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1.
PLoS Pathog ; 19(10): e1011682, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37782657

RESUMEN

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.


Asunto(s)
Citomegalovirus , Transducción de Señal , Animales , Ratones , Humanos , Citomegalovirus/fisiología , Latencia del Virus , Diferenciación Celular , Células Madre Hematopoyéticas
2.
J Virol ; 97(10): e0124123, 2023 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-37772824

RESUMEN

IMPORTANCE: CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/ß-catenin pathway as an important component of viral reactivation. We further define that pUL8 and ß-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with ß-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.


Asunto(s)
Antígenos CD34 , Citomegalovirus , Proteínas Dishevelled , Células Madre Hematopoyéticas , Proteínas Virales , Activación Viral , beta Catenina , Humanos , Antígenos CD34/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Citomegalovirus/genética , Citomegalovirus/fisiología , Proteínas Dishevelled/química , Proteínas Dishevelled/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/virología , Dominios PDZ , Proteínas Virales/química , Proteínas Virales/metabolismo , Latencia del Virus/genética
4.
PLoS Pathog ; 13(3): e1006219, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28278237

RESUMEN

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.


Asunto(s)
Infección por el Virus Zika/patología , Infección por el Virus Zika/virología , Animales , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Hibridación in Situ , Macaca mulatta , Masculino , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Viremia/virología , Virus Zika
5.
bioRxiv ; 2024 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-38826434

RESUMEN

HCMV genes UL135 and UL138 play opposing roles regulating latency and reactivation in CD34+ human progenitor cells (HPCs). Using the THP-1 cell line model for latency and reactivation, we designed an RNA sequencing study to compare the transcriptional profile of HCMV infection in the presence and absence of these genes. The loss of UL138 results in elevated levels of viral gene expression and increased differentiation of cell populations that support HCMV gene expression and genome synthesis. The loss of UL135 results in diminished viral gene expression during an initial burst that occurs as latency is established and no expression of eleven viral genes from the ULb' region even following stimulation for differentiation and reactivation. Transcriptional network analysis revealed host transcription factors with potential to regulate the ULb' genes in coordination with pUL135. These results reveal roles for UL135 and UL138 in regulation of viral gene expression and potentially hematopoietic differentiation.

6.
PLoS One ; 15(1): e0227676, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31935257

RESUMEN

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.


Asunto(s)
Modelos Animales de Enfermedad , Infección por el Virus Zika/veterinaria , Virus Zika/patogenicidad , Animales , Cardiomiopatías/virología , Femenino , Feto/virología , Macaca mulatta , Microcefalia/virología , Embarazo , Complicaciones Infecciosas del Embarazo/veterinaria , Complicaciones Infecciosas del Embarazo/virología , Primer Trimestre del Embarazo , Convulsiones/virología , Infección por el Virus Zika/virología
7.
Antiviral Res ; 155: 12-19, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29709563

RESUMEN

Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions and cause significant morbidity and mortality worldwide. Although a partially effective vaccine is in use in several countries in which DENV are endemic, no antiviral therapeutics are approved for combating DENV-associated disease. Herein, we report the characterization of novel small molecule inhibitors of DENV replication, VGTI-A3 and VGTI-A3-03, as well as structure-activity relationship analysis of the molecules using a panel of chemical analogs. VGTI-A3 and VGTI-A3-03 are highly virus-specific, with greatest activity against DENV serotype 2. Further analysis revealed that treatment of infected cells with VGTI-A3-03 does not inhibit viral RNA replication or secretion of viral particles. Rather, the infectivity of secreted particles from A3-03 treated cells is significantly diminished compared to particles secreted from control cells. Elicitation of VGTI-A3-03-resistant mutants demonstrated a clear binding pocket in the capsid molecule at the dimerization interface. Additionally, we show that VGTI-A3-03 is incorporated into virus particles released from infected cells. In summary, these data provide detailed analysis of a potentially useful class of anti-DENV inhibitors and further identify a region of the viral capsid protein as a druggable target for other therapeutic approaches.


