RESUMEN
Previously we reported that the HSV-1 latency associated transcript (LAT) specifically upregulates the cellular herpesvirus entry mediator (HVEM) but no other known HSV-1 receptors. HSV-1 glycoprotein D (gD) binds to HVEM but the effect of this interaction on latency-reactivation is not known. We found that the levels of latent viral genomes were not affected by the absence of gD binding to HVEM. However, reactivation of latent virus in trigeminal ganglia explant cultures was blocked in the absence of gD binding to HVEM. Neither differential HSV-1 replication and spread in the eye nor levels of latency influenced reactivation. Despite similar levels of latency, reactivation in the absence of gD binding to HVEM correlated with reduced T cell exhaustion. Our results indicate that HVEM-gD signaling plays a significant role in HSV-1 reactivation but not in ocular virus replication or levels of latency. The results presented here identify gD binding to HVEM as an important target that influences reactivation and survival of ganglion resident T cells but not levels of latency. This concept may also apply to other herpesviruses that engages HVEM.
Asunto(s)
Herpesvirus Humano 1 , Herpesvirus Humano 1/fisiología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismo , Ojo , Replicación Viral , Latencia del Virus/fisiologíaRESUMEN
The presence of immunosuppressive immune cells in tumors is a significant barrier to the generation of therapeutic immune responses. Similarly, in vivo triple-negative breast cancer (TNBC) models often contain prevalent, immunosuppressive tumor-associated macrophages in the tumor microenvironment (TME), resulting in breast cancer initiation, invasion, and metastasis. Here, we test systemic chemoimmunotherapy using small-molecule agents, paclitaxel (PTX), and colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX3397, to enhance the adaptive T cell immunity against TNBCs in immunocompetent mouse TNBC models. We use high-capacity poly(2-oxazoline) (POx)-based polymeric micelles to greatly improve the solubility of insoluble PTX and PLX3397 and widen the therapeutic index of such drugs. The results demonstrate that high-dose PTX in POx, even as a single agent, exerts strong effects on TME and induces long-term immune memory. In addition, we demonstrate that the PTX and PLX3397 combination provides consistent therapeutic improvement across several TNBC models, resulting from the repolarization of the immunosuppressive TME and enhanced T cell immune response that suppress both the primary tumor growth and metastasis. Overall, the work emphasizes the benefit of drug reformulation and outlines potential translational path for both PTX and PTX with PLX3397 combination therapy using POx polymeric micelles for the treatment of TNBC.
RESUMEN
The HSV-1 latency-associated transcript (LAT) locus contains two small noncoding RNA (sncRNA) sequences (sncRNA1 and sncRNA2) that are not microRNAs (miRNAs). We recently reported that sncRNA1 is more important for in vitro activation of the herpesvirus entry mediator than sncRNA2, but its in vivo function is not known. To determine the role, if any, of sncRNA1 during herpes simplex virus 1 (HSV-1) infection in vivo, we deleted the 62-bp sncRNA1 sequence in HSV-1 strain McKrae using dLAT2903 (LAT-minus) virus, creating ΔsncRNA1 recombinant virus. Deletion of the sncRNA1 in ΔsncRNA1 virus was confirmed by complete sequencing of ΔsncRNA1 virus and its parental virus (i.e., McKrae). Replication of ΔsncRNA1 virus in tissue culture or in the eyes of infected mice was similar to that of HSV-1 strain McKrae and dLAT2903 viruses. However, the absence of sncRNA1 significantly reduced the levels of ICP0, ICP4, and gB but not LAT transcripts in infected rabbit skin cells in vitro. In contrast, the absence of sncRNA1 did reduce LAT expression in trigeminal ganglia (TG), but not in corneas, by day 5 postinfection (p.i.) in infected mice. Levels of eye disease in mice infected with ΔsncRNA1 or McKrae virus were similar, and despite reduced LAT levels in TG during acute ΔsncRNA1 infection, McKrae and ΔsncRNA1 viruses did not affect latency or reactivation on day 28 p.i. However, mice infected with ΔsncRNA1 virus were more susceptible to ocular infection than their wild-type (WT) counterparts. Expression of host immune response genes in corneas and TG of infected mice during primary infection showed reduced expression of beta interferon (IFNß) and IFNγ and altered activation of key innate immune pathways, such as the JAK-STAT pathway in ΔsncRNA1 virus compared with parental WT virus. Our results reveal novel functions for sncRNA1 in upregulating the host immune response and suggest that sncRNA1 has a protective role during primary ocular HSV-1 infection. IMPORTANCE HSV-1 latency-associated transcript (LAT) plays a major role in establishing latency and reactivation; however, the mechanism by which LAT controls these processes is largely unknown. In this study, we sought to establish the role of the small noncoding RNA1 (sncRNA1) encoded within LAT during HSV-1 ocular infection. Our results suggest that sncRNA1 has a protective role during acute ocular infection by modulating the innate immune response to infection.
