RESUMEN
The tryptophan metabolite, kynurenine, is known to be produced at elevated levels within human cytomegalovirus (HCMV)-infected fibroblasts. Kynurenine is an endogenous aryl hydrocarbon receptor (AhR) ligand. Here we show that the AhR is activated following HCMV infection, and pharmacological inhibition of AhR or knockdown of AhR RNA reduced the accumulation of viral RNAs and infectious progeny. RNA-seq analysis of infected cells following AhR knockdown showed that the receptor alters the levels of numerous RNAs, including RNAs related to cell cycle progression. AhR knockdown alleviated the G1/S cell cycle block that is normally instituted in HCMV-infected fibroblasts, consistent with its known ability to regulate cell cycle progression and cell proliferation. In sum, AhR is activated by kynurenine and perhaps other ligands produced during HCMV infection, it profoundly alters the infected-cell transcriptome, and one outcome of its activity is a block to cell cycle progression, providing mechanistic insight to a long-known element of the virus-host cell interaction.
Asunto(s)
Ciclo Celular , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Fase G1 , Receptores de Hidrocarburo de Aril/metabolismo , División Celular , Infecciones por Citomegalovirus/genética , Interacciones Huésped-Patógeno , Humanos , Quinurenina/metabolismo , LigandosRESUMEN
Pericentromeric human satellite II (HSATII) repeats are normally silent but can be actively transcribed in tumor cells, where increased HSATII copy number is associated with a poor prognosis in colon cancer, and in human cytomegalovirus (HCMV)-infected fibroblasts, where the RNA facilitates viral replication. Here, we report that HCMV infection or treatment of ARPE-19 diploid epithelial cells with DNA-damaging agents, etoposide or zeocin, induces HSATII RNA expression, and a kinase-independent function of ATM is required for the induction. Additionally, various breast cancer cell lines growing in adherent, two-dimensional cell culture express HSATII RNA at different levels, and levels are markedly increased when cells are infected with HCMV or treated with zeocin. High levels of HSATII RNA expression correlate with enhanced migration of breast cancer cells, and knockdown of HSATII RNA reduces cell migration and the rate of cell proliferation. Our investigation links high expression of HSATII RNA to the DNA damage response, centered on a noncanonical function of ATM, and demonstrates a role for the satellite RNA in tumor cell proliferation and movement.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias de la Mama/genética , Infecciones por Citomegalovirus/genética , ADN Satélite/genética , ARN no Traducido/genética , Bleomicina/farmacología , Bleomicina/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/virología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Daño del ADN/efectos de los fármacos , Reparación del ADN , Progresión de la Enfermedad , Etopósido/farmacología , Etopósido/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Aptitud Genética/efectos de los fármacos , Humanos , ARN no Traducido/metabolismo , RNA-Seq , Secuencias Repetitivas de Ácidos Nucleicos , Activación Transcripcional/efectos de los fármacosRESUMEN
Human cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. Our study reveals that levels of several components of the purinergic signaling system, including the P2Y2 and P2X5 receptors, are elevated in HCMV-infected fibroblasts. Knockdown and drug treatment experiments demonstrated that P2Y2 enhances the yield of virus, whereas P2X5 reduces HCMV production. The HCMV IE1 protein induces P2Y2 expression; and P2Y2-mediated signaling is important for efficient HCMV gene expression, DNA synthesis, and the production of infectious HCMV progeny. P2Y2 cooperates with the viral UL37x1 protein to regulate cystolic Ca2+ levels. P2Y2 also regulates PI3K/Akt signaling and infected cell motility. Thus, P2Y2 functions at multiple points within the viral replication cycle to support the efficient production of HCMV progeny, and it may facilitate in vivo viral spread through its role in cell migration.