Asunto(s)
Antivirales/química , Proteínas de la Cápside/metabolismo , Virus del Dengue/efectos de los fármacos , Virión/efectos de los fármacos , Antivirales/farmacología , Cápside/efectos de los fármacos , Dengue/virología , Células HEK293 , Humanos , Mutagénesis , Unión Proteica , ARN Viral , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos
8.
Nat Commun ; 9(1): 263, 2018 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-29343712

RESUMEN

Zika virus (ZIKV) infection during pregnancy leads to an increased risk of fetal growth restriction and fetal central nervous system malformations, which are outcomes broadly referred to as the Congenital Zika Syndrome (CZS). Here we infect pregnant rhesus macaques and investigate the impact of persistent ZIKV infection on uteroplacental pathology, blood flow, and fetal growth and development. Despite seemingly normal fetal growth and persistent fetal-placenta-maternal infection, advanced non-invasive in vivo imaging studies reveal dramatic effects on placental oxygen reserve accompanied by significantly decreased oxygen permeability of the placental villi. The observation of abnormal oxygen transport within the placenta appears to be a consequence of uterine vasculitis and placental villous damage in ZIKV cases. In addition, we demonstrate a robust maternal-placental-fetal inflammatory response following ZIKV infection. This animal model reveals a potential relationship between ZIKV infection and uteroplacental pathology that appears to affect oxygen delivery to the fetus during development.


Asunto(s)
Placenta/metabolismo , Circulación Placentaria , Complicaciones Infecciosas del Embarazo/inmunología , Infección por el Virus Zika/inmunología , Inmunidad Adaptativa , Animales , Encéfalo/embriología , Encéfalo/patología , Citocinas/sangre , Modelos Animales de Enfermedad , Femenino , Desarrollo Fetal , Feto/patología , Inmunidad Innata , Macaca mulatta , Imagen por Resonancia Magnética , Oxígeno/metabolismo , Permeabilidad , Placenta/inmunología , Placenta/patología , Placenta/virología , Embarazo , Complicaciones Infecciosas del Embarazo/metabolismo , Complicaciones Infecciosas del Embarazo/patología , Complicaciones Infecciosas del Embarazo/fisiopatología , Carga Viral , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/patología , Infección por el Virus Zika/fisiopatología
9.
Sci Rep ; 6: 21674, 2016 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-26876974

RESUMEN

Ebolaviruses pose significant public health problems due to their high lethality, unpredictable emergence, and localization to the poorest areas of the world. In addition to implementation of standard public health control procedures, a number of experimental human vaccines are being explored as a further means for outbreak control. Recombinant cytomegalovirus (CMV)-based vectors are a novel vaccine platform that have been shown to induce substantial levels of durable, but primarily T-cell-biased responses against the encoded heterologous target antigen. Herein, we demonstrate the ability of rhesus CMV (RhCMV) expressing Ebola virus (EBOV) glycoprotein (GP) to provide protective immunity to rhesus macaques against lethal EBOV challenge. Surprisingly, vaccination was associated with high levels of GP-specific antibodies, but with no detectable GP-directed cellular immunity.


Asunto(s)
Citomegalovirus/genética , Portadores de Fármacos , Vacunas contra el Virus del Ébola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Vacunas contra el Virus del Ébola/administración & dosificación , Vacunas contra el Virus del Ébola/genética , Femenino , Macaca mulatta , Masculino , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genética
10.
Vaccine ; 33(19): 2261-2266, 2015 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-25820063

RESUMEN

Ebola virus (Zaire ebolavirus; EBOV) is a highly lethal hemorrhagic disease virus that most recently was responsible for two independent 2014 outbreaks in multiple countries in Western Africa, and the Democratic Republic of the Congo, respectively. Herein, we show that a cytomegalovirus (CMV)-based vaccine provides durable protective immunity from Ebola virus following a single vaccine dose. This study has implications for human vaccination against ebolaviruses, as well as for development of a 'disseminating' vaccine to target these viruses in wild African great apes.


Asunto(s)
Citomegalovirus/genética , Ebolavirus/inmunología , Vectores Genéticos , Fiebre Hemorrágica Ebola/prevención & control , Vacunación/métodos , Vacunas Virales/inmunología , Animales , Peso Corporal , Modelos Animales de Enfermedad , Femenino , Fiebre Hemorrágica Ebola/inmunología , Ratones Endogámicos C57BL , Análisis de Supervivencia , Vacunas Virales/administración & dosificación , Vacunas Virales/genética
11.
Vaccine ; 30(20): 3047-52, 2012 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-22414558

RESUMEN

The current commercially available vaccine used to prevent tetanus disease following infection with the anaerobic bacterium Clostridium tetani is safe and effective. However, tetanus remains a major source of mortality in developing countries. In 2008, neonatal tetanus was estimated to have caused >59,000 deaths, accounting for 1% of worldwide infant mortality, primarily in poorer nations. The cost of multiple vaccine doses administered by injection necessary to achieve protective levels of anti-tetanus toxoid antibodies is the primary reason for low vaccine coverage. Herein, we show that a novel vaccine strategy using a cytomegalovirus (CMV)-based vaccine platform induces protective levels of anti-tetanus antibodies that are durable (lasting >13 months) in mice following only a single dose. This study demonstrates the ability of a 'single-dose' CMV-based vaccine strategy to induce durable protection, and supports the potential for a tetanus vaccine based on CMV to impact the incidence of tetanus in developing countries.


