatively; 7 were inconclusive. Therapies patients perceived to be helpful included dietary management and pain management adjuncts such as physical therapy, heat and cold, meditation and relaxation, acupuncture, stress reduction, exercise, and sleep hygiene. Many therapies worked better for those diagnosed recently than for those diagnosed long before. CONCLUSIONS: Randomized, placebo-controlled studies are needed to demonstrate which therapies may indeed control IC symptoms and help send research in new and productive directions.

Different mechanisms of recognition and ER retention by transmembrane transcription factors CREB-H and ATF6.

CREB-H and activating transcription factor 6 (ATF6) are transmembrane transcription factors that, in response to endoplasmic reticulum (ER) stress, traffic to the Golgi where they are cleaved by specific proteases, producing the N-terminal domains that effect appropriate transcriptional responses. We show that unlike in ATF6 whose lumenal tail binds BiP and contains determinants for stress sensing and Golgi transport, in CREB-H the lumenal tail is not involved in ER retention, not required for Golgi transport and does not bind BiP. The main determinant for CREB-H ER retention resides in a membrane-proximal cytoplasmic determinant that is conserved in related members of the CREB-H family, but lacking in ATF6. We refine requirements within the ER-retention motif (ERM) and show that ERM-ve variants exhibited constitutive Golgi localization and constitutive cleavage by the Golgi protease, S1P. The ERM also conferred ER retention on a heterologous protein. Furthermore, deletion of the lumenal tail of CREB-H had no effect on ER retention of parental CREB-H or Golgi localization of ERM-ve variants. Importantly, when the lumenal tail of ATF6 was transferred into an ERM-ve variant, the chimera was now retained in the ER. Together, these data demonstrate novel and qualitatively distinct mechanisms of trafficking and stress signalling in CREB-H compared to ATF6.

Raman spectroscopic monitoring of the osteogenic differentiation of human mesenchymal stem cells.

The differentiation of stem cells into multi-lineages is essential to aid the development of tissue engineered materials that replicate the functionality of their tissue of origin. For this study, Raman spectroscopy was used to monitor the formation of a bone-like apatite mineral during the differentiation of human mesenchymal stem cells (hMSCs) towards an osteogenic lineage. Raman spectroscopy observed dramatic changes in the region dominated by the stretching of phosphate groups (950-970 cm(-1)) during the period of 7-28 days. Changes were also seen at 1030 cm(-1) and 1070 cm(-1), which are associated with the P-O symmetric stretch of PO(4)(3-) and the C-O vibration in the plane stretch of CO(3)(2-). Multivariate factor analysis revealed the presence of various mineral species throughout the 28 day culture period. Bone mineral formation was observed first at day 14 and was identified as a crystalline, non-substituted apatite. During the later stages of culture, different mineral species were observed, namely an amorphous apatite and a carbonate, substituted apatite, all of which are known to be Raman markers for a bone-like material. Band area ratios revealed that both the carbonate-to-phosphate and mineral-to-matrix ratios increased with age. When taken together, these findings suggest that the osteogenic differentiation of hMSCs at early stages resembles endochondral ossification. Due to the various mineral species observed, namely a disordered amorphous apatite, a B-type carbonate-substituted apatite and a crystalline non-substituted hydroxyapatite, it is suggested that the bone-like mineral observed here can be compared to native bone. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analyses for monitoring the various mineral species, degree of mineralisation and the crystallinity of hMSCs as they differentiate into osteoblasts.

Differing roles of inner tegument proteins pUL36 and pUL37 during entry of herpes simplex virus type 1.

Studies with herpes simplex virus type 1 (HSV-1) have shown that secondary envelopment and virus release are blocked in mutants deleted for the tegument protein gene UL36 or UL37, leading to the accumulation of DNA-containing capsids in the cytoplasm of infected cells. The failure to assemble infectious virions has meant that the roles of these genes in the initial stages of infection could not be investigated. To circumvent this, cells infected at a low multiplicity were fused to form syncytia, thereby allowing capsids released from infected nuclei access to uninfected nuclei without having to cross a plasma membrane. Visualization of virus DNA replication showed that a UL37-minus mutant was capable of transmitting infection to all the nuclei within a syncytium as efficiently as the wild-type HSV-1 strain 17(+) did, whereas infection by UL36-minus mutants failed to spread. Thus, these inner tegument proteins have differing functions, with pUL36 being essential during both the assembly and uptake stages of infection, while pUL37 is needed for the formation of virions but is not required during the initial stages of infection. Analysis of noninfectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.

HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection.

Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.

Nuclear pore composition and gating in herpes simplex virus-infected cells.

The mechanism by which herpes simplex virus (HSV) exits the nucleus remains a matter of controversy. The generally accepted route proposes that capsids exit via primary envelopment at the inner nuclear membrane and subsequent fusion of this primary particle with the outer nuclear membrane to gain capsid entry to the cytoplasm. However, recent observations indicate that HSV may induce gross morphological alterations of nuclear pores, resulting in the loss of normal pores and the appearance of dilated gaps in the nuclear membrane of up to several 100 nm. On this basis, it was proposed that a main route of capsid exit from the nucleus is directly through these altered pores. Here, we examine the biochemical composition of some of the major nuclear pore components in uninfected and HSV-infected cells. We show that total levels of major nucleoporins and their sedimentation patterns in density gradients remain largely unchanged up to 18 h after HSV infection. Some alteration in modification of one nucleoporin, Nup358/RanBP2, was observed during enrichment with anti-nucleoporin antibody and probing for O glycosylation. In addition, we examine functional gating within the nucleus in live cells, using microinjection of labeled dextran beads and a recombinant virus expressing GFP-VP16 to track the progress of infection. The nuclear permeability barrier for molecules bigger than 70 kDa remained intact throughout infection. Thus, in a functional assay in live cells, we find no evidence for gross perturbation to the gating of nuclear pores, although this might not exclude a small population of modified pores.

CREB4, a transmembrane bZip transcription factor and potential new substrate for regulation and cleavage by S1P.

Regulated intramembrane proteolysis of the factors SREBP and ATF6 represents a central control mechanism in sterol homeostasis and stress response within the endoplasmic reticulum. Here, we compare localization of ATF6-related bZip factors CREB4, CREB-H, Luman, and OASIS. These factors contain the defining features of a bZip domain, a predicted transmembrane domain and an adjacent cleavage site for the Golgi protease S1P, with conserved features which indicate that it represents a specific subclass of S1P sites. Each factor localizes to the endoplasmic reticulum (ER), but a population of CREB4 was also observed in the Golgi. Deletion of the transmembrane domain in CREB4 resulted in efficient nuclear accumulation. An N-terminal variant of CREB4 containing the BZIp domain potently activated expression from a target gene containing ATF6 binding sites and from the promoter for the ER chaperone GRP78/BIP. CREB4 was cleaved in a site-specific manner in response to brefeldin A disruption of the Golgi or by coexpression with S1P but only after deletion or substitution of its C-terminal lumenal domain. Thus, S1P cleavage of CREB4 may be suppressed by a determinant in the C-terminal region. Dithiothreitol induced more complete transport of CREB4 to the Golgi, but not cleavage. Together, the data identify at least one additional bZip factor whose localization responds to ER stress, and we propose a model based on these results that indicates additional levels of control of this novel class of transcription factors.

Transmembrane bZIP transcription factors in ER stress signaling and the unfolded protein response.

Regulated intramembrane proteolysis (RIP) of the transmembrane transcription factor ATF6 represents a key step in effecting adaptive response to the presence of unfolded or malfolded protein in the endoplasmic reticulum. Recent studies have highlighted new ATF6-related transmembrane transcription factors. It is likely that current models for ER stress signaling are incomplete and that the expansion of the bZIP transmembrane family reflects selectivity in many aspects of these responses, including the type and duration of any particular stress, the cell type in which it occurs, and the integration with other aspects of cell-type-specific organization or additional intrinsic pathways, and the integration and communication between these pathways, not only in a cell-type-specific manner, but also between different tissues and organs. This review summarizes current information on the bZIP-transmembrane proteins and discusses outstanding questions on the elucidation of the stress signals, the repertoire of components involved in regulating different aspects of the forward transport, cleavage, nuclear import, transcriptional activity, and turnover of each of these factors, and dissection of the integration of the various outputs into broad coordinated responses.

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.

GSK-3-mediated phosphorylation couples ER-Golgi transport and nuclear stabilization of the CREB-H transcription factor to mediate apolipoprotein secretion.

CREB-H, an ER-anchored transcription factor, plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes, including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases, releasing the N-terminal product, which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif termed the P-motif, located in the N-terminus between serines 73 and 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation, promoting ER retention, while mutation of target serines and drug inhibition of GSK-3 activity coordinately induce both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations, which regulate stability by targeting the protein to the SCFFbw1a E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilization of the active form in the nucleus.