Asunto(s)
Calcio/metabolismo , Movimiento Celular , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Receptores Purinérgicos P2Y2/metabolismo , Línea Celular , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/patología , ADN Viral/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virología , Expresión Génica , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Antagonistas del Receptor Purinérgico P2/farmacología , Receptores Purinérgicos P2X5/genética , Receptores Purinérgicos P2X5/metabolismo , Receptores Purinérgicos P2Y2/genética , Transducción de Señal , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Human cytomegalovirus (HCMV) infects peripheral blood monocytes and triggers biological changes that promote viral dissemination and persistence. We have shown that HCMV induces a proinflammatory state in infected monocytes, resulting in enhanced monocyte motility and transendothelial migration, prolonged monocyte survival, and differentiation toward a long-lived M1-like macrophage phenotype. Our data indicate that HCMV triggers these changes, in the absence of de novo viral gene expression and replication, through engagement and activation of epidermal growth factor receptor (EGFR) and integrins on the surface of monocytes. We previously identified that HCMV induces the upregulation of multiple proinflammatory gene ontologies, with the interferon-associated gene ontology exhibiting the highest percentage of upregulated genes. However, the function of the HCMV-induced interferon (IFN)-stimulated genes (ISGs) in infected monocytes remained unclear. We now show that HCMV induces the enhanced expression and activation of a key ISG transcriptional regulator, signal transducer and activator of transcription (STAT1), via an IFN-independent but EGFR- and integrin-dependent signaling pathway. Furthermore, we identified a biphasic activation of STAT1 that likely promotes two distinct phases of STAT1-mediated transcriptional activity. Moreover, our data show that STAT1 is required for efficient early HCMV-induced enhanced monocyte motility and later for HCMV-induced monocyte-to-macrophage differentiation and for the regulation of macrophage polarization, suggesting that STAT1 may serve as a molecular convergence point linking the biological changes that occur at early and later times postinfection. Taken together, our results suggest that HCMV reroutes the biphasic activation of a traditionally antiviral gene product through an EGFR- and integrin-dependent pathway in order to help promote the proviral activation and polarization of infected monocytes.IMPORTANCE HCMV promotes multiple functional changes in infected monocytes that are required for viral spread and persistence, including their enhanced motility and differentiation/polarization toward a proinflammatory M1 macrophage. We now show that HCMV utilizes the traditionally IFN-associated gene product, STAT1, to promote these changes. Our data suggest that HCMV utilizes EGFR- and integrin-dependent (but IFN-independent) signaling pathways to induce STAT1 activation, which may allow the virus to specifically dictate the biological activity of STAT1 during infection. Our data indicate that HCMV utilizes two phases of STAT1 activation, which we argue molecularly links the biological changes that occur following initial binding to those that continue to occur days to weeks following infection. Furthermore, our findings may highlight a unique mechanism for how HCMV avoids the antiviral response during infection by hijacking the function of a critical component of the IFN response pathway.
Asunto(s)
Movimiento Celular , Infecciones por Citomegalovirus/genética , Citomegalovirus/patogenicidad , Monocitos/citología , Factor de Transcripción STAT1/genética , Diferenciación Celular , Polaridad Celular , Células Cultivadas , Infecciones por Citomegalovirus/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Redes Reguladoras de Genes , Humanos , Integrinas/genética , Integrinas/metabolismo , Monocitos/metabolismo , Monocitos/virología , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Activación Transcripcional , Regulación hacia ArribaRESUMEN
UNLABELLED: Monocytes play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short life span of monocytes and their antiviral proapoptotic response to infection. We have shown that virally induced upregulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 h postinfection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary antiapoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was upregulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist ABT-199 only reduced the prosurvival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was upregulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is upregulated through binding/activation of epidermal growth factor receptor [EGFR]), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular upregulation of proapoptotic Bax, as small interfering RNA (siRNA)-mediated knockdown of Bax reduced the upregulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a biphasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection. IMPORTANCE: Hematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the establishment of persistent infection and for viral spread to additional hosts. Our system of infected primary human blood monocytes provides us with an opportunity to answer specific questions about viral spread and persistence in in vivo-relevant myeloid cells that cannot be addressed with the more traditionally used replication-permissive cells. Our goal in examining the mechanisms whereby HCMV reprograms infected monocytes to promote viral dissemination is to uncover new targets for therapeutic intervention that would disrupt key viral survival and persistence strategies. Because of this important role in maintaining survival of HCMV-infected monocytes, our new data on the role of Bcl-2 regulation during viral infection represents a promising molecular target for mitigating viral spread and persistence.
