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Articles by Andrew D. Yurochko 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 Andrew D. Yurochko on PubMed

Human Cytomegalovirus-caused Damage to Placental Trophoblasts Mediated by Immediate-early Gene-induced Tumor Necrosis Factor-alpha

Infection of the fetal epithelium (trophoblast) lining the villous placenta by human cytomegalovirus (HCMV) accompanies placental inflammations and fetal intrauterine growth restriction. However, the consequences of infection on the villous trophoblast have not been explored. We show that HCMV infection of primary immature (cytotrophoblast-like) or mature (syncytiotrophoblast-like) cultures results in loss of half of the cells within 24 hours of virus challenge. Two-color immunofluorescence of HCMV immediate early (IE) gene expression and apoptosis (terminal dUTP nick-end labeling) revealed apoptosis only in uninfected cells. Antibody to tumor necrosis factor (TNF)-alpha completely inhibited infection-induced trophoblast apoptosis and cell loss, as did co-incubation with epidermal growth factor, known to inhibit trophoblast apoptosis. Transfection with HCMV immediate early- (IE)1-72 and IE2-86, but not IE2-55, expression plasmids induced paracrine trophoblast apoptosis inhibitable by epidermal growth factor or antibody to TNF-alpha. These results show that HCMV infection of villous trophoblasts leads to rapid loss of neighboring cells mediated by viral IE protein-induced TNF-alpha secretion. We propose that HCMV infection damages the placental trophoblast barrier by accelerating trophoblast turnover and decreasing its capacity for renewal.

DC-SIGN and L-SIGN Can Act As Attachment Receptors for Alphaviruses and Distinguish Between Mosquito Cell- and Mammalian Cell-derived Viruses

C-type lectins such as DC-SIGN and L-SIGN, which bind mannose-enriched carbohydrate modifications of host and pathogen proteins, have been shown to bind glycoproteins of several viruses and facilitate either cis or trans infection. DC-SIGN and L-SIGN are expressed in several early targets of arbovirus infection, including dendritic cells (DCs) and cells of the reticuloendothelial system. In the present study, we show that DC-SIGN and L-SIGN can function as attachment receptors for Sindbis (SB) virus, an arbovirus of the Alphavirus genus. Human monocytic THP-1 cells stably transfected with DC-SIGN or L-SIGN were permissive for SB virus replication, while untransfected controls were essentially nonpermissive. The majority of control THP-1 cells were permissive when attachment and entry steps were eliminated through electroporation of virus transcripts. Infectivity for the DC-SIGN/L-SIGN-expressing cells was largely blocked by yeast mannan, EDTA, or a DC-SIGN/L-SIGN-specific monoclonal antibody. Infection of primary human DCs by SB virus was also dependent upon SIGN expression by similar criteria. Furthermore, production of virus particles in either C6/36 mosquito cells or CHO mammalian cells under conditions that limited complex carbohydrate content greatly increased SB virus binding to and infection of THP-1 cells expressing these lectins. C6/36-derived virus also was much more infectious for primary human DCs than CHO-derived virus. These results suggest that (i) lectin molecules such as DC-SIGN and L-SIGN may represent common attachment receptor molecules for arthropod-borne viruses, (ii) arbovirus particles produced in and delivered by arthropod vectors may preferentially target vertebrate host cells bearing these or similar lectin molecules, and (iii) a cell line has been identified that can productively replicate alphaviruses but is deficient in attachment receptors.

Human Cytomegalovirus Induces Monocyte Differentiation and Migration As a Strategy for Dissemination and Persistence

Human cytomegalovirus (HCMV) pathogenesis is characterized by multiple organ system involvement due to viral spread to host organs after a cell-associated viremia. The cell type responsible for HCMV dissemination is unknown. Monocytes are the most likely candidate since they are the predominant cell type infected in the blood. However, monocytes are not productive for viral replication and are abortively infected. The results presented here provide a potential answer to this conundrum. We report that primary HCMV infection of monocytes induces transendothelial migration and monocyte-to-macrophage differentiation and that these HCMV-differentiated macrophages are productive for viral replication. Together, our data suggest a novel mechanism for HCMV pathogenesis; HCMV induces cellular changes in monocytes to promote viral replication and spread to host organs.

Activation of the NF-kappaB Pathway in Human Cytomegalovirus-infected Cells is Necessary for Efficient Transactivation of the Major Immediate-early Promoter

