A Permanently Growing Human Endothelial Cell Line Supports Productive Infection With Human Cytomegalovirus Under Conditional Cell Growth Arrest

Lieber, Diana; Hochdorfer, Daniel; Stoehr, Dagmar; Schubert, Axel; Lotfi, Ramin; May, Tobias; Wirth, Dagmar; Sinzger, Christian

doi:10.2144/000114326

Cited by 24 publications

(32 citation statements)

References 28 publications

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“…We previously showed that EA.hy926 cells support entry and expression of immediate early antigens of HCMV strains with a broad cell tropism, such as TB40/E, but not those of strains with narrow cell tropism, thus resembling primary endothelial cells with regard to the initial phase of infection (41). Therefore, EA.hy926 cells qualify for analysis of the viral entry mechanism in endothelial cells.…”

Section: Rnai Screen For Host Factors In Hcmv Infectionmentioning

confidence: 99%

“…Proliferation of HEC-LTT cells can be controlled by switching between silent and active states of transgenes encoding the SV40 T antigen and human telomerase. Recently, we adopted the cell line as a cell culture model for endothelial cell infection by HCMV (41). Like primary HUVEC, HEC-LTTs are susceptible to highly endotheliotropic virus strains and release comparable amounts of infectious virus, but they scarcely allow infection by poorly endotheliotropic strains (41).…”

Section: Rnai Screen For Host Factors In Hcmv Infectionmentioning

confidence: 99%

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Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection

Hochdorfer¹,

Florin

Sinzger³

et al. 2016

J Virol

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Human cytomegalovirus (HCMV), a betaherpesvirus, can cause life-threatening disease in immunocompromised individuals. Viral envelope glycoproteins that mediate binding to and penetration into target cells have been identified previously. In contrast, cellular proteins supporting HCMV during entry are largely unknown. In order to systematically identify host genes affecting initial steps of HCMV infection, a targeted RNA interference screen of 96 cellular genes was performed in endothelial cells by use of a virus strain expressing the full set of known glycoprotein H and L (gH/gL) complexes. The approach yielded five proviral host factors from different protein families and eight antiviral host factors, mostly growth factor receptors. The tetraspanin CD151 was uncovered as a novel proviral host factor and was analyzed further. Like endothelial cells, fibroblasts were also less susceptible to HCMV infection after CD151 depletion. Virus strains with different sets of gH/gL complexes conferring either broad or narrow cell tropism were equally impaired. Infection of CD151-depleted cells by a fluorescent virus with differentially labeled capsid and envelope proteins revealed a role of CD151 in viral penetration but not in adsorption to the cell. In conclusion, the tetraspanin CD151 has emerged as a novel host factor in HCMV entry and as a putative antiviral target. IMPORTANCEAt present, the events at the virus-cell interface and the cellular proteins involved during the HCMV entry steps are scarcely understood. In this study, several host factors with putative roles in this process were identified. The tetraspanin CD151 was discovered as a previously unrecognized proviral host factor for HCMV and was found to support viral penetration into the target cells. The findings of this study shed light on the cellular contribution during the initial steps of HCMV infection and open a new direction in HCMV research.

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Section: Rnai Screen For Host Factors In Hcmv Infectionmentioning

confidence: 99%

Section: Rnai Screen For Host Factors In Hcmv Infectionmentioning

confidence: 99%

Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection

Hochdorfer¹,

Florin

Sinzger³

et al. 2016

J Virol

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“…HEC-LTT cells originate from human umbilical vein endothelial cells that contain doxycycline-inducible expression cassettes for SV40 large T antigen and the human telomerase catalytic subunit [23], and were maintained in endothelial growth medium (EGM BulletKit, Lonza) with 2 μg/ml doxycycline (Sigma-Aldrich) culture vessels coated with 0.1% gelatin for 30 min (Sigma-Aldrich). Infection was performed 1 d after doxycycline withdrawal [24]. MV9G cells [22] were cultured in minimum essential medium (MEM) supplemented with GlutaMAX (Life Technologies), 10% fetal bovine serum (PAA), 50 μg/ml G-418 (GE Healthcare) and 100 μg/ml gentamicin (Life Technologies) as previously described [25].…”