Asunto(s)
Infecciones del Ojo , Herpes Simple , Herpesvirus Humano 1 , Inmunidad , ARN Pequeño no Traducido , Virulencia , Animales , Células Cultivadas , Infecciones del Ojo/inmunología , Infecciones del Ojo/virología , Regulación de la Expresión Génica/inmunología , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , Inmunidad/genética , Ratones , ARN Pequeño no Traducido/metabolismo , Conejos , Transducción de Señal/genética , Virulencia/genética , Activación Viral/genética , Latencia del Virus/genéticaRESUMEN
Metabolic dysregulation is a prominent feature in breast cancer, but it remains poorly characterized in patient tumors. In this study, untargeted metabolomics analysis of triple-negative breast cancer (TNBC) and patient with estrogen receptor (ER)-positive breast cancer samples, as well as TNBC patient-derived xenografts (PDX), revealed two major metabolic groups independent of breast cancer histologic subtypes: a "Nucleotide/Carbohydrate-Enriched" group and a "Lipid/Fatty Acid-Enriched" group. Cell lines grown in vivo more faithfully recapitulated the metabolic profiles of patient tumors compared with those grown in vitro. Integrated metabolic and gene expression analyses identified genes that strongly correlate with metabolic dysregulation and predict patient prognosis. As a proof of principle, targeting Nucleotide/Carbohydrate-Enriched TNBC cell lines or PDX xenografts with a pyrimidine biosynthesis inhibitor or a glutaminase inhibitor led to therapeutic efficacy. In multiple in vivo models of TNBC, treatment with the pyrimidine biosynthesis inhibitor conferred better therapeutic outcomes than chemotherapeutic agents. This study provides a metabolic stratification of breast tumor samples that can guide the selection of effective therapeutic strategies targeting breast cancer subsets. In addition, we have developed a public, interactive data visualization portal (http://brcametab.org) based on the data generated from this study to facilitate future research. SIGNIFICANCE: A multiomics strategy that integrates metabolic and gene expression profiling in patient tumor samples and animal models identifies effective pharmacologic approaches to target rapidly proliferating breast tumor subtypes.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Metabolómica/métodos , Terapia Molecular Dirigida/métodos , Neoplasias de la Mama Triple Negativas/genética , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Compuestos de Bifenilo/farmacología , Carboplatino/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/genética , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Paclitaxel/administración & dosificación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2-). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER+ tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.
Asunto(s)
Neoplasias de la Mama , Diferenciación Celular , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
High rates of wild-type (WT) herpes simplex virus 1 (HSV-1) latency reactivation depend on the anti-apoptotic activities of latency-associated transcript (LAT). Replacing LAT with the baculovirus inhibitor of apoptosis protein (cpIAP) or cellular FLIP (FLICE-like inhibitory protein) gene restored the WT latency reactivation phenotype to that of a LAT-minus [LAT(-)] virus, while similar recombinant viruses expressing interleukin-4 (IL-4) or interferon gamma (IFN-γ) did not. However, HSV-1 recombinant virus expressing cpIAP did not restore all LAT functions. Recently, we reported that a similar recombinant virus expressing CD80 in place of LAT had higher latency reactivation than a LAT-null virus. The present study was designed to determine if this CD80-expressing recombinant virus can restore all LAT functions as observed with WT virus. Our results suggest that overexpression of CD80 fully rescues LAT function in latency reactivation, apoptosis, and immune exhaustion, suggesting that LAT and CD80 have multiple overlapping functions.IMPORTANCE Recurring ocular infections caused by HSV-1 can cause corneal scarring and blindness. A major function of the HSV-1 latency-associated transcript (LAT) is to establish high levels of latency and reactivation, thus contributing to the development of eye disease. Here, we show that the host CD80 T cell costimulatory molecule functions similarly to LAT and can restore the ability of LAT to establish latency, reactivation, and immune exhaustion as well as induce the expression of caspase 3, caspase 8, caspase 9, and Bcl2. Our results suggest that, in contrast to several other previously tested genes, CD80-expressing virus can completely compensate for all known and tested LAT functions.