Asunto(s)
Citomegalovirus/fisiología , Interacciones Huésped-Patógeno , Monocitos/fisiología , Monocitos/virología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Regulación hacia Arriba , Supervivencia Celular , Células Cultivadas , Humanos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
We have established that HCMV acts as a specific ligand engaging and activating cellular integrins on monocytes. As a result, integrin signaling via Src activation leads to the functional activation of paxillin required for efficient viral entry and for the biological changes in monocytes needed for viral dissemination. These biological/molecular changes allow HCMV to use monocytes as "vehicles" for systemic spread and the establishment of lifelong persistence. However, it remains unresolved how HCMV specifically induces this observed monocyte activation. It was previously demonstrated that the HCMV gH/gL/UL128-131 glycoprotein complex facilitates viral entry into biologically relevant cell types. Nevertheless, the mechanism by which the gH/gL/UL128-131 complex promotes this process is unknown. We now show that only HCMV virions possessing the gH/gL/UL128-131 complex are capable of activating integrin/Src/paxillin-signaling in monocytes. In fibroblasts, this signaling is reversed, such that virus lacking the gH/gL/UL128-131 complex is the only virus able to induce the paxillin activation cascade. The presence of the gH/gL/UL128-131 complex also may have an inhibitory effect on integrin-mediated signaling pathway in fibroblasts. Furthermore, we demonstrate that the presence of the gH/gL/UL128-131 complex on the viral envelope, through its activation of the integrin/Src/paxillin pathway, is necessary for efficient HCMV internalization into monocytes and that appropriate actin and dynamin regulation is critical for this entry process. Importantly, productive infection in monocyte-derived macrophages was seen only in cells exposed to HCMV expressing the gH/gL/UL128-131 complex. From our data, the HCMV gH/gL/U128-131 complex emerges as the specific ligand driving the activation of the receptor-mediated signaling required for the regulation of the actin cytoskeleton and, consequently, for efficient and productive internalization of HCMV into monocytes. To our knowledge, our studies demonstrate a possible molecular mechanism for why the gH/gL/UL128-131 complex dictates HCMV tropism and why the complex is lost as clinical isolates are passaged in the laboratory.
Asunto(s)
Citomegalovirus/inmunología , Glicoproteínas de Membrana/metabolismo , Monocitos/inmunología , Transducción de Señal , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Línea Celular , Células Cultivadas , Citomegalovirus/metabolismo , Dinaminas/antagonistas & inhibidores , Dinaminas/genética , Dinaminas/metabolismo , Endocitosis , Fibroblastos/metabolismo , Fibroblastos/virología , Mutación del Sistema de Lectura , Humanos , Cadenas beta de Integrinas/metabolismo , Glicoproteínas de Membrana/genética , Monocitos/citología , Monocitos/metabolismo , Monocitos/virología , Paxillin/antagonistas & inhibidores , Paxillin/genética , Paxillin/metabolismo , Multimerización de Proteína , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Regulación hacia Arriba , Proteínas del Envoltorio Viral/genéticaRESUMEN
