Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

Straschewski, Sarah; Warmer, Martin; Frascaroli, Giada; Hohenberg, Heinz; Mertens, Thomas; Winkler, Michael

doi:10.1371/journal.pone.0009174

Cited by 25 publications

(29 citation statements)

References 65 publications

(70 reference statements)

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“…4A). To examine whether HCMV-infected M showed morphological signs of activation, we used the recombinant fluorescent TB4-IE2-EYFP (19) and correlated the fluorescence and SEM signals (26). As shown in tants obtained from HCMV-infected M. As a positive control, monocytes were directly stimulated with LPS plus IFN-␥.…”

Section: Resultsmentioning

confidence: 99%

“…The HCMV endotheliotropic strains TB40E (kindly provided by C. Sinzger, University of Ulm, Germany) and TB4-IE2-EYFP (19) were produced by infected human foreskin fibroblasts (HFF) cultivated in minimal essential medium (MEM) with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. Infectious supernatants were recovered at maximum cytopathic effect and cleared of cellular debris by centrifugation (7,000 ϫ g for 10 min).…”

Section: Methodsmentioning

confidence: 99%

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Human Cytomegalovirus Infection of M1 and M2 Macrophages Triggers Inflammation and Autologous T-Cell Proliferation

Bayer

Varani

Wang

et al. 2013

J Virol

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H uman cytomegalovirus (HCMV) (1) is a herpesvirus that persistently infects the majority of the human population. After primary infection, HCMV remains lifelong in its host, being able to avoid clearance from the immune system. Whether HCMV persists in a truly latent state (defined as persistence in the absence of detectable infectious virus particles) or in a continuous lowlevel replication state is not clear (2, 3). However, the observation that around 10% of CD8 ϩ and CD4 ϩ T cells in the peripheral blood of healthy seropositive persons are committed to anti-HCMV responses (4) argues for continuous restimulation of T cells with antigens produced during phases of viral reactivation or low-grade active replication. Antigen recognition and T-cell activation are defined by the tightly regulated interaction between the T-cell receptor (TCR) and antigenic peptides that are presented in the context of class I or class II major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC). A number of studies have shown that the most potent APC, i.e., dendritic cells (DC), are severely impaired by HCMV in their antigen presentation, migration, and T-cell activation capabilities (reviewed in reference 5). How APC that are altered in their function can trigger and maintain a massive HCMVspecific T-cell repertoire is difficult to explain. Due to their dual nature of being permissive to HCMV infection (6-9) and being professional APC (10), macrophages (M) would represent the ideal site for antigen production, processing, and presentation to the adaptive branch of the immune system during HCMV infection.We and others have shown that M are highly susceptible to HCMV infection in vitro and that these cells produce viral progeny (11)(12)(13)(14)(15). Nevertheless, the majority of previous studies did not take into account that, in the context of immunity and inflammation, M acquire different activation states. For the sake of simplicity, M have been classified along what could be viewed as a linear scale, in which M1 M represent one extreme and M2 M the other (16). In this classification, the M1 designation refers to classically activated M, namely, cells that are capable of sustaining the immune response to pathogens through release of proinflammatory factors as well as efficient antigen presentation and T-cell stimulation. The M2 designation refers to alternatively activated M, namely, a very heterogeneous group of cells contributing to resolution of inflammation, tissue repair, extracellular matrix remodeling, and pathogen scavenging. Recent evidence indicates different susceptibilities of M1 and M2 M to HCMV infection (17,18). Nevertheless, the course of HCMV infection in these two types of M as well as the M-specific contribution to the adaptive immune response against HCMV still remains elusive.In this study, we addressed how M polarization defines HCMV susceptibility and how HCMV infection modifies M activation. We also determined the capability of HCMV-infected M to present antigen to ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Human Cytomegalovirus Infection of M1 and M2 Macrophages Triggers Inflammation and Autologous T-Cell Proliferation

Bayer

Varani

Wang

et al. 2013

J Virol

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“…The analysis software usually contains a set of readymade solutions for standard applications of the high-content imager such as quantifying the translocation of proteins from the cytoplasm to the nucleus (Borchert et al, 2005;Dull et al, 2010;Granas et al, 2006;Kau et al, 2003;Link et al, 2009;Straschewski et al, 2010;Xu et al, 2008;Zanella et al, 2007;Zanella et al, 2008). The combination of high-content imagers and integrated analysis programs, has contributed to many applications of HCS in both basic biology and in drug discovery (reviewed by Dragunow, 2008;Bullen, 2008).…”