Asunto(s)
Apoptosis/inmunología , Antígeno B7-1/inmunología , Herpesvirus Humano 1/fisiología , MicroARNs/inmunología , ARN Viral/inmunología , Activación Viral/inmunología , Latencia del Virus/inmunología , Animales , Apoptosis/genética , Antígeno B7-1/genética , Ratones , MicroARNs/genética , ARN Viral/genética , Activación Viral/genética , Latencia del Virus/genéticaRESUMEN
This study identifies mechanisms mediating responses to immune checkpoint inhibitors using mouse models of triple-negative breast cancer. By creating new mammary tumor models, we find that tumor mutation burden and specific immune cells are associated with response. Further, we developed a rich resource of single-cell RNA-seq and bulk mRNA-seq data of immunotherapy-treated and non-treated tumors from sensitive and resistant murine models. Using this, we uncover that immune checkpoint therapy induces T follicular helper cell activation of B cells to facilitate the anti-tumor response in these models. We also show that B cell activation of T cells and the generation of antibody are key to immunotherapy response and propose a new biomarker for immune checkpoint therapy. In total, this work presents resources of new preclinical models of breast cancer with large mRNA-seq and single-cell RNA-seq datasets annotated for sensitivity to therapy and uncovers new components of response to immune checkpoint inhibitors.
Asunto(s)
Linfocitos B/inmunología , Inmunoterapia , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/inmunología , Mutación/genética , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígeno CTLA-4/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Ingeniería Genética , Genoma , Humanos , Inmunoglobulina G/metabolismo , Activación de Linfocitos/inmunología , Neoplasias Mamarias Animales/terapia , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/terapiaRESUMEN
The Wnt gene family encodes an evolutionarily conserved group of proteins that regulate cell growth, differentiation and stem cell self-renewal. Aberrant Wnt signaling in human breast tumors has been proposed as a driver of tumorigenesis, especially in the basal-like tumor subtype where canonical Wnt signaling is both enriched and predictive of poor clinical outcomes. The development of effective Wnt-based therapeutics, however, has been slowed in part by a limited understanding of the context-dependent nature with which these aberrations influence breast tumorigenesis. We previously reported that MMTV-Wnt1 mice, an established model for studying Wnt signaling in breast tumors, develop two subtypes of tumors by gene expression classification: Wnt1-EarlyEx and Wnt1-LateEx Here, we extend this initial observation and show that Wnt1-EarlyEx tumors exhibit high expression of canonical Wnt, non-canonical Wnt, and EGFR signaling pathway signatures. Therapeutically, Wnt1-EarlyEx tumors showed a dynamic reduction in tumor volume when treated with an EGFR inhibitor. Wnt1-EarlyEx tumors had primarily Cd49fpos/Epcamneg FACS profiles, but it was not possible to serially transplant these tumors into wild-type FVB female mice. Conversely, Wnt1-LateEx tumors had a bloody gross pathology, which was highlighted by the presence of 'blood lakes' identified by H&E staining. These tumors had primarily Cd49fpos/Epcampos FACS profiles, but also contained a secondary Cd49fpos/Epcamneg subpopulation. Wnt1-LateEx tumors were enriched for activating Hras1 mutations and were capable of reproducing tumors when serially transplanted into wild-type FVB female mice. This study definitively shows that the MMTV-Wnt1 mouse model produces two phenotypically distinct subtypes of mammary tumors that differ in multiple biological aspects including sensitivity to an EGFR inhibitor.