Monocytes are primary targets for human cytomegalovirus (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Biologically, monocytes have a short life span of 48 h in the circulation, a period of time during which monocytes must make a cell fate decision on whether to undergo apoptosis or differentiate into a macrophage. We have previously shown that HCMV infection stimulates monocyte-to-macrophage differentiation; however, the mechanism(s) by which HCMV-infected monocytes simultaneously navigate the 48-h "viability gate" and undergo macrophagic differentiation has remained elusive. Studies have demonstrated that the level of caspase 3 and 8 activities in monocytes may mediate the delicate balance between apoptosis and macrophage colony-stimulating factor (M-CSF)-induced myeloid differentiation. Here, we show that HCMV infection, unlike M-CSF treatment, does not induce caspase 8 activity to promote myeloid differentiation. However, HCMV infection does induce a temporal activation of caspase 3, with only a low level of active caspase 3 being observed after the 48-h viability checkpoint. Consistent with the role of a time-dependent activation of caspase 3 in promoting myeloid differentiation, the inhibition of caspase 3 blocked HCMV-induced monocyte-to-macrophage differentiation. Temporal transcriptome and functional analyses identified heat shock protein 27 (HSP27) and Mcl-1, two known regulators of caspase 3 activation, as being upregulated prior to the 48-h viability gate following HCMV infection. Using small interfering RNAs (siRNAs), we demonstrate that HCMV targets the rapid induction of HSP27 and Mcl-1, which cooperatively function to precisely control caspase 3 activity in order to allow for HCMV-infected monocytes to successfully traverse the 48-h cell fate decision checkpoint and commence macrophage maturation. Overall, this study highlights a unique regulatory mechanism employed by HCMV to tightly modulate the caspase 3 activity needed to promote myeloid differentiation, a key process in the viral dissemination and persistence strategy.
Asunto(s)
Caspasa 3/metabolismo , Citomegalovirus/metabolismo , Macrófagos/virología , Monocitos/virología , Caspasa 8/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula , Humanos , Pulmón/citología , Pulmón/embriología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/citología , Monocitos/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
We have established that human cytomegalovirus (HCMV) infection modulates the biology of target primary peripheral blood monocytes, allowing HCMV to use monocytes as "vehicles" for its systemic spread. HCMV infection of monocytes results in rapid induction of phosphatidylinositol-3-kinase [PI(3)K] and NF-κB activities. Integrins, which are upstream of the PI(3)K and NF-κB pathways, were shown to be involved in HCMV binding to and entry into fibroblasts, suggesting that receptor ligand-mediated signaling following viral binding to integrins on monocytes could trigger the functional changes seen in infected monocytes. We now show that integrin engagement and the activation of the integrin/Src signaling pathway are essential for the induction of HCMV-infected monocyte motility. To investigate how integrin engagement by HCMV triggers monocyte motility, we examined the infected-monocyte transcriptome and found that the integrin/Src signaling pathway regulates the expression of paxillin, which is an important signal transducer in the regulation of actin rearrangement during cell adhesion and movement. Functionally, we observed that paxillin is activated via the integrin/Src signaling pathway and is required for monocyte motility. Because motility is intimately connected to cellular cytoskeletal organization, a process that is also important in viral entry, we investigated the role paxillin regulation plays in the process of viral entry into monocytes. New results confirmed that HCMV entry into target monocytes was significantly reduced in cells deficient in paxillin expression or the integrin/Src/paxillin signaling pathway. From our data, HCMV-cell interactions emerge as an essential trigger for the cellular changes that allow for HCMV entry and hematogenous dissemination.
