RESUMEN
Of the many research challenges posed by the study of human cytomegalovirus (HCMV) latency, one of the most notable is the requirement for the use of primary hematopoietic cell culture. Culturing hematopoietic progenitor subpopulations requires that consideration be given to maintaining their physiological relevance. We describe a long-standing primary CD34+ hematopoietic progenitor cell (HPC) system as an in vitro model to study HCMV latent infection. Key aspects of the model include infection of primary human CD34+ HPCs prior to ex vivo expansion, a long-term culture with a stromal cell support designed to maintain the ability of stem cells to support hematopoietic reconstitution, and an assay to quantify infectious centers produced prior to and following a reactivation stimulus. Importantly, this system has been used to identify a number of viral determinants of latency or reactivation and findings have been recapitulated in vivo using a humanized mouse model for HCMV latency. Therefore, this system offers a powerful approach to defining virus-host interactions and mechanisms important for HCMV latency and reactivation.
Asunto(s)
Citomegalovirus/metabolismo , Cultivo Primario de Células/métodos , Latencia del Virus/fisiología , Antígenos CD34/metabolismo , Diferenciación Celular , Infecciones por Citomegalovirus/virología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Transducción de Señal , Proteínas Virales , Tropismo Viral/genética , Tropismo Viral/fisiología , Activación Viral/genética , Activación Viral/fisiologíaRESUMEN
Blood monocytes mediate the hematogenous dissemination of human cytomegalovirus (HCMV) in the host. However, monocytes have a short 48-hour (h) lifespan and are not permissive for viral replication. We previously established that HCMV infection drives differentiation of monocytes into long-lived macrophages to mediate viral dissemination, though the mechanism was unclear. Here, we found that HCMV infection promoted monocyte polarization into distinct macrophages by inducing select M1 and M2 differentiation markers and that Akt played a central role in driving differentiation. Akt's upstream positive regulators, PI3K and SHIP1, facilitated the expression of the M1/M2 differentiation markers with p110δ being the predominant PI3K isoform inducing differentiation. Downstream of Akt, M1/M2 differentiation was mediated by caspase 3, whose activity was tightly regulated by Akt in a temporal manner. Overall, this study highlights that HCMV employs the PI3K/SHIP1/Akt pathway to regulate caspase 3 activity and drive monocyte differentiation into unique macrophages, which is critical for viral dissemination.
Asunto(s)
Diferenciación Celular , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Infecciones por Citomegalovirus/fisiopatología , Citomegalovirus/fisiología , Macrófagos/citología , Monocitos/citología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/genética , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Humanos , Macrófagos/metabolismo , Monocitos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de SeñalRESUMEN
Human cytomegalovirus (HCMV) is a beta herpesvirus that establishes a life-long persistence in the host, like all herpesviruses, by way of a latent infection. During latency, viral genomes are maintained in a quieted state. Virus replication can be reactivated from latency in response to changes in cellular signaling caused by stress or differentiation. The past decade has brought great insights into the molecular basis of HCMV latency. Here, we review the complex persistence of HCMV with consideration of latent reservoirs, viral determinants and their host interactions, and host signaling and the control of cellular and viral gene expression that contributes to the establishment of and reactivation from latency.
Asunto(s)
Citomegalovirus/genética , Regulación Viral de la Expresión Génica , Genoma Viral , Interacciones Huésped-Patógeno , Activación Viral/genética , Latencia del Virus/genética , Células de la Médula Ósea/virología , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/virología , Células Epiteliales/virología , Humanos , Macrófagos/virología , Transducción de Señal , Replicación Viral/genéticaRESUMEN
Primary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. In order to facilitate viral spread, HCMV extends the naturally short 48-h lifespan of monocytes by stimulating a non-canonical activation of Akt during viral entry, which leads to the increased expression of a specific subset of antiapoptotic proteins. In this study, global analysis of the Akt signaling network showed HCMV induced a more robust activation of the entire network when compared to normal myeloid growth factors. Furthermore, we found a unique interplay between HCMV-activated Akt and the stress response transcription heat shock factor 1 (HSF1) that allowed for the synthesis of both cap- and internal ribosome entry site (IRES)-containing antiapoptotic mRNAs such as myeloid cell leukemia-1 (Mcl-1) and X-linked inhibitor of apoptosis (XIAP), respectively. As generally a switch from cap-dependent to IRES-mediated translation occurs during cellular stress, the ability of HCMV to concurrently drive both types of translation produces a distinct milieu of prosurvival proteins needed for the viability of infected monocytes. Indeed, we found inhibition of XIAP led to death of â¼99% of HCMV-infected monocytes while having minimal effect on the viability of uninfected cells. Taken together, these data indicate that the aberrant activation of the Akt network by HCMV induces the upregulation of a unique subset of antiapoptotic proteins specifically required for the survival of infected monocytes. Consequently, our study highlights the possibility of exploiting these virus-induced changes to prevent viral spread in immunocompromised patients at high-risk for HCMV exposure.