Asunto(s)
Antineoplásicos/uso terapéutico , Receptores ErbB/antagonistas & inhibidores , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/patología , Virus del Tumor Mamario del Ratón/patogenicidad , Proteína Wnt1/fisiología , Animales , Antineoplásicos/farmacología , Carcinogénesis , Separación Celular , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Genes ras , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/virología , Ratones , Fenotipo , Vía de Señalización Wnt , Proteína Wnt1/metabolismoRESUMEN
The herpes simplex virus (HSV-1) latency-associated transcript (LAT) has been shown to inhibit apoptosis via inhibiting activation of proapoptotic caspases. However, the mechanism of LAT control of apoptosis is unclear, because LAT is not known to encode a functional protein, and the LAT transcript is found largely in the nucleus. We hypothesized that LAT inhibits apoptosis by regulating expression of genes that control apoptosis. Consequently, we sought to establish the molecular mechanism of antiapoptosis functions of LAT at a transcriptional level during latent HSV-1 ocular infection in mice. Our results suggest the following. (i) LAT likely inhibits apoptosis via upregulation of several components of the type I interferon (IFN) pathway. (ii) LAT does not inhibit apoptosis via the caspase cascade at a transcriptional level or via downregulating Toll-like receptors (TLRs). (iii) The mechanism of LAT antiapoptotic effect is distinct from that of the baculovirus inhibitor of apoptosis (cpIAP) because replacement of LAT with the cpIAP gene resulted in a different gene expression pattern than in either LAT+ or LAT- viruses. (iv) Replacement of LAT with the cpIAP gene does not cause upregulation of CD8 or markers of T cell exhaustion despite their having similar levels of latency, further supporting that LAT and cpIAP function via distinct mechanisms.IMPORTANCE The HSV-1 latency reactivation cycle is the cause of significant human pathology. The HSV-1 latency-associated transcript (LAT) functions by regulating latency and reactivation, in part by inhibiting apoptosis. However, the mechanism of this process is unknown. Here we show that LAT likely controls apoptosis via downregulation of several components in the JAK-STAT pathway. Furthermore, we provide evidence that immune exhaustion is not caused by the antiapoptotic activity of the LAT.
Asunto(s)
Interferón Tipo I/metabolismo , MicroARNs/metabolismo , Latencia del Virus/fisiología , Animales , Apoptosis/genética , Regulación hacia Abajo , Ojo/virología , Infecciones Virales del Ojo/metabolismo , Infecciones Virales del Ojo/virología , Femenino , Regulación Viral de la Expresión Génica/genética , Herpes Simple/metabolismo , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidad , Evasión Inmune/genética , Evasión Inmune/fisiología , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Interferón Tipo I/fisiología , Ratones , Ratones Endogámicos C57BL , MicroARNs/fisiología , Activación Viral/genética , Latencia del Virus/genética , Replicación Viral/genéticaRESUMEN
PURPOSE AND METHODS: In human basal-like breast cancer, mutations and deletions in TP53 and BRCA1 are frequent oncogenic events. Thus, we interbred mice expressing the CRE-recombinase with mice harboring loxP sites at TP53 and BRCA1 (K14-Cre; p53f/f Brca1f/f) to test the hypothesis that tissue-specific deletion of TP53 and BRCA1 would give rise to tumors reflective of human basal-like breast cancer. RESULTS: In support of our hypothesis, these transgenic mice developed tumors that express basal-like cytokeratins and demonstrated intrinsic gene expression features similar to human basal-like tumors. Array comparative genomic hybridization revealed a striking conservation of copy number alterations between the K14-Cre; p53f/f Brca1f/f mouse model and human basal-like breast cancer. Conserved events included MYC amplification, KRAS amplification, and RB1 loss. Microarray analysis demonstrated that these DNA copy number events also led to corresponding changes in signatures of pathway activation including high proliferation due to RB1 loss. K14-Cre; p53f/f Brca1f/f also matched human basal-like breast cancer for a propensity to have immune cell infiltrates. Given the long latency of K14-Cre; p53f/f Brca1f/f tumors (~ 250 days), we created tumor syngeneic transplant lines, as well as in vitro cell lines, which were tested for sensitivity to carboplatin and paclitaxel. These therapies invoked acute regression, extended overall survival, and resulted in gene expression signatures of an anti-tumor immune response. CONCLUSION: These findings demonstrate that this model is a valuable preclinical resource for the study of human basal-like breast cancer.