Asunto(s)
Movimiento Celular , Citomegalovirus/inmunología , Citomegalovirus/patogenicidad , Interacciones Huésped-Patógeno , Monocitos/virología , Paxillin/biosíntesis , Acoplamiento Viral , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Integrinas/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
Monocytes are a primary target for human CMV (HCMV) infection and are a key cell type responsible for hematogenous dissemination of the virus. Biologically, these cells have a short lifespan of 1-3 d in the circulation, yet infected cells remain viable for weeks despite the lack of viral antiapoptotic gene expression during this period. To understand the mechanism by which HCMV inhibits the initial phase of monocyte apoptosis, we focused on the viral modulation of early prosurvival cell signaling events after infection. We demonstrate in this study that the viral upregulation of the PI3K pathway promotes an early block in apoptosis after infection. Temporal transcriptome and protein analyses revealed Mcl-1, a member of the Bcl-2 family, was transiently induced in a PI3K-dependent manner during the early stages of HCMV infection. In accord with the survival studies, virally induced levels of Mcl-1 expression dissipated to mock levels by 72 h postinfection. Through the use of Mcl-1-specific small interfering RNA, we confirmed the functional role that Mcl-1 plays as a key early regulator of apoptosis in monocytes. Lastly, we showed that HCMV engagement and activation of the epidermal growth factor receptor during viral binding triggered the upregulation of Mcl-1. Overall, our data indicates that activation of the epidermal growth factor receptor/PI3K signaling pathway, via the PI3K-dependent upregulation of Mcl-1, is required to circumvent apoptosis in naturally short-lived monocytes during the early stages of HCMV infection, thus ensuring the early steps in the viral persistence strategy.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Supervivencia Celular/inmunología , Citomegalovirus/inmunología , Receptores ErbB/fisiología , Macrófagos/inmunología , Monocitos/inmunología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/fisiología , Muerte Celular/inmunología , Células Cultivadas , Citomegalovirus/patogenicidad , Inducción Enzimática/inmunología , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/fisiología , Inmunofenotipificación , Macrófagos/citología , Macrófagos/virología , Monocitos/citología , Monocitos/enzimología , Monocitos/virología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Regulación hacia Arriba/inmunología , Virulencia/inmunologíaRESUMEN
Human cytomegalovirus (HCMV) rapidly induces a mobile and functionally unique proinflammatory monocyte following infection that is proposed to mediate viral spread. The cellular pathways used by HCMV to initiate these biological changes remain unknown. Here, we document the expression of the epidermal growth factor receptor (EGFR) on the surface of human peripheral blood monocytes but not on other blood leukocyte populations. Inhibition of EGFR signaling abrogated viral entry into monocytes, indicating that EGFR can serve as a cellular tropism receptor. Moreover, HCMV-activated EGFR was required for the induction of monocyte motility and transendothelial migration, two biological events required for monocyte extravasation into peripheral tissue, and thus viral spread. Transcriptome analysis revealed that HCMV-mediated EGFR signaling up-regulated neural Wiskott-Aldrich syndrome protein (N-WASP), an actin nucleator whose expression and function are normally limited in leukocytes. Knockdown of N-WASP expression blocked HCMV-induced but not phorbol 12-myristate 13-acetate (PMA)-induced monocyte motility, suggesting that a switch to and/or the distinct use of a new actin nucleator controlling motility occurs during HCMV infection of monocytes. Together, these data provide evidence that EGFR plays an essential role in the immunopathobiology of HCMV by mediating viral entry into monocytes and stimulating the aberrant biological activity that promotes hematogenous dissemination.
Asunto(s)
Movimiento Celular/fisiología , Citomegalovirus/fisiología , Citomegalovirus/patogenicidad , Receptores ErbB/fisiología , Monocitos/fisiología , Monocitos/virología , Internalización del Virus , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/fisiopatología , Infecciones por Citomegalovirus/virología , Endotelio Vascular/patología , Endotelio Vascular/virología , Receptores ErbB/química , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno/fisiología , Humanos , Técnicas In Vitro , Monocitos/efectos de los fármacos , Monocitos/patología , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Regulación hacia Arriba , Proteína Neuronal del Síndrome de Wiskott-Aldrich/antagonistas & inhibidores , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Human cytomegalovirus (HCMV) is a betaherpesvirus with a global seroprevalence of 60-90%. HCMV is the leading cause of congenital infections and poses a great health risk to immunocompromised individuals. Although HCMV infection is typically asymptomatic in the immunocompetent population, infection can result in mononucleosis and has also been associated with the development of certain cancers, as well as chronic inflammatory diseases such as various cardiovascular diseases. In immunocompromised patients, including AIDS patients, transplant recipients, and developing fetuses, HCMV infection is associated with increased rates of morbidity and mortality. Currently there is no vaccine for HCMV and there is a need for new pharmacological treatments. Ongoing research seeks to further define the complex aspects of HCMV pathogenesis, which could potentially lead to the generation of new therapeutics to mitigate the disease states associated with HCMV infection. The following chapter reviews the advancements in our understanding of HCMV pathogenesis in the immunocompetent and immunocompromised hosts.