We previously demonstrated that human cytomegalovirus (HCMV) infection induced the activation of the cellular transcription factor NF-kappaB. Here, we investigate the mechanism for the HCMV-induced NF-kappaB activation and the role that the induced NF-kappaB plays in transactivation of the major immediate-early promoter (MIEP) and production of immediate-early (IE) proteins. Using a dominant-negative inhibitor of NF-kappaB, the IkappaB-superrepressor, we demonstrated that active NF-kappaB is critical for transactivation of the HCMV MIEP. Investigation of the mechanisms of NF-kappaB activation following HCMV infection showed a rapid and sustained decrease in the inhibitors of NF-kappaB, IkappaBalpha and IkappaBbeta. Because the IkappaB kinases (IKKs) regulate the degradation of the IkappaBs, virus-mediated changes in the IKKs were examined next. Using dominant-negative forms of the IKKs, we showed significant decreases in transactivation of the MIEP in the presence of these mutants. In addition, protein levels of members of the IKK complex and IKK kinase activity were upregulated throughout the time course of infection. Lastly, the role NF-kappaB plays in HCMV IE mRNA and protein production during infection was examined. Using aspirin and MG-132, we demonstrated that production of IE protein and mRNA was significantly decreased and delayed in infected cells treated with these drugs. Together, the results of these studies suggest that virus-mediated NF-kappaB activation, through the dysregulation of the IKK complex, plays a primary role in the initiation of the HCMV gene cascade in fibroblasts and may provide new targets for therapeutic intervention.

HCMV Activates PI(3)K in Monocytes and Promotes Monocyte Motility and Transendothelial Migration in a PI(3)K-dependent Manner

Human cytomegalovirus (HCMV) is a leading cause of morbidity and mortality in immunocompromised hosts. In immunocompetent hosts, HCMV is associated with chronic inflammatory diseases including atherosclerosis. Monocytes and macrophages are proposed to play key roles in HCMV dissemination to host tissue, and their infection provides a biological link between the lifecycle of HCMV and disease pathology. We hypothesize that viral spread occurs via a mechanism in which infected peripheral blood monocytes, which are nonpermissive for viral replication, extravasate into host tissue and subsequently differentiate into permissive macrophages. Supporting this hypothesis, we recently showed that HCMV specifically induced the differentiation of monocytes into macrophages that become permissive for viral replication. To expand our understanding of HCMV pathogenesis, we next examined monocyte activation and migration, the first events in viral pathogenesis. We show here that HCMV up-regulates phosphatidylinositol 3,4,5 triphosphate kinase [PI(3)K] activity and that this increased PI(3)K activity is essential for infected monocyte-transendothelial migration. This increase in migration occurs through the up-regulation of cell motility in a PI(3)K-dependent process. Last, we show that these activated monocytes express a number of inflammatory mediators via PI(3)K signaling. We propose that the up-regulation of monocyte migration and immune mediators by HCMV infection is required for the hematogenous dissemination of the virus and as a consequence, could promote chronic inflammatory diseases associated with HCMV infection.

Human Cytomegalovirus IE1-72 Activates Ataxia Telangiectasia Mutated Kinase and a P53/p21-mediated Growth Arrest Response

Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type (p53(+/+)) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19(Arf) and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G(1) to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.

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.

Human Cytomegalovirus (HCMV) Infection of Endothelial Cells Promotes Naive Monocyte Extravasation and Transfer of Productive Virus to Enhance Hematogenous Dissemination of HCMV

Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of naïve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased naïve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.

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.

Roles of Phosphatidylinositol 3-kinase and NF-kappaB in Human Cytomegalovirus-mediated Monocyte Diapedesis and Adhesion: Strategy for Viral Persistence

Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-kappaB activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the beta(1) integrins, the beta(2) integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-kappaB affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-kappaB signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.

Transcriptome Analysis of NF-kappaB- and Phosphatidylinositol 3-kinase-regulated Genes in Human Cytomegalovirus-infected Monocytes

Human cytomegalovirus induces a proinflammatory monocyte following infection, and we have evidence that NF-kappaB and phosphatidylinositol 3-kinase [PI(3)K] are key mediators in this early activation. To begin to address how these signaling pathways are responsible for the rapid activation of infected monocytes, we examined the role that these pathways played in the transcriptome of infected monocytes. Global transcriptional profiling using cDNA microarrays revealed that a significant number of genes, including inflammatory genes, were regulated in an NF-kappaB- and/or PI(3)K-dependent manner, identifying the NF-kappaB and PI(3)K pathways as key cellular control points in the conversion of monocytes to an activated proinflammatory state following HCMV infection.

Human CMV Infection of Endothelial Cells Induces an Angiogenic Response Through Viral Binding to EGF Receptor and Beta1 and Beta3 Integrins

Human cytomegalovirus (HCMV) infection is associated with atherosclerosis, transplant vascular sclerosis, and coronary restenosis. A common theme in these vascular diseases is an increased rate of angiogenesis. Angiogenesis is a complex biological process mediated by endothelial cell (EC) proliferation, migration, and morphogenesis. Although angiogenesis is a normal process in the host, its dysregulation, after viral infection or injury to the vessel wall, is associated with plaque development in atherosclerotic patients. We now document that HCMV infection results in increased EC proliferation, motility, and capillary tube formation. The observed HCMV-induced angiogenic response depended on viral binding to and signaling through the beta(1) and beta(3) integrins and the epidermal growth factor receptor, via their ability to activate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase signaling pathways. Because a proangiogenic response drives the neovascularization observed in atherosclerotic disease, our findings identify a possible mechanism for how HCMV infection contributes to vascular disease.