Asunto(s)
Modelos Animales de Enfermedad , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Animales , Proteína BRCA1 , Femenino , Humanos , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: Based on promising phase II data, the histone deacetylase inhibitor entinostat is in phase III trials for patients with metastatic estrogen receptor-positive breast cancer. Predictors of sensitivity and resistance, however, remain unknown. METHODS: A total of eight cell lines and nine mouse models of breast cancer were treated with entinostat. Luminal cell lines were treated with or without entinostat at their IC50 doses, and MMTV/Neu luminal mouse tumors were untreated or treated with entinostat until progression. We investigated these models using their gene expression profiling by microarray and copy number by arrayCGH. We also utilized the network-based DawnRank algorithm that integrates DNA and RNA data to identify driver genes of resistance. The impact of candidate drivers was investigated in The Cancer Genome Atlas and METABRIC breast cancer datasets. RESULTS: Luminal models displayed enhanced sensitivity to entinostat as compared to basal-like or claudin-low models. Both in vitro and in vivo luminal models showed significant downregulation of Myc gene signatures following entinostat treatment. Myc gene signatures became upregulated on tumor progression in vivo and overexpression of Myc conferred resistance to entinostat in vitro. Further examination of resistance mechanisms in MMTV/Neu tumors identified a portion of mouse chromosome 4 that had DNA copy number loss and low gene expression. Within this region, Jun was computationally identified to be a driver gene of resistance. Jun knockdown in cell lines resulted in upregulation of Myc signatures and made these lines more resistant to entinostat. Jun-deleted samples, found in 17-23% of luminal patients, had significantly higher Myc signature scores that predicted worse survival. CONCLUSIONS: Entinostat inhibited luminal breast cancer through Myc signaling, which was upregulated by Jun DNA loss to promote resistance to entinostat in our models. Jun DNA copy number loss, and/or high MYC signatures, might represent biomarkers for entinostat responsiveness in luminal breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Inhibidores de Histona Desacetilasas/farmacología , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-myc/genética , Piridinas/farmacología , Animales , Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/genética , Ratones Endogámicos BALB C , Ratones Transgénicos , Piridinas/uso terapéuticoRESUMEN
UL20, an essential herpes simplex virus 1 (HSV-1) protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein [GODZ]). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in blocking palmitoylation of UL20 and altered localization and expression of UL20 and glycoprotein K (gK); the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1-infected GODZ-/- mice was significantly lower than in HSV-1-infected wild-type (WT) mice. The levels of UL20, gK, and gB transcripts in the corneas of HSV-1-infected GODZ-/- mice on day 5 postinfection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis are critically dependent on GODZ-mediated palmitoylation of viral UL20.IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70 to 90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection, as well as reactivation, in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results strongly suggest that binding of UL20 to GODZ promotes virus infectivity in vitro and viral pathogenesis in vivo.
Asunto(s)
Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Virales/metabolismo , Latencia del Virus , Replicación Viral , Animales , Línea Celular , Córnea/virología , Citoplasma/virología , Femenino , Herpesvirus Humano 1/genética , Lipoilación , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Ganglio del Trigémino/virología , Proteínas Virales/genéticaRESUMEN
Herpes simplex virus 1 (HSV-1) UL20 plays a crucial role in the envelopment of the cytoplasmic virion and its egress. It is a nonglycosylated envelope protein that is regulated as a γ1 gene. Two-hybrid and pulldown assays demonstrated that UL20, but no other HSV-1 gene-encoded proteins, binds specifically to GODZ (also known as DHHC3), a cellular Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein. A catalytically inactive dominant-negative GODZ construct significantly reduced HSV-1 replication in vitro and affected the localization of UL20 and glycoprotein K (gK) and their interactions but not glycoprotein C (gC). GODZ is involved in palmitoylation, and we found that UL20 is palmitoylated by GODZ using a GODZ dominant-negative plasmid. Blocking of palmitoylation using 2-bromopalmitate (2-BP) affected the virus titer and the interaction of UL20 and gK but did not affect the levels of these proteins. In conclusion, we have shown that binding of UL20 to GODZ in the Golgi apparatus regulates trafficking of UL20 and its subsequent effects on gK localization and virus replication. We also have demonstrated that GODZ-mediated UL20 palmitoylation is critical for UL20 membrane targeting and thus gK cell surface expression, providing new mechanistic insights into how UL20 palmitoylation regulates HSV-1 infectivity.IMPORTANCE HSV-1 UL20 is a nonglycosylated essential envelope protein that is highly conserved among herpesviruses. In this study, we show that (i) HSV-1 UL20 binds to GODZ (also known as DHHC3), a Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein; (ii) a GODZ dominant-negative mutant and an inhibitor of palmitoylation reduced HSV-1 titers and altered the localization of UL20 and glycoprotein K; and (iii) UL20 is palmitoylated by GODZ, and this UL20 palmitoylation is required for HSV-1 infectivity. Thus, blocking of the interaction of UL20 with GODZ, using a GODZ dominant-negative mutant or possibly GODZ shRNA, should be considered a potential alternative therapy in not only HSV-1 but also other conditions in which GODZ processing is an integral component of pathogenesis.