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Citomegalovirus/metabolismo , Citomegalovirus/fisiología , Citomegalovirus/genética , Citomegalovirus/patogenicidad , ADN Viral/genética , Humanos , Huésped Inmunocomprometido/inmunología , Estudios SeroepidemiológicosRESUMEN
Tandem satellite repeats account for 3% of the human genome. One of them, Human Satellite II (HSATII), is highly expressed in several epithelial cancers and cancer cell lines. Here we report an acute induction of HSATII RNA in human cells infected with two herpes viruses. We show that human cytomegalovirus (HCMV) IE1 and IE2 proteins cooperate to induce HSATII RNA affecting several aspects of the HCMV replication cycle, viral titers and infected-cell processes. HSATII RNA expression in tissue from two chronic HCMV colitis patients correlates with the strength of CMV antigen staining. Thus, endogenous HSATII RNA synthesis after herpesvirus infections appears to have functionally important consequences for viral replication and may provide a novel insight into viral pathogenesis. The HSATII induction seen in both infected and cancer cells suggests possible convergence upon common HSATII-based regulatory mechanisms in these seemingly disparate diseases.
Asunto(s)
Secuencias Repetitivas Esparcidas/fisiología , Línea Celular , Movimiento Celular , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Herpesviridae , Genética Humana , Humanos , Hibridación in Situ , ARN/genética , ARN/metabolismo , Regulación hacia ArribaRESUMEN
Human cytomegalovirus (HCMV) is a human pathogen that infects greater than 50 % of the human population. HCMV infection is usually asymptomatic in most individuals. That is, primary infection or reactivation of latent virus is generally clinically silent. HCMV infection, however, is associated with significant morbidity and mortality in the immunocompromised and chronic inflammatory diseases in the immunocompetent. In immunocompromised individuals (acquired immune deficiency syndrome and transplant patients, developing children (in utero), and cancer patients undergoing chemotherapy), HCMV infection increases morbidity and mortality. In those individuals with a normal immune system, HCMV infection is also associated with a risk of serious disease, as viral infection is now considered to be a strong risk factor for the development of various vascular diseases and to be associated with some types of tumor development. Intense research is currently being undertaken to better understand the mechanisms of viral pathogenesis that are briefly discussed in this chapter.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/patología , Infecciones por Citomegalovirus/patología , Citomegalovirus/patogenicidad , Biología Molecular/métodos , Síndrome de Inmunodeficiencia Adquirida/virología , Infecciones por Citomegalovirus/virología , Humanos , Huésped Inmunocomprometido , Trasplante/efectos adversos , Activación Viral/genéticaRESUMEN
HCMV pathogenesis is a direct consequence of the hematogenous dissemination of the virus to multiple host organ sites. The presence of infected monocytes in the peripheral blood and organs of individuals exhibiting primary HCMV infection have long suggested that these blood sentinels are responsible for mediating viral spread. Despite monocytes being "at the right place at the right time", their short lifespan and the lack of productive viral infection in these cells complicate this scenario of a monocyte-driven approach to viral dissemination by HCMV. However, our laboratory has provided evidence that HCMV infection is able to induce a highly controlled polarization of monocytes toward a unique and long-lived proinflammatory macrophage, which we have demonstrated to be permissive for viral replication. These observations suggest that HCMV has evolved as a distinct mechanism to induce select proinflammatory characteristics that provide infected monocytes with the necessary tools to mediate viral spread following a primary infection. In the absence of viral gene products during the early stages of infection, the process by which HCMV "tunes" the inflammatory response in infected monocytes to promote viral spread and subsequently, viral persistence remains unclear. In this current review, we focus on the viral entry process of HCMV and the potential role of receptor-ligand interactions in modulating monocyte biology. Specifically, we examine the signaling pathways initiated by the distinct combination of cellular receptors simultaneously engaged and activated by HCMV during viral entry and how the acquisition of this distinct signalsome results in a nontraditional activation of monocytes leading to the induction of the unique, functional attributes observed in monocytes following HCMV infection.
