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Articles by Maciej T. Nogalski in JoVE

 JoVE Immunology and Infection

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

1Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 2Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, 3Department of Microbiology and Immunology, SUNY Upstate Medical University, 4Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center


JoVE 4165

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Other articles by Maciej T. Nogalski on PubMed

Prolonged Activation of NF-kappaB by Human Cytomegalovirus Promotes Efficient Viral Replication and Late Gene Expression

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.

The Human Cytomegalovirus Virion Possesses an Activated Casein Kinase II That Allows for the Rapid Phosphorylation of the Inhibitor of NF-kappaB, IkappaBalpha

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.

Activation of EGFR on Monocytes is Required for Human Cytomegalovirus Entry and Mediates Cellular Motility

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.

PI3K-dependent Upregulation of Mcl-1 by Human Cytomegalovirus is Mediated by Epidermal Growth Factor Receptor and Inhibits Apoptosis in Short-lived Monocytes

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.

Human Cytomegalovirus-regulated Paxillin in Monocytes Links Cellular Pathogenic Motility to the Process of Viral Entry

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.

Human Cytomegalovirus Induction of a Unique Signalsome During Viral Entry into Monocytes Mediates Distinct Functional Changes: a Strategy for Viral Dissemination

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.

Human Cytomegalovirus Stimulates Monocyte-to-macrophage Differentiation Via the Temporal Regulation of Caspase 3

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.

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