Asunto(s)
Citomegalovirus/fisiología , Monocitos/virología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular , Supervivencia Celular , Proteínas de Choque Térmico HSP27/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , ARN Mensajero , Regulación hacia Arriba , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
UNLABELLED: Human cytomegalovirus (HCMV) is a pervasive herpesvirus responsible for significant morbidity and mortality among immunodeficient/naive hosts. Following a primary HCMV infection, circulating blood monocytes mediate the systemic spread of the virus. Extending the short 48-h life span of monocytes is critical to the viral dissemination process, as these blood-borne cells are nonpermissive for virus replication until they are fully differentiated into macrophages. Here, we show that HCMV glycoprotein gB binding to cellular epidermal growth factor receptor (EGFR) during HCMV entry initiated a rapid (within 15 min) activation of the apoptosis suppressor Akt, which was maintained through 72 h. The virus-induced activation of Akt was more robust than that with the normal myeloid growth factor macrophage colony-stimulating factor (M-CSF) and was essential for infected monocytes to bypass the 48-h viability checkpoint. Activation of phosphoinositide 3-kinase (PI3K) following EGFR engagement by HCMV mediated the phosphorylation of Akt. Moreover, HCMV entry drove a switch away from the PI3K p110δ isoform, which was required for the viability of uninfected monocytes, to the p110ß isoform in order to facilitate the Akt-dependent prosurvival state within infected cells. Simultaneously, in contrast to M-CSF, HCMV promoted a rapid increase in SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) expression, leading to signaling through a noncanonical Akt activation pathway. To ensure maximum Akt activity, HCMV also induced an early phosphorylation-dependent inactivation of the negative regulator phosphatase and tensin homolog. Overall, our data indicate that HCMV hijacks the upstream Akt signaling network to induce a nontraditional activation of Akt and subsequently a prosurvival decision at the 48-h cell fate checkpoint, a vital step for HCMV's dissemination and persistence strategy. IMPORTANCE: HCMV is found throughout the world with a prevalence of 55 to 100% within the human population. HCMV infection is generally asymptomatic in immunocompetent or naive individuals but is a significant cause of morbidity and mortality among the immunocompromised. Widespread organ inflammation is associated with symptomatic infections, which is a direct consequence of the viral dissemination strategy. Inflammatory peripheral blood monocytes facilitate the spread of HCMV. However, HCMV must subvert the naturally short life span of monocytes. In this work, we demonstrate that HCMV induces the activation of Akt, an antiapoptotic protein, in a manner distinct from that of normal myeloid growth factors. Moreover, we decipher how HCMV dysregulates the upstream Akt signaling network during viral entry to promote an Akt-dependent prosurvival state following infection. Delineation of the virus-specific mechanisms that regulate cellular prosurvival pathways in order to drive the survival of HCMV-infected monocytes is important to identifying new anti-HCMV therapeutic targets.
Asunto(s)
Citomegalovirus , Factor Estimulante de Colonias de Macrófagos/metabolismo , Monocitos/virología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Internalización del Virus , Células Cultivadas , Interacciones Huésped-Patógeno , Humanos , Monocitos/metabolismo , Monocitos/patología , Fosforilación , Transducción de SeñalRESUMEN
UNLABELLED: Primary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. To facilitate viral spread, we have previously shown HCMV to extend the short 48-h life span of monocytes. Mechanistically, HCMV upregulated two specific cellular antiapoptotic proteins, myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the two proteolytic cleavages necessary for the formation of fully active caspase 3 and the subsequent initiation of apoptosis. We now show that HCMV more robustly upregulated Mcl-1 than normal myeloid growth factors and that Mcl-1 was the only myeloid survival factor to rapidly induce HSP27 prior to the 48-h cell fate checkpoint. We determined that HCMV glycoproteins gB and gH signal through the cellular epidermal growth factor receptor (EGFR) and αvß3 integrin, respectively, during viral entry in order to drive the increase of Mcl-1 and HSP27 in an Akt-dependent manner. Although Akt is known to regulate protein stability and transcription, we found that gB- and gH-initiated signaling preferentially and cooperatively stimulated the synthesis of Mcl-1 and HSP27 through mTOR-mediated translation. Overall, these data suggest that the unique signaling network generated during the viral entry process stimulates the upregulation of select antiapoptotic proteins allowing for the differentiation of short-lived monocytes into long-lived macrophages, a key step in the viral dissemination strategy. IMPORTANCE: Human cytomegalovirus (HCMV) infection is endemic within the human population. Although primary infection is generally asymptomatic in immunocompetent individuals, HCMV is a significant cause of morbidity and mortality in the immunocompromised. The multiorgan inflammatory diseases associated with symptomatic HCMV infection are a direct consequence of the monocyte-mediated systemic spread of the virus. In order for peripheral blood monocytes to facilitate viral dissemination, HCMV subverts the short 48-h life span of monocytes by inducing the expression of cellular antiapoptotic proteins Mcl-1 and HSP27. Here, we demonstrate that the rapid and simultaneous upregulation of Mcl-1 and HSP27 is a distinctive feature of HCMV-induced monocyte survival. Moreover, we decipher the signaling pathways activated during viral entry needed for the robust synthesis of Mcl-1 and HSP27. Identifying the virus-specific mechanisms used to upregulate select cellular factors required for the survival of HCMV-infected monocytes is important to the development of new classes of anti-HCMV drugs.
Asunto(s)
Citomegalovirus/fisiología , Proteínas de Choque Térmico HSP27/biosíntesis , Interacciones Huésped-Patógeno , Monocitos/virología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Activación Transcripcional , Internalización del Virus , Supervivencia Celular , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Transducción de Señal , Factores de TiempoRESUMEN
Herpesviruses, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus, establish latency by modulating or mimicking antiapoptotic Bcl-2 proteins to promote survival of carrier cells. BH3 profiling, which assesses the contribution of Bcl-2 proteins towards cellular survival, was able to globally determine the level of dependence on individual cellular and viral Bcl-2 proteins within latently infected cells. Moreover, BH3 profiling predicted the sensitivity of infected cells to small-molecule inhibitors of Bcl-2 proteins.