Asunto(s)
Anticuerpos Antibacterianos/sangre , Antitoxinas/sangre , Citomegalovirus/genética , Vectores Genéticos , Fragmentos de Péptidos/inmunología , Toxina Tetánica/inmunología , Toxoide Tetánico/inmunología , Tétanos/prevención & control , Animales , Modelos Animales de Enfermedad , Ratones , Fragmentos de Péptidos/genética , Toxina Tetánica/genética , Toxoide Tetánico/administración & dosificación , Toxoide Tetánico/genética
12.
J Immunother ; 35(5): 390-9, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22576344

RESUMEN

Cytomegalovirus (CMV) is a highly immunogenic virus that results in a persistent, life-long infection in the host typically with no ill effects. Certain unique features of CMV, including its capacity to actively replicate in the presence of strong host CMV-specific immunity, may give CMV an advantage compared with other virus-based vaccine delivery platforms. In the present study, we tested the utility of mouse CMV (mCMV)-based vaccines expressing human prostate-specific antigen (PSA) for prostate cancer immunotherapy in double-transgenic mice expressing PSA and HLA-DRB1*1501 (DR2bxPSA F1 mice). We assessed the capacity of 2 mCMV-based vectors to induce PSA-specific CD8 T-cell responses and affect the growth of PSA-expressing Transgenic Adenocarcinoma of the Mouse Prostate tumors (TRAMP-PSA). In the absence of tumor challenge, immunization with mCMV vectors expressing either a H2-D(b)-restricted epitope PSA(65-73) (mCMV/PSA(65-73)) or the full-length gene for PSA (mCMV/PSA(FL)) induced comparable levels of CD8 T-cell responses that increased (inflated) with time. Upon challenge with TRAMP-PSA tumor cells, animals immunized with mCMV/PSA(65-73) had delay of tumor growth and increased PSA-specific CD8 T-cell responses, whereas animals immunized with mCMV/PSA(FL) showed progressive tumor growth and no increase in number of splenic PSA(65-73)-specific T cells. The data show that a prototype CMV-based prostate cancer vaccine can induce an effective antitumor immune response in a "humanized" double-transgenic mouse model. The observation that mCMV/PSA(FL) is not effective against TRAMP-PSA is consistent with our previous findings that HLA-DRB1*1501-restricted immune responses to PSA are associated with suppression of effective CD8 T-cell responses to TRAMP-PSA tumors.


Asunto(s)
Adenocarcinoma/prevención & control , Vacunas contra el Cáncer/inmunología , Citomegalovirus/inmunología , Epítopos de Linfocito T/inmunología , Cadenas HLA-DRB1/inmunología , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/prevención & control , Adenocarcinoma/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Proliferación Celular , Citomegalovirus/genética , Modelos Animales de Enfermedad , Epítopos de Linfocito T/genética , Expresión Génica , Cadenas HLA-DRB1/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Sistemas de Lectura Abierta , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/inmunología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Carga Tumoral , Vacunación
13.
PLoS Negl Trop Dis ; 5(8): e1275, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21858240

RESUMEN

BACKGROUND: Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP(CTL)). MCMV/ZEBOV-NP(CTL) induced high levels of long-lasting (>8 months) CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.


Asunto(s)
Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/inmunología , Epítopos de Linfocito T/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Muromegalovirus/genética , Nucleoproteínas/inmunología , Vacunas Sintéticas/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Portadores de Fármacos , Vacunas contra el Virus del Ébola/administración & dosificación , Ebolavirus/genética , Epítopos de Linfocito T/genética , Femenino , Vectores Genéticos , Ratones , Ratones Endogámicos C57BL , Muromegalovirus/crecimiento & desarrollo , Nucleoproteínas/genética , Linfocitos T Citotóxicos/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación
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