Asunto(s)
Aciltransferasas/metabolismo , Aparato de Golgi/virología , Herpesvirus Humano 1/crecimiento & desarrollo , Proteínas Virales/metabolismo , Línea Celular Tumoral , Células HeLa , Herpes Simple/virología , Humanos , Lipoilación/efectos de los fármacos , Palmitatos/farmacología , Unión Proteica , Técnicas del Sistema de Dos Híbridos , Replicación Viral/fisiologíaRESUMEN
We have established two mouse models of central nervous system (CNS) demyelination that differ from most other available models of multiple sclerosis (MS) in that they represent a mixture of viral and immune triggers. In the first model, ocular infection of different strains of mice with a recombinant HSV-1 that expresses murine IL-2 constitutively (HSV-IL-2) causes CNS demyelination. In the second model, depletion of macrophages causes CNS demyelination in mice that are ocularly infected with wild-type (WT) HSV-1. In the present study, we found that the demyelination in macrophage-intact mice infected with HSV-IL-2 was blocked by depletion of FoxP3-expressing cells, while concurrent depletion of macrophages restored demyelination. In contrast, demyelination was blocked in the macrophage-depleted mice infected with wild-type HSV-1 following depletion of FoxP3-expressing cells. In macrophage-depleted HSV-IL-2-infected mice, demyelination was associated with the activity of both CD4+ and CD8+ T cells, whereas in macrophage-depleted mice infected with WT HSV-1, demyelination was associated with CD4+ T cells. Macrophage depletion or infection with HSV-IL-2 caused an imbalance of T cells and TH1 responses as well as alterations in IL-12p35 and IL-12p40 but not other members of the IL-12 family or their receptors. Demyelination was blocked by adoptive transfer of macrophages that were infected with HSV-IL-12p70 or HSV-IL-12p40 but not by HSV-IL-12p35. These results indicate that suppression of IL-12p70 formation by IL-2 or following macrophage depletion causes T-cell autoreactivity leading to CNS demyelination in HSV-1-infected mice.
Asunto(s)
Sistema Nervioso Central/inmunología , Herpesvirus Humano 1/fisiología , Interleucina-12/inmunología , Interleucina-2/inmunología , Macrófagos/citología , Esclerosis Múltiple/inmunología , Vaina de Mielina/metabolismo , Linfocitos T/inmunología , Animales , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Herpesvirus Humano 1/genética , Humanos , Interleucina-12/genética , Interleucina-2/genética , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/virología , Vaina de Mielina/inmunologíaRESUMEN
UNLABELLED: We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT((-)) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation. IMPORTANCE: Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation.