Transcriptome Analysis Reveals Human Cytomegalovirus Reprograms Monocyte Differentiation Toward an M1 Macrophage

Monocytes are primary targets for human CMV (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Monocytes acquire different functional traits during polarization to the classical proinflammatory M1 macrophage or the alternative antiinflammatory M2 macrophage. We hypothesized that HCMV induced a proinflammatory M1 macrophage following infection to promote viral dissemination because, biologically, a proinflammatory state provides the tools to drive infected monocytes from the blood into the tissue. To test this hypothesis of monocyte conversion from a normal quiescent phenotype to an inflammatory phenotype, we used Affymetrix Microarray to acquire a transcriptional profile of infected monocytes at a time point our data emphasized is a key temporal regulatory point following infection. We found that HCMV significantly up-regulated 583 (5.2%) of the total genes and down-regulated 621 (5.5%) of the total genes>or=1.5-fold at 4 h postinfection. Further ontology analysis revealed that genes implicated in classical M1 macrophage activation were stimulated by HCMV infection. We found that 65% of genes strictly associated with M1 polarization were up-regulated, while only 4% of genes solely associated with M2 polarization were up-regulated. Analysis of the monocyte chemokinome at the transcriptional level showed that 44% of M1 and 33% of M2 macrophage chemokines were up-regulated. Proteomic analysis using chemokine Ab arrays confirmed the secretion of these chemotactic proteins from HCMV-infected monocytes. Overall, the results identify that the HCMV-infected monocyte transcriptome displayed a unique M1/M2 polarization signature that was skewed toward the classical M1 activation phenotype.

NF-kappaB and Phosphatidylinositol 3-kinase Activity Mediates the HCMV-induced Atypical M1/M2 Polarization of Monocytes

Human cytomegalovirus infection of monocytes stimulates a unique monocyte differentiation reprogramming resulting in polarization towards an M1 pro-inflammatory macrophage that simultaneously exhibits characteristics of an M2 anti-inflammatory macrophage. Our laboratory has previously shown that HCMV infection stimulates monocyte NF-kappaB and PI(3)K activities and now provides evidence that these cellular factors are essential for the HCMV-induced polarization of infected monocytes/macrophages. We find that the induction of NF-kappaB and PI(3)K activities following HCMV infection was required for the initiation of monocyte-to-macrophage differentiation. HCMV-infected monocytes treated with Bay11-7802 (an inhibitor of NF-kappaB activity) or LY294002 [an inhibitor of PI(3)K activity] prior to infection exhibited a small, round and monocyte-like undifferentiated morphology and the lack of CD68 upregulation (a macrophage differentiation marker). Detailed transcriptome analysis revealed 48%, 7% and 31% of HCMV-induced M1-associated genes were dependent on NF-kappaB, PI(3)K or both activities, respectively; while 100% of HCMV-induced M2-associated genes required both NF-kappaB and PI(3)K activities. Functionally, we demonstrated that NF-kappaB and PI(3)K activities were critical for the production of M1- and M2-associated cytokines/chemokines, in HCMV-induced differentiating monocytes. Supernatant from HCMV-infected monocytes pretreated with Bay11-7802 or LY294002 exhibited an 80% and 67% reduction in cell motility-inducing activity. Overall, these data show that HCMV usurps monocyte NF-kappaB and PI(3)K signal transduction pathways to induce the unique polarization of HCMV-infected monocytes needed for the earliest steps in the viral dissemination and persistence strategy.

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.

Cytomegalovirus Infection Leads to Microvascular Dysfunction and Exacerbates Hypercholesterolemia-induced Responses

Cytomegalovirus (CMV) persistently infects more than 60% of the worldwide population. In immunocompetent hosts, it has been implicated in several diseases, including cardiovascular disease, possibly through the induction of inflammatory pathways. Cardiovascular risk factors promote an inflammatory phenotype in the microvasculature long before clinical disease is evident. This study determined whether CMV also impairs microvascular homeostasis and synergizes with hypercholesterolemia to exaggerate these responses. Intravital microscopy was used to assess endothelium-dependent and -independent arteriolar vasodilation and venular leukocyte and platelet adhesion in mice after injection with either mock inoculum or murine CMV (mCMV). Mice were fed a normal (ND) or high-cholesterol (HC) diet beginning at 5 weeks postinfection (p.i.), or a HC diet for the final 4 weeks of infection. mCMV-ND mice exhibited impaired endothelium-dependent vasodilation versus mock-ND at 9 and 12 weeks and endothelium-independent arteriolar dysfunction by 24 weeks. Transient mild leukocyte adhesion occurred in mCMV-ND venules at 7 and 21 weeks p.i. HC alone caused temporary arteriolar dysfunction and venular leukocyte and platelet recruitment, which were exaggerated and prolonged by mCMV infection. The time of introduction of HC after mCMV infection determined whether mCMV+HC led to worse venular inflammation than either factor alone. These findings reveal a proinflammatory influence of persistent mCMV on the microvasculature, and suggest that mCMV infection enhances microvasculature susceptibility to both inflammatory and thrombogenic responses caused by hypercholesterolemia.

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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