Section: Methodsmentioning

confidence: 99%

“…Tropism assays were performed as previously described [24]. Briefly, cells were seeded 1 day prior to infection at a density of 1x10 4 cells/well in duplicates in 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

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A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line

Maier¹,

Jung²,

Villinger

et al. 2017

PLoS ONE

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Widely used methods for quantification of human cytomegalovirus (HCMV) infection in cell culture such as immunoblotting or plaque reduction assays are generally restricted to low throughput and require time-consuming evaluation. Up to now, only few HCMV reporter cell lines have been generated to overcome these restrictions and they are afflicted with other limitations because permanently expandable cell lines are normally not fully permissive to HCMV. In this work, a previously existing epithelial cell line hosting a luciferase gene under control of a Varicella-zoster virus promoter was adopted to investigate HCMV infection. The cells were susceptible to different HCMV strains at infection efficiencies that corresponded to their respective degree of epithelial cell tropism. Expression of early and late viral antigens, formation of nuclear inclusions, release of infectious virus progeny, and focal growth indicated productive viral replication. However, viral release and spread occurred at lower levels than in primary cell lines which appears to be due to a malfunction of virion morphogenesis during the nuclear stage. Expression of the luciferase reporter gene was specifically induced in HCMV infected cultures as a function of the virus dose and dependent on viral immediate early gene expression. The level of reporter activity accurately reflected infection efficiencies as determined by viral antigen immunostaining, and hence could discriminate the cell tropism of the tested virus strains. As proof-of-principle, we demonstrate that this cell line is applicable to evaluate drug resistance of clinical HCMV isolates and the neutralization capacity of human sera, and that it allows comparative and simultaneous analysis of HCMV and human herpes simplex virus type 1. In summary, the permanent epithelial reporter cell line allows robust, rapid and objective quantitation of HCMV infection and it will be particularly useful in higher throughput analyses as well as in comparative analyses of different human herpesviruses.

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Relationship of human cytomegalovirus‐infected endothelial cells and circulating leukocytes in the pathogenesis of disseminated human cytomegalovirus infection: A narrative review

Gerna,

Lilleri,

Fornara

et al. 2023

Reviews in Medical Virology

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Among the leucocyte subpopulations circulating in peripheral blood of immune‐compromised patients with disseminated Human cytomegalovirus (HCMV) infection, polymorphonuclear leuckocytes (PMNL) and M/M may carry infectious virus. While only in PMNL early HCMV replicative events do occur, monocytes are susceptible to complete virus replication when they enter human organs, where as macrophages become a site of active complete virus replication. In vivo leucocytes and endothelial cells interact continuously, as suggested by several in vitro experimental findings showing the bidirectional HCMV transmission from leucocytes to and from endothelial cells with the critical aid of adhesion molecules. Recently, the neutralising antibody response in sera from subjects with primary HCMV infection was reported to be much higher and earlier than in human embryonic lung fibroblasts (HELF) cells when measured in endothelial cells and epithelial cells, where virus entry is mediated mostly by the pentamer complex gH/gL/pUL128/pUL130/pUL131, whereas it was much lower and delayed when determined in HELF, where virus entry is mediated mostly by the trimer complex gH/gL/gO. Thus, these results suggested that products of UL128L were the molecules primary responsible for the differential neutralising antibody response. This conclusion was confirmed by a series of polyclonal and monoclonal antibodies directed to the components of pUL128L. Very recently, based on two sets of experiments including inhibition and immunoblotting assays, the pentamer complex/trimer complex ratio has been finally identified as the main factor of the neutralising antibody response. This ratio may change with the virus suspension producer and target cell system as well as number of cell culture passages.

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A Permanently Growing Human Endothelial Cell Line Supports Productive Infection With Human Cytomegalovirus Under Conditional Cell Growth Arrest

Cited by 24 publications

References 28 publications

Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection

Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection

A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line

Relationship of human cytomegalovirus‐infected endothelial cells and circulating leukocytes in the pathogenesis of disseminated human cytomegalovirus infection: A narrative review

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