Asunto(s)
Herpes Simple/virología , Herpesvirus Humano 1/genética , Ganglio del Trigémino/virología , Activación Viral/genética , Latencia del Virus/genética , Replicación Viral/genética , Animales , Córnea/virología , ADN Viral/genética , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Carga Viral/métodosRESUMEN
UNLABELLED: Based on an explant reactivation model, it has been proposed that CD8(+) T cells maintain latency in trigeminal ganglia (TG) of mice latently infected with herpes simplex virus 1 (HSV-1) [T. Liu, K. M. Khanna, X. Chen, D. J. Fink, and R. L. Hendricks, J Exp Med 191:1459-1466, 2000, doi:10.1084/jem.191.9.1459; K. M. Khanna, R. H. Bonneau, P. R. Kinchington, and R. L. Hendricks, Immunity 18:593-603, 2003, doi:10.1016/S1074-7613(03)00112-2]. In those studies, BALB/c mice were ocularly infected with an avirulent HSV-1 strain (RE) after corneal scarification. However, in our studies, we typically infect mice with a virulent HSV-1 strain (McKrae) that does not require corneal scarification. Using a combination of knockout mice, adoptive transfers, and depletion studies, we recently found that CD8α(+) dendritic cells (DCs) contribute to HSV-1 latency and reactivation in TG of ocularly infected mice (K. R. Mott, S. J. Allen, M. Zandian, B. Konda, B. G. Sharifi, C. Jones, S. L. Wechsler, T. Town, and H. Ghiasi, PLoS One 9:e93444, 2014, doi:10.1371/journal.pone.0093444). This suggested that CD8(+) T cells might not be the major regulators of HSV-1 latency in the mouse TG. To investigate this iconoclastic possibility, we used a blocking CD8 antibody and CD8(+) T cells in reactivated TG explants from mice latently infected with (i) the avirulent HSV-1 strain RE following corneal scarification or (ii) the virulent HSV-1 strain McKrae without corneal scarification. Independently of the strain or approach, our results show that CD8α(+) DCs, not CD8(+) T cells, drive latency and reactivation. In addition, adoptive transfer of CD8(+) T cells from wild-type (wt) mice to CD8α(-/-) mice did not restore latency to the level for wt mice or wt virus. In the presence of latency-associated transcript (LAT((+)); wt virus), CD8(+) T cells seem to play a bystander role in the TG. These bystander T cells highly express PD-1, most likely due to the presence of CD8α(+) DCs. Collectively, these results support the notion that CD8(+) T cells do not play a major role in maintaining HSV-1 latency and reactivation. SIGNIFICANCE: This study addresses a fundamentally important and widely debated issue in the field of HSV latency-reactivation. In this article, we directly compare the effects of anti-CD8 antibody, CD8(+) T cells, LAT, and CD8α(+) DCs in blocking explant reactivation in TG of mice latently infected with avirulent or virulent HSV-1. Our data suggest that CD8(+) T cells are not responsible for an increase or maintenance of latency in ocularly infected mice. However, they seem to play a bystander role that correlates with the presence of LAT, higher subclinical reactivation levels, and higher PD-1 expression levels.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Herpesvirus Humano 1/fisiología , Queratitis Herpética/inmunología , Queratitis Herpética/virología , Ganglio del Trigémino/virología , Latencia del Virus , Animales , Células Dendríticas/química , Ojo/virología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptor de Muerte Celular Programada 1/genética , Activación ViralRESUMEN
Recently, we have reported that CD8α(+) DCs, rather than CD8(+) T cells, are involved in the establishment and maintenance of HSV-1 latency in the trigeminal ganglia (TG) of ocularly infected mice. In the current study, we investigated whether similar results can be obtained using Batf3(-/-) mice that previously were reported to lack CD8α(+) DCs. However, our results demonstrate that Batf3(-/-) mice, without any known infection, express CD8α(+) DCs. Consequently, due to the presence of CD8α(+) DCs, no differences were detected in the level of HSV-1 latency between Batf3(-/-) mice compared with wild type control mice.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Antígenos CD8/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Proteínas Represoras/deficiencia , Animales , Antígenos CD8/genética , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Herpes Simple/genética , Herpes Simple/inmunología , Herpes Simple/metabolismo , Herpesvirus Humano 1/inmunología , Inmunohistoquímica , Inmunofenotipificación , Ratones , Ratones Noqueados , Fenotipo , ConejosRESUMEN
UNLABELLED: To investigate the role of the signal sequences of herpes simplex virus 1 (HSV-1) gK on virus replication and viral pathogenesis, we constructed recombinant viruses with or without mutations within the signal sequences of gK. These recombinant viruses expressed two additional copies of the mutated (MgK) or native (NgK) form of the gK gene in place of the latency-associated transcript with a myc epitope tag to facilitate detection at their 3' ends. The replication of MgK virus was similar to that of NgK both in vitro and in vivo, as well as in the trigeminal ganglia (TG) of latently infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 postinfection were markedly virus and time dependent, as well as tissue specific. Mutation in the signal sequence of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin cells, increased 50% lethal dose, and decreased corneal scarring in ocularly infected mice compared to the NgK or revertant (RgK) virus. MgK and NgK viruses, and not the RgK virus, showed a reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose. However, the time of reactivation was not affected by overexpression of the different forms of gK. Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice. IMPORTANCE: In this study, we show for the first time that mutations within the signal sequence of gK blocked cell surface expression of inserted recombinant gK in vitro. Furthermore, this blockage in cell surface expression was correlated with higher 50% lethal dose and less corneal scarring in vivo. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity.