Section: Analysis Softwarementioning

confidence: 99%

Getting the whole picture: combining throughput with content in microscopy

Rimon

Schuldiner

2011

Journal of Cell Science

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Commentary 3743Introduction Technological developments in the past years have led to the emergence of high-throughput methods in cell biology (Box 1), where multiple perturbations of a system are automatically repeated in a controlled and identical fashion. These capabilities allow the acquisition of vast amounts of data on a biological system. Evaluating all aspects of such data using computational and mathematical tools can then lead to an unbiased systematic representation of the process studied (Ahn et al., 2006). Technical breakthroughs, often driven by the pharmaceutical industry, have pushed this field forwards and have led to major advances in drug screening (reviewed by Zanella et al., 2010) and numerous discoveries in basic biology.The need for systematic and unbiased approaches has always been at the core of scientific efforts. However, the technology that is required for such efforts often displays an inverse relationship between throughput (speed) and content (biological information). Sydney Brenner referred to this phenomenon by saying that highthroughput experiments are in danger of creating "low-input, high-throughput, no-output biology" (Brenner, 2008). Therefore, it remains a major goal to develop high-throughput science that will give all the advantages of being systematic, accurate, fast and unbiased without giving up the requirement to provide profound and highly informative data.Among the variety of high-throughput approaches available to date (Box 1), one of the methods that holds the promise to bridge the gap between these expectations is high-throughput microscopy, often also referred to as high-content screening (HCS). This is mainly owing to the ability of microscopy methods to focus on single cells at a subcellular resolution, in a time-dependent manner and to measure a large number of parameters in each frame. In this Commentary, we focus on the current frontiers of HCS by presenting the wide range of tools that are utilized and the biological questions that can be tackled using such approaches. We also discuss possible ways to increase content while maintaining throughput at all levels of the screen -from sample design and preparation through to the acquisition and analysis capabilities of the high-content imager system. The power of high-throughput microscopyHigh-throughput microscopy can be employed to address a wide spectrum of biological questions. At the basis of this capability lies the ever-growing variety of labeling methods (discussed below) that allow visualization of cellular architecture and function, as well as developmental or behavioral processes (Fig. 1). In addition, the technological advance of biological tools and microscopic platforms now enables screens to be performed in an array of genetic backgrounds, under different growth conditions and at various time points, as well as allowing the comparison of multiple tissues, cell lines or organisms. Combining the richness of visualization approaches with various experimental strategies creates nearly endless options ...

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“…Such strains have also been used to examine the kinetics and localization of viral protein expression during CMV and herpes simplex virus 1 (HSV-1) infections (12). Viral strains that express fluorescent proteins alone or as a viral chimera are also useful for measuring virus infectivity, testing the antiviral properties of small molecules and the neutralizing capability of monoclonal anti-CMV antibodies, and elucidating early steps in viral binding and entry (13)(14)(15).In this study, we employed a CMV strain that ectopically expresses a yellow fluorescent protein (YFP) fused to the IE2 transcript, AD169 IE2-YFP , to establish a high-throughput cell-based assay that can quantify viral entry into human cells by measuring fluorescence of infected cells. The high-throughput format of the assay offers an easy and rapid approach for evaluating viral growth kinetics and, as we demonstrate, can be employed to test the virusneutralizing capability of monoclonal antibodies and human serum from CMV-positive patients.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Gardner

Bolovan-Fritts

Teng

et al. 2013

Clin Vaccine Immunol

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e Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169 IE2-YFP ) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169 IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.T he coevolution of human herpesviruses with their hosts over the past millions of years has led to the development of complex strategies of immune evasion that allow persistent viral infection despite the presence of an active immune response (1). A comprehensive understanding of cytomegalovirus (CMV) infection is essential to delineate the molecular and cellular interactions necessary for priming a targeted humoral immune response and how a pathogen may coopt these processes to establish a persistent and lifelong infection (2). Furthermore, a more complete comprehension of CMV entry, replication, and immune evasion is paramount in developing strategies to diagnose and alleviate CMV disease in immunocompromised patients such as transplant recipients, AIDS patients, and neonates.Analysis of viral protein expression during CMV infection can be useful in studying viral entry, cellular manipulation, and egress. The replication cycle of CMV is temporally controlled and regulated by different segments of the viral genome. The replicative cycle is divided into immediate-early (IE), early (E), and late (L) phases of replication. CMV IE proteins are produced first and appear within 6 h postinfection (hpi). IE proteins are potent transactivators that stimulate the transcription of E genes (3, 4). IE1 and IE2 are the best-characterized IE gene products and are essential for viral replication a...

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Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

Cited by 25 publications

References 65 publications

Human Cytomegalovirus Infection of M1 and M2 Macrophages Triggers Inflammation and Autologous T-Cell Proliferation

Human Cytomegalovirus Infection of M1 and M2 Macrophages Triggers Inflammation and Autologous T-Cell Proliferation

Getting the whole picture: combining throughput with content in microscopy

Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

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