Asunto(s)
Citomegalovirus/patogenicidad , Monocitos/virología , Transducción de Señal/fisiología , Internalización del Virus , Infecciones por Citomegalovirus/etiología , Receptores ErbB/análisis , Receptores ErbB/fisiología , Humanos , Integrinas/fisiología , Monocitos/fisiología , Receptores Virales/fisiologíaRESUMEN
Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles(1,2,3). Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread(4,5). Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell(6,7,8). However, many biological questions about cell migration remain unanswered. The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers. The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip(9,10). With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells(11,12), fibroblasts(9), neutrophils(13), skeletal muscle cells(14), keratinocytes(15), trophoblasts(16), endothelial cells(17), and monocytes(10,18-22). The protocol involves the creation of slides coated with gold nanoparticles (Au°) that are generated by a reduction of chloroauric acid (Au(3+)) by sodium citrate. This method was developed by Turkevich et al. in 1951(23) and then improved in the 1970s by Frens et al.(24,25). As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses(9,26,27). In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.
Asunto(s)
Movimiento Celular/fisiología , Técnicas Citológicas/métodos , Fagocitos/citología , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/sangre , Oro Coloide/química , Humanos , Nanopartículas del Metal/química , Monocitos/citología , Monocitos/virologíaRESUMEN
We documented that the NF-kappaB signaling pathway was rapidly induced following human cytomegalovirus (HCMV) infection of human fibroblasts and that this induced NF-kappaB activity promoted efficient transactivation of the major immediate-early promoter (MIEP). Previously, we showed that the major HCMV envelope glycoproteins, gB and gH, initiated this NF-kappaB signaling event. However, we also hypothesized that there were additional mechanisms utilized by the virus to rapidly upregulate NF-kappaB. In this light, we specifically hypothesized that the HCMV virion contained IkappaBalpha kinase activity, allowing for direct phosphorylation of IkappaBalpha following virion entry into infected cells. In vitro kinase assays performed on purified HCMV virion extract identified bona fide IkappaBalpha kinase activity in the virion. The enzyme responsible for this kinase activity was identified as casein kinase II (CKII), a cellular serine-threonine protein kinase. CKII activity was necessary for efficient transactivation of the MIEP and IE gene expression. CKII is generally considered to be a constitutively active kinase. We suggest that this molecular characteristic of CKII represents the biologic rationale for the viral capture and utilization of this kinase early after infection. The packaging of CKII into the HCMV virion identifies that diverse molecular mechanisms are utilized by HCMV for rapid NF-kappaB activation. We propose that HCMV possesses multiple pathways to increase NF-kappaB activity to ensure that the correct temporal regulation of NF-kappaB occurs following infection and that sufficient threshold levels of NF-kappaB are reached in the diverse array of cells, including monocytes and endothelial cells, infected in vivo.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Citomegalovirus/fisiología , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Western Blotting , Células Cultivadas , Citomegalovirus/química , Citomegalovirus/enzimología , Citomegalovirus/inmunología , Genes Inmediatos-Precoces , Humanos , Inmunoprecipitación , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Activación Transcripcional , Proteínas Virales/biosíntesis , Virión/química , Virión/enzimología , Virión/fisiologíaRESUMEN
Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-kappaB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498-4507). Because a second, sustained increase in NF-kappaB activity following the initial phase of NF-kappaB activation was also observed, we investigated the role that this prolonged NF-kappaB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-kappaB activation was blocked by pretreatment with NF-kappaB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-kappaB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-kappaB in the regulation of the later class of viral genes.