Asunto(s)
Herpes Simple/virología , Herpesvirus Humano 1/crecimiento & desarrollo , Proteínas de la Membrana/metabolismo , Mutación , Señales de Clasificación de Proteína , Proteínas Virales/metabolismo , Animales , Encéfalo/virología , Células Cultivadas , Córnea/virología , Modelos Animales de Enfermedad , Expresión Génica , Herpes Simple/patología , Herpesvirus Humano 1/genética , Lagomorpha , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de Tiempo , Ganglio del Trigémino/virología , Proteínas Virales/genética , Virulencia , Replicación ViralRESUMEN
The development of immunization strategies to protect against ocular infection with herpes simplex virus 1 (HSV-1) must address the issue of the effects of the strategy on the establishment of latency in the trigeminal ganglia (TG). It is the reactivation of this latent virus that can cause recurrent disease and corneal scarring. CD8(+) T cells and dendritic cells (DCs) have been implicated in the establishment and maintenance of latency through several lines of inquiry. The objective of the current study was to use CD8α(-/-) and CD8ß(-/-) mice to further evaluate the contributions of CD8(+) T cells and the CD8α(+) and CD8α(-) subpopulations of DCs to the protection afforded against ocular infection by immunization against HSV-1 and their potential to increase latency. Neutralizing antibody titers were similar in immunized CD8α(-/-), CD8ß(-/-), and wild-type (WT) mice, as was virus replication in the eye. However, on day 3 postinfection (p.i.), the copy number of HSV-1 glycoprotein B (gB) was higher in the corneas and TG of CD8α(-/-) mice than those of WT mice, whereas on day 5 p.i. it was lower. As would be anticipated, the lack of CD8α(+) or CD8ß(+) cells affected the levels of type I and type II interferon transcripts, but the effects were markedly time dependent and tissue specific. The levels of latent virus in the TG, as estimated by measurement of LAT transcripts and in vitro explant reactivation assays, were lower in the immunized, ocularly challenged CD8α(-/-) and WT mice than in their CD8ß(-/-) counterparts. Immunization reduced the expression of PD-1, a marker of T-cell exhaustion, in the TG of ocularly challenged mice, and mock-immunized CD8α(-/-) mice had lower levels of PD-1 expression and latency than mock-immunized WT or CD8ß(-/-) mice. The expansion of the CD8α(-) subpopulation of DCs through injection of WT mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA reduced the amount of latency and PD-1 expression in the TG of infected mice. In contrast, injection of FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which expanded both subpopulations, was less effective. Our results suggest that the absence of both CD8α(+) T cells and CD8α(+) DCs does not reduce vaccine efficacy, either directly or indirectly, in challenged mice and that administration of GM-CSF appears to play a beneficial role in reducing latency and T-cell exhaustion. Importance: In the past 2 decades, two large clinical HSV vaccine trials were performed, but both vaccine studies failed to reach their goals. Thus, as an alternative to conventional vaccine studies, we have used a different strategy to manipulate the host immune responses in an effort to induce greater protection against HSV infection. In lieu of the pleiotropic effect of CD8α(+) DCs in HSV-1 latency, in this report, we show that the absence of CD8α(+) T cells and CD8α(+) DCs has no adverse effect on vaccine efficacy. In line with our hypothesis, we found that pushing DC subpopulations from CD8α(+) DCs toward CD8α(-) DCs by injection of GM-CSF reduced the amount of latent virus and T-cell exhaustion in TG. While these studies point to the lack of a role for CD8α(+) T cells in vaccine efficacy, they in turn point to a role for GM-CSF in reducing HSV-1 latency.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Herpesvirus Humano 1/inmunología , Vacunas contra Herpesvirus/inmunología , Queratitis Herpética/prevención & control , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Córnea/virología , Vacunas contra Herpesvirus/administración & dosificación , Queratitis Herpética/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ganglio del Trigémino/virología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology.