UNIVERSIDAD DE COSTA RICA SISTEMA DE ESTUDIOS DE POSGRADO DEVELOPMENT AND VALIDATION OF FLUORESCENCE LIVE-CELL IMAGING APPROACHES TO STUDY FLAVIVIRUS INFECTION KINETICS IN ANIMAL CELLS Tesis sometida a la consideración de la Comisión del Programa de Doctorado en Ciencias para optar al grado y título de Doctorado Académico en Ciencias JORGE LUIS ARIAS ARIAS Ciudad Universitaria Rodrigo Facio, Costa Rica 2021 ii Dedicatoria A mi madre, por su ejemplo, empatía y amor incondicional A mis hermanas por el apoyo constante Al destino por traerlas a mi vida iii Agradecimientos Mi más sincero agradecimiento al Centro de Investigación en Enfermedades Tropicales y a la Facultad de Microbiología de la Universidad de Costa Rica por abrirme las puertas para realizar labores de investigación con infinita libertad y confianza. A todo el personal docente y administrativo de dichas instancias que con su valiosa labor posibilitan el trabajo de investigación, en especial a Teresita Solano Díaz, Erick López Sánchez, Norman Rojas Campos, Erick Guerrero Torres y Susan Chavarría Montenegro. A mi tutor Rodrigo Mora Rodríguez por su invaluable consejo científico, apoyo con herramientas biocomputacionales y en la consecución de fondos que posibilitaron este trabajo. Su perspectiva desde la investigación en cáncer me permitió amalgamar la biología celular con la virología de una manera más armoniosa y moderna. A la profesora Jeanne A. Hardy del Departamento de Química de la Universidad de Massachusetts, Amherst por recibirme en su laboratorio y permitirme trabajar con entera libertad conceptual y financiera durante la fructífera colaboración establecida. A Francisco Vega Aguilar por su amistad y valiosísimo consejo metodológico que hicieron posible esta investigación. A Eugenia Corrales Aguilar, Carlos Chacón Díaz, Steve Quirós Barrantes, Cesar Rodríguez Sánchez y Esteban Chaves Olarte por la camaradería, consejo científico, apoyo emocional y aliento durante este proceso. A Gilbert David Loría Masís por la perspectiva científica brindada durante la primera etapa de este proceso. A Jose María Gutiérrez Gutiérrez por la revisión crítica y constructiva del primer artículo publicado durante esta investigación. iv A Laya Hun Opfer, Sandra Silva de la Fuente y Marielos Mora López por la confianza depositada en mi persona y por creer en mi trabajo en los buenos y malos momentos. A los todos aquellos estudiantes y compañeros que me brindaron su apoyo en distintas etapas de este proceso, en especial a Marvin Durán Delgado, Ana Laura Rodríguez Hidalgo, Víctor González Calderón, Vanessa López Li, Sofía Herrera Agüero, Beatriz Aragón Chamberlain, Katherine Benavides Mayorga, Juan Diego Romero Carpio, Catalina Porras Silesky, Norman Brenes Cordero, David Vargas Díaz, Jose Arturo Molina Mora, Iveth Jiménez Badilla, Ramezi Araya Rivera, Isaac Quirós Fernández, Silvia Elena Molina Castro, Dayana Jiménez Araya, Claudio Soto Garita, Shirley Camacho Vargas, María Carolina Castro Peña, Javier Mora Rodríguez, Alonso Saavedra Coles y Ginger Monge Jiménez. A Mayra Lizeth Taylor Castillo (ƚ) que en espíritu siempre estuvo presente acompañándome y motivándome durante las largas jornadas de trabajo. v “Esta tesis fue aceptada por la Comisión del Programa de Doctorado en Ciencias de la Universidad de Costa Rica, como requisito parcial para optar al grado y título de Doctorado Académico en Ciencias” __________________________ Dr. Javier Mora Rodríguez Representante del Decano Sistema de Estudios de Posgrado __________________________ Dr. Rodrigo Mora Rodríguez Profesor Guía __________________________ Dr. Steve Quirós Barrantes Lector __________________________ Dra. Eugenia Corrales Aguilar Lectora __________________________ Dr. Esteban Chaves Olarte Representante de la Directora Programa de Doctorado en Ciencias __________________________ Jorge Luis Arias Arias Sustentante vi Tabla de contenidos Dedicatoria .............................................................................................................................. ii Agradeciminetos .................................................................................................................... iii Hoja de aprobación ................................................................................................................. v Tabla de contenidos ............................................................................................................... vi Resumen ............................................................................................................................... vii Abstract ................................................................................................................................ viii List of figures .......................................................................................................................... ix Aims ........................................................................................................................................ 1 Hypothesis .............................................................................................................................. 2 Prologue .................................................................................................................................. 3 Chapter 1 ................................................................................................................................ 4 Chapter 2 .............................................................................................................................. 27 Chapter 3 .............................................................................................................................. 68 Concluding remarks .............................................................................................................. 91 References ............................................................................................................................ 92 vii Resumen El género Flavivirus de la familia Flaviviridae incluye muchos virus de importancia médica, como el virus del dengue (DENV), el virus Zika (ZIKV) y el virus de la fiebre amarilla (YFV). La búsqueda de blancos terapéuticos para combatir las afecciones causadas por flavivirus requiere un mejor entendimiento de la cinética de interacción virus-célula durante las infecciones con cepas virales silvestres. Sin embargo, esto se ve obstaculizado por las limitaciones de los sistemas celulares actuales para monitorear la infección por flavivirus mediante imagenología de células vivas. La presente tesis describe el desarrollo y validación de sensores fluorescentes activables para detectar la actividad de la serin proteasa flaviviral NS2B-NS3 en células vivas. El sistema consta de reporteros basados en la proteína verde fluorescente (GFP) que activan la fluorescencia al ser cortados por proteasas recombinantes de DENV-2/ZIKV in vitro. Tras la infección por DENV-2/ZIKV, una versión de este sensor que contiene el sitio de corte interno de la proteína NS3 de flavivirus (AAQRRGRIG) reportó la mayor activación de fluorescencia en células de mamífero transducidas de manera estable. La activación de la fluorescencia correlacionó con la actividad de la proteasa viral. Además, una versión de color rojo lejano de este sensor de flavivirus presentó la mejor relación señal/ruido en un ensayo de placas de Dulbecco fluorescentes, lo que llevó a la construcción de una plataforma multireportero que combina el sensor de flavivirus con sondas fluorescentes de intercalado en el ADN para la detección de condensación de cromatina y muerte celular inducida por el virus (marcaje del efecto citopático). Esto permitió realizar estudios de formación de placas virales con resolución a nivel de células individuales. Dicho abordaje para el marcaje del efecto citopático fue conceptualizado y validado durante el presente trabajo. Finalmente, la aplicación de la plataforma multireportero también posibilitó el estudio de la cinética de infección a nivel de subpoblaciones celulares, así como de la inducción del efecto citopático por DENV-2, ZIKV y YFV. Anticipamos que estudios futuros de la cinética de infección viral con nuestros sistemas reporteros permitirán investigaciones básicas de la interacción virus-célula huésped y facilitarán el tamizaje de fármacos antivirales para controlar las infecciones por flavivirus. viii Abstract The genus Flavivirus in the family Flaviviridae comprises many clinically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of the virus-cell interplay during infections with wild-type viral strains. Nevertheless, this is hindered by limitations of the current cell-based systems for monitoring flavivirus infection by live-cell imaging. The present dissertation describes the development and validation of fluorescence-activatable sensors to detect the activity of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of green fluorescent protein (GFP)-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus internal NS3 cleavage site linker (AAQRRGRIG) reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. The onset of fluorescence correlated with viral protease activity. Moreover, a far-red version of this flavivirus sensor presented the best signal-to-noise ratio in a fluorescent Dulbecco’s plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with DNA fluorescent dyes for the detection of virus-induced chromatin condensation and cell death (cytophatic effect labeling). This enabled studies of viral plaque formation with a single-cell resolution. This cytopathic effect labeling approach was conceptualized and validated during the present work. Finally, the application of the multireporter platform also enabled the study of kinetics of infection and cytophatic effect induction by DENV-2, ZIKV, and YFV in cell-subpopulations. We anticipate that future studies of viral infection kinetics with our reporter systems will enable basic investigations of virus-host cell interactions and will also facilitate the screening of antiviral drugs to manage flavivirus infections. ix List of figures Figure 1 ................................................................................................................................... 8 Figure 2 ................................................................................................................................. 13 Figure 3 ................................................................................................................................. 15 Figure 4 ................................................................................................................................. 16 Autorización para digitalización y comunicación pública de Trabajos Finales de Graduación del Sistema de Estudios de Posgrado en el Repositorio Institucional de la Universidad de Costa Rica. Yo, Jorge Luis Arias Arias , con cédula de identidad,______2-0604-0936_______________en mi condición de autor del TFG titulado __Development and Validation of Fluorescence Live-Cell Imaging Approaches to Study Flavivirus Infection Kinetics in Animal Cells________________________________ Autorizo a la Universidad de Costa Rica para digitalizar y hacer divulgación pública de forma gratuita de dicho TFG a través del Repositorio Institucional u otro medio electrónico, para ser puesto a disposición del público según lo que establezca el Sistema de Estudios de Posgrado. SI X NO * *En caso de la negativa favor indicar el tiempo de restricción: año (s). Este Trabajo Final de Graduación será publicado en formato PDF, o en el formato que en el momento se establezca, de tal forma que el acceso al mismo sea libre, con el fin de permitir la consulta e impresión, pero no su modificación. Manifiesto que mi Trabajo Final de Graduación fue debidamente subido al sistema digital Kerwá y su contenido corresponde al documento original que sirvió para la obtención de mi título, y que su información no infringe ni violenta ningún derecho a terceros. El TFG además cuenta con el visto bueno de mi Director (a) de Tesis o Tutor (a) y cumplió con lo establecido en la revisión del Formato por parte del Sistema de Estudios de Posgrado. INFORMACIÓN DEL ESTUDIANTE: Nombre Completo:___Jorge Luis Arias Arias______________________________________________________. Número de Carné:____A30449______Número de cédula:_____2-0640-0936_____________________________. Correo Electrónico:___jorgeluis.arias@ucr.ac.cr____________________________________________________. Fecha:____12/10/2021__________. Número de teléfono:___8515-0440_________________________________. Nombre del Director (a) de Tesis o Tutor (a):____Rodrigo Mora Rodríguez______________________________. FIRMA ESTUDIANTE Nota: El presente documento constituye una declaración jurada, cuyos alcances aseguran a la Universidad, que su contenido sea tomado como cierto. Su importancia radica en que permite abreviar procedimientos administrativos, y al mismo tiempo genera una responsabilidad legal para que quien declare contrario a la verdad de lo que manifiesta, puede como consecuencia, enfrentar un proceso penal por delito de perjurio, tipificado en el artículo 318 de nuestro Código Penal. Lo anterior implica que el estudiante se vea forzado a realizar su mayor esfuerzo para que no sólo incluya información veraz en la Licencia de Publicación, sino que también realice diligentemente la gestión de subir el documento correcto en la plataforma digital Kerwá. https://es.wikipedia.org/wiki/Responsabilidad https://es.wikipedia.org/wiki/Perjurio 1 Aims General To develop and validate fluorescence live-cell imaging approaches to study flavivirus infection kinetics in animal cells. Specific To evaluate and validate the usage of DNA fluorescent dyes to label and monitor the kinetics of flavivirus-induced cytopathic effect by live-cell imaging in single animal cells. To develop and validate a kinetic flavivirus plaque assay to track the cytopathic effect by fluorescent labeling and live-cell imaging. To design, elaborate and biochemically validate a genetic construct codifying for a fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity. To establish a stable animal cell line expressing homogenous levels of a genetic construct codifying for a fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity and validate its performance to monitor flavivirus infection in single cells. To establish a multireporter fluorescent plaque assay combining a fluorescence-activatable reporter of flaviviral NS2B-NS3 protease activity and DNA fluorescent dyes to monitor flavivirus replication and cytopathic effect by live-cell imaging and validate it against standard virological methods. 2 Hypothesis The kinetics of flavivirus infection in animal cells can be studied and monitored by the application of live-cell imaging approaches based on molecular reporters of the viral NS2B- NS3 protease activity and/or the fluorescent labeling of virus-induced cytopathic effect. 3 Prologue The present dissertation is divided in three chapters. Chapter 1 serves as an introduction to the fluorescence imaging techniques applied in flavivirus research and also address our previous work in the field that guided us into the live-cell imaging methodologies. Chapter 2 represents the core of the work in the form of a research article with a detail description about the development and validation of two cell-based molecular approaches for the study of flavivirus infection kinetics by live-cell imaging. Finally, chapter 3 contains a deep description of the protocols for live-cell imaging of flavivirus infection that were developed during the present research work. 4 Chapter 1 An overview of the fluorescence imaging approaches in flavivirus research Summary The genus Flavivirus within the family Flaviviridae contains many arthropod-borne infectious agents of medical relevance such as dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV), among others, that can cause epidemics of hemorrhagic fevers and encephalitis for which there are no antiviral treatments and effective vaccines yet available. Fluorescence imaging is a powerful and versatile research tool for the study of flaviviral diseases. This tool can be complemented with biochemical and molecular methods to gain insight into the mechanisms of flavivirus infection and immunity, in order to develop feasible prophylactic and therapeutic interventions to lower these viruses impact on public health. The present introductory chapter addresses the basic aspects of the fluorescence imaging techniques currently employed in flavivirus research, including immunofluorescence assay (IFA), fluorescence in situ hybridization (FISH), fluorescence- labeled viral particles, fluorescent labeling of cytopathic effect (CPE), subgenomic reporter replicons (SRRs) / reporter virus particles (RVPs), and cell-based molecular reporters (CBMRs). This chapter also includes a published article (Arias-Arias et al., 2018), where we developed and applied a rapid IFA protocol for DENV that can be easily adapted to other flaviviruses. This protocol is described in detail at the end of the chapter as its validation was our starting point in the field of flavivirus fluorescence imaging. Background The genus Flavivirus within the family Flaviviridae contains more than 70 species of arthropod-borne viruses, transmitted to animals and humans by the bite of infected 5 mosquitoes or ticks, including the clinically relevant species DENV, ZIKV, YFV, WNV, JEV, TBEV, and SLEV, among others (Gould and Solomon, 2008). Flaviviruses possess small enveloped icosahedral particles of about 50 nm in diameter, which harbor a positive sense RNA genome of approximately 11 kb in length. This genome encodes three structural proteins: capsid (C), membrane precursor (prM), and envelope (E), and seven nonstructural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, that form a precursor polyprotein (Figure 3A) which must be cleaved by both cellular and viral proteases in order to generate the individual viral proteins (Lindenbach et al., 2007). During the last seven decades, flaviviruses have continuously emerged and re-emerged, constituting a global threat as causes of epidemics of hemorrhagic fevers and encephalitis for which there are no specific treatments more than life support upon hospitalization. This highlights the critical need for a detailed understanding of the biology of flavivirus, the interplay between the virus and the host cell, and the immunological responses elicited, in order to develop feasible prophylactic and therapeutic approaches to lower their impact on public health (Lindenbach et al., 2007; Pierson and Diamond, 2020). In this scenario, fluorescence imaging techniques represent powerful and versatile research tools for visualization and study of flavivirus infected cells, that can be complemented with biochemical and molecular methods in order to gain knowledge about the mechanisms of infection and to test candidate antiviral drugs and vaccines to combat flaviviral diseases (Chong et al., 2014). Among them, immunofluorescence assay (IFA), fluorescence in situ hybridization (FISH), fluorescent labeling of cytopathic effect (CPE), fluorescence-labeled viral particles, subgenomic reporter replicons (SRRs) / reporter virus particles (RVPs), and cell-based molecular reporters (CBMRs), have been applied in flavivirus research and will be individually discussed in the present introductory chapter. 6 Immunofluorescence assay (IFA) IFA is the most popular and widely applied fluorescence imaging approach for flaviviruses, since this method has been used in diagnostics and research of flaviviral infections for more than 50 years (Atchison et al., 1966). Indeed, virus presence and cellular localization can be visualized inside infected cells by means of fluorophore-tagged antibodies directed against either structural or non-structural flavivirus antigens (Chong et al., 2014). Zuza and collaborators observed a perinuclear localization of the SLEV proteins upon immuno- labeling of infected astrocytes with a polyclonal ascitic fluid from immunized mice (Zuza et al., 2016). Likewise, Miorin and collaborators reported that the TBEV E, prM, and NS1 proteins were localized at the perinuclear region and within irregularly shaped foci of infected BHK-21 cells (Miorin et al., 2013). This is a common fluorescence pattern observed among other members of the genus, including DENV 1-4, ZIKV, YFV, JEV, and WNV (Ledizet et al., 2007; Ricciardi-Jorge et al., 2017; Slon Campos et al., 2017), since flaviviruses replication and assembly occurs on the cytosolic side of the endoplasmic reticulum (ER) membrane (Rothan and Kumar, 2019). IFA also remains as an excellent method to evaluate and visualize the permissiveness of cell lineages to flavivirus infection. Růžek and collaborators tested the susceptibility of different human neural cell lines and the TBEV-induced cytopathic effect using an anti-E antibody (Růžek et al., 2009). In our work, we applied immunostaining of E and NS3 proteins as part of our experiments to demonstrate the permissiveness of primary human umbilical artery smooth muscle cells (HUASMC) to clinical isolates of both DENV-2 and DENV-3 (Figure 1C) (Arias-Arias et al., 2018). Others also employed immunolabeling to evaluate the susceptibility of JEG-3 and hCMEC/D3 cell lines, as well as Sertoli cells in mouse testis to ZIKV infection (Chiu et al., 2020; Sheng et al., 2017). IFA also enables the study of colocalization as a first screening approach of protein-protein interactions by the combination of different antibodies directed against both host and viral antigens, as these interactions play important roles during flavivirus infection. Hung and 7 collaborators employed IFA colocalization of host secreted heat-shock protein 90 beta (Hsp90β) and viral E protein during JEV infection (Hung et al., 2011). They performed a subsequent validation with sucrose-density fractionation and Western blot analysis to demonstrate that this interaction is required for JEV infectivity in BHK-21 cells. In addition, using IFA and cryoimmunoelectron microscopy colocalization with monospecific antibodies, NS3 and NS2B proteins were found to be present in WNV-induced membrane structures in Vero cells (Westaway et al., 1997). One of the greatest methodological advantages of applying IFA in flavivirus research, is the wide commercial availability of group cross-reactive and type-specific antibodies. This is based on the fact that some flaviviral proteins possess both conserved and variable antigens, e.g., the fusion loop at the extremity of domain II of protein E carries the flavivirus group conserved epitopes, whereas domains I and III contain the variable antigens (Lai et al., 2008). This observation guided the in vitro production of the hybridoma clone D1-4G2- 4-15, which produces the most popular flavivirus group monoclonal antibody (4G2), used in diagnostics for the screening of viral isolates from clinical samples and in research for the monitoring of the infection by agents like DENV, WNV, JEV, YFV, and ZIKV (Garg et al., 2020; Göertz et al., 2017; Lai et al., 2008; Martins et al., 2019), among other flaviviruses. At the end of this chapter, I describe in detail an IFA protocol for the immunostaining of DENV/ZIKV with commercial antibodies, that was applied in the first published article from this dissertation (Arias-Arias et al., 2018, attached article) and for the generation of the images depicted in Figure 1. 8 Figure 1. Immunofluorescence assay (IFA) in DENV/ZIKV-infected cell cultures. Epifluorescence images of ZIKV-infected (A) and mock-infected (B) Vero cells after immunostaining with an anti-ZIKV E protein monoclonal antibody (green) and cytoplasmic (Evans blue, red) - nuclear (Hoechst 33342, blue) counterstains (total magnification of 100 X; scale bar = 200 µm). DENV-infected HUASMC (C) and LLC-MK2 (B) cells immunostained with an anti-DENV 1-4 E protein monoclonal antibody (green) and counterstained with 9 cytoplasmic (Evans blue, red) and nuclear (Hoechst 33342, blue) fluorescent dyes (total magnification of 400 X and 100 X; scale bars = 20 µm and 50 µm, respectively). Immunolabeling of DENV in BHK-21 cell with an anti-DENV NS3 protein polyclonal antibody (orange) and a nuclear counterstain (Hoechst 33342, blue, total magnification of 600 X; scale bar = 30 µm). Images by Jorge L. Arias-Arias, Universidad de Costa Rica. Fluorescence in situ hybridization (FISH) FISH is a classical cytogenetic technique used to detect both RNA and DNA within tissues and cells by the application of fluorochrome-labelled probes that are complementary to the sequence of interest, with their subsequent visualization by fluorescence microscopy (Rudkin and Stollar, 1977). One of the mayor goals in RNA viruses research is the understanding of the coordination of the intracellular trafficking of viral RNA and proteins during the assembly of virions, as well as the deciphering of the involved interactions between the viral genome and other components of the host and the virus itself (Vyboh et al., 2012). A combination of FISH with other cellular and molecular techniques has been applied in recent years to tackle the above-mentioned challenges in flavivirus infection research. FISH is the method of choice to visualize the localization, transcription, and replication of flavivirus RNA inside infected cells. Raquin and collaborators developed a set of highly specific oligonucleotide probes that hybridize to the viral RNA from a broad range of DENV isolates including all the four serotypes, but not to the closely related YFV and WNV genomes. They used those probes to label DENV RNA in vitro on infected C6/36 cells and in vivo with dissected salivary glands from infected Aedes albopictus specimens (Raquin et al., 2012). Similar FISH approaches have been used for the observation of ZIKV (Hou et al., 2017; Liu et al., 2019; Martinez-Lopez et al., 2019) and YFV genome replication (Sinigaglia et al., 2018), and for the visualization of WNV noncoding RNAs (Roby et al., 2014), among other flaviviruses. 10 When combined with IF, FISH serves as a useful starting point to study protein-viral RNA interactions. Hirano and collaborators applied FISH/IF and immunoprecipitation/RT-PCR to demonstrate that neuronal granules, involved in the transportation and local translation of dendritic mRNAs, also transport the TBEV genomic RNA (Hirano et al., 2017). Viral RNA interacts with a RNA-binding protein present in the neuronal granules and impairs the transport of dendritic mRNAs, which seems to be involved in the neuropathogenesis of the TBEV infection. Also, Hou and collaborators observed co-localization of ZIKV E protein with its own viral RNA by FISH/IF and demonstrated its interaction via an RNA chromatin immunoprecipitation (RNA-ChIP) assay, implying that the E protein may have a role in ZIKV replication (Hou et al., 2017). The availability of online bioinformatic tools and databases assisting probe design (e.g., https://www.arb-silva.de/fish-probes/probe-design/), together with the custom probe synthesis services offered by many biotech companies, is increasing the feasible application of RNA FISH in the field of virology, including flavivirus research. For a detailed and versatile FISH protocol specially standardized for RNA viruses, please refer to the work of Lindquist and Schmaljohn (Lindquist and Schmaljohn, 2018). Fluorescence-labeled viral particles In recent years, with the improvements in confocal and super-resolution fluorescence microscopy, several researchers have exploited the direct labeling of virions for the visualization of the early events in virus-cell interactions, even at a single particle level (Sakin et al., 2016). To assess viral membrane fusion, the use of lipophilic dyes that get inserted into the lipid bilayer membrane of virions and are released in the endosomal membrane after the fusion event, have been reported for some flaviviruses (Hoffmann et al., 2018). Nour and collaborators applied octadecyl rhodamine B chloride (R18) labeling of JEV and YFV particles to study the kinetics of viral membrane fusion and nucleocapsid delivery into the cytoplasm (Nour et al., 2013). Moreover, labeling of virions with 1,1'-dioctadecyl- 3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) was used to https://www.arb-silva.de/fish-probes/probe-design/ 11 analyze the cellular entry of WNV (Makino et al., 2014) and to demonstrate the involvement of the autophagy machinery during the early stages of DENV infection (Chu, 2013). Furthermore, Zhang and collaborators developed a simple and efficient method to covalently tag the amino free groups of DENV E protein with the dye Alexa Fluor 594 succinimidyl ester, a useful labeling approach employed to monitor virus binding, uptake, and intracellular trafficking (Zhang et al., 2010). A similar procedure was used to label ZIKV particles with the dye Atto647N-NHS ester, which enabled the visualization of viral transcytosis through both the placental and the blood brain barriers (Chiu et al., 2020). Labeling of virions is an advantageous technique since the above-mentioned dyes show bright fluorescence, high photostability, are suitable to be applied in both fixed and live-cell imaging protocols, and exist in a broad range of colors over the ultraviolet-visible spectrum (Hoffmann et al., 2018). The work of Zhang and collaborators describes a detailed protocol for the fluorescence-labeling of flavivirus particles (Zhang et al., 2011). A comprehensive description of labeling procedures applied to a broader repertory of viruses is discussed by Hoffmann and collaborators (Hoffmann et al., 2018). Fluorescent labeling of cytopathic effect (CPE) Detection of CPE as an indirect way to monitor viral infections has been largely exploited since the early days of virology and constitutes a fundamental part of the principle behind classical virological methods such as Dulbecco plaque assay (Dulbecco, 1952). For example, monitoring the morphological changes on flavivirus infected cells is generally accomplished by bright-field light microscopy, directly on living cells or after fixation and staining with conventional dyes as crystal violet or hematoxylin-eosin (Bakonyi et al., 2005; Chong et al., 2014). Taking into account that in many cases the CPE is a result of virus–cell interactions leading to cell demise by host-encoded programs like programmed necrosis and apoptosis (Agol, 12 2012), during the present research work we envisaged a simple but effective way to label and monitor CPE in real-time by fluorescence live-cell imaging (chapter 2). Using fluorescent DNA dyes commonly employed in cell biology and cancer research such as nuclei stains (Hoechst 33342) and cell dead markers (propidium iodide, SYTOX green, TO-PRO-3 iodide), we were able to visualize early (chromatin condensation) and late (membrane permeabilization) events of the virus elicited CPE correlating with cell damage and cell death, respectively. We successfully applied the above-mentioned approach to perform kinetics of CPE detection and real-time plaque assays on living cells infected with DENV, ZIKV, and YFV (Figure 2). This allowed us to analyze the viral plaques growth over time at a single-cell level using an image analysis software, as exposed in the second published article from this thesis (Arias-Arias et al., 2020, attached in chapter 2). A detailed protocol for kinetic CPE labeling and monitoring in flavivirus infected cells is described in chapter 3. Subgenomic reporter replicons (SRRs) and reporter virus particles (RVPs) Flaviviruses harbor positive strand RNA genomes that are per se infectious. Thus, transfection of RNA produced by in vitro transcription from a cDNA clone containing the reverse transcribed full-length flavivirus genome results in the production of infectious recombinant viral particles (Figure 3A). In contrast, flavivirus subgenomic replicons possess all the essential genetic elements for self- replication and production of nonstructural proteins, but lack the complete encoding sequences of the structural C-prM-E proteins (Figure 3B) and consequently do not allow the generation of virions. Using such replicons as templates, SRRs are established by the introduction of reporter genes that code for bioluminescent or fluorescent proteins in the position of the deleted structural genes (Figure 3C) (Kümmerer, 2018). This enables the easy tracking of the replication/translation of subgenomic replicons and the screening of antiviral compounds by the direct visualization and measurement of the signal produced by the reporter proteins (Kato and Hishiki, 2016). Flavivirus SRRs have been developed and validated for YFV (Jones et al., 13 2005), JEV (Li et al., 2013), WNV (Shi et al., 2002), ZIKV (Mutso et al., 2017), and DENV (Pang et al., 2001; Usme-Ciro et al., 2017), among others. Figure 2. Fluorescent labeling of cytopathic effect (CPE) in flavivirus-infected cell cultures. A. CPE imaging kinetics in YFV-infected BHK-21 cells with the DNA staining dyes Hoechst 33342 (cells with condensed chromatin, saturated blue) and TO-PRO-3 iodide (dead cells, red) at a total magnification of 200 X (scale bar = 100 µm). B. Dulbecco’s plaque assay on unfixed Vero cells by CPE labeling with the nucleic acid dyes Hoechst 33342 (DNA/chromatin 14 condensation, blue) and SYTOX green (cell death, green), at 96 hours post-infection with ZIKV (total magnification of 40 X; scale bar = 1000 µm). Images by Jorge L. Arias-Arias, Universidad de Costa Rica. SRRs are also used for the generation of single-round infectious RVPs, by providing the deleted C-prM-E genes in trans with another genetic construct (Figure 3D). Such single- round RVPs are extremely useful as surrogate pseudoviruses in studies of BSL3-handling of flaviviruses such as JEV (Lu et al., 2017) and WNV (Li et al., 2017; Velado Fernández et al., 2014). Single-round infectious RVPs have also been used on the development of easy fluorescent neutralization assays for the detection of flavivirus-specific antibodies in the serum of individuals and the assessment of the humoral immune response elicited by candidate vaccines, as shown for DENV (Mattia et al., 2011), TBEV (Yoshii et al., 2009), ZIKV (Garg et al., 2017), and WNV (Pierson et al., 2006). However, SRRs and single-round infectious RVPs cannot be used to study the pathogenesis, transmission, and dynamics of the complete virus replication cycle, as well as for the screening of antivirals targeting the structural proteins (Kato and Hishiki, 2016). For such applications, whole genome RVPs have been engineered by the insertion of reporter genes into full-length flavivirus cDNA clones (full-length reporter cDNA clone, Figure 3E), as described for JEV (Jia et al., 2016), DENV (Schmid et al., 2015; Schoggins et al., 2012; Suphatrakul et al., 2018), WNV (Pierson et al., 2005), and ZIKV (Gadea et al., 2016), among others. As an example, Schmid and collaborators developed and characterized a far-red DENV-2 reporter virion that allows the monitoring of the viral infection kinetics in animal cells by live imaging (Schmid et al., 2015). For further details, the work by Kümmerer describes in detail the molecular genetics, development and applications of flavivirus subgenomic and full-length replicons (Kümmerer, 2018). 15 Figure 3. Schematic presentation of genetic constructs encoding flavivirus full-length cDNA clones (A), subgenomic replicons (B), subgenomic reporter replicons (SRRs, C), and full- length reporter cDNA clones (E), which enable the production of reporter virus particles (RVPs, D). UTR: untranslated region; Ub: ubiquitin coding sequence; 2A: ribosomal skipping 2A peptide. Modified and adapted from Kümmerer, 2018. Cell-based molecular reporters (CBMRs) SRRs and RVPs are valuable tools to perform kinetic studies by live-cell imaging, but their development is expensive, time consuming, and limited only to the pre-selected molecular clones derived from specific flavivirus strains, which precludes the direct work with clinical isolates and wild-type virus strains. To overcome this limitation, in recent years a few articles have outlined the use of CBMRs as an alternative to carry out kinetics of infection with wild-type flaviviruses in living cells (Arias-Arias et al., 2020). So far, all the published flavivirus CBMRs are based on the monitoring of the proteolytic activity of the flaviviral NS2B-NS3 serine protease. Medin and collaborators devised a DENV 1-4 plasmid-based reporter system containing the cleavage site between the NS4B and NS5 16 proteins attached to an EGFP by a nuclear localization sequence (NLS) (Medin et al., 2015). Upon cleavage the EGFP relocalizes from the cytoplasm to the nucleus of infected cells. The same principle was adapted by McFadden and collaborators for imaging ZIKV infection in living Huh7 cells (McFadden et al., 2018). In addition, a modification of this approach by Hsieh and collaborators exploited the DENV NS3 cleavage between NS4B/NS5 to activate a Cre recombinase-based nuclear reporter (Hsieh et al., 2017). This system showed superior performance than traditional methods for DENV 1-4 titration. Moreover, during the present research work we developed a fluorescence-activatable reporter of flavivirus infection by the modification of previously published caspase 7 reporters (Wu et al., 2013). Instead of the caspase 7 cleavage sequence, we inserted the internal NS3 cleavage site (conserved among many members of the Flavivirus genus) between a fluorescent protein and a quenching peptide (QP, Figure 4A). This reporter system was used to generate a BHK-21 reporter cell line suitable for monitoring the kinetics of infection by DENV, ZIKV, and YFV both in viral plaques and at a single-cell level using live- cell imaging (Figure 4B) (Arias-Arias et al., 2020). Figure 4. The flavivirus cell-based molecular reporter. A. The flavivirus-activatable GFP reporter (FlaviA-GFP) contains a GFP with a C-terminal quenching peptide (QP) joined by a linker composed by a cleavage site of the flaviviral NS3 protease. When the protease cuts 17 the linker, the quenching peptide is removed, and the GFP adopts the fluorescent conformation. B. DENV-2 infection kinetics in BHK-21 reporter cells. An automated image analysis protocol was programmed in CellProfiler 2.0 for the quantification of activated FlaviA-GFP fluorescent cells (green), live cells (white outline) and dead cells (red outline). Total magnification of 200 X; scale bar = 100 µm. Adapted from Arias-Arias et al., 2020. The details about the development and validation of our flaviviral CBMR system are addressed in chapter 2 and the exact protocol for the generation of the above-mentioned reporter cell line is described in the third published article from this dissertation (Arias-Arias et al., 2021, attached in chapter 3). Published article Arias-Arias, J.L., Vega-Aguilar, F., Corrales-Aguilar, E., Hun, L., Loría, G.D., Mora-Rodríguez, R., 2018. Dengue virus infection of primary human smooth muscle cells. Am. J. Trop. Med. Hyg. 99, 1451–1457. https://doi.org/10.4269/ajtmh.18-0175 https://doi.org/10.4269/ajtmh.18-0175 Am. J. Trop. Med. Hyg., 99(6), 2018, pp. 1451–1457 doi:10.4269/ajtmh.18-0175 Copyright © 2018 by The American Society of Tropical Medicine and Hygiene Dengue Virus Infection of Primary Human Smooth Muscle Cells Jorge L. Arias-Arias, Francisco Vega-Aguilar, Eugenia Corrales-Aguilar, Laya Hun, Gilbert D. Lorı́a, and Rodrigo Mora-Rodrı́guez* Centro de Investigación en Enfermedades Tropicales (CIET), Facultad de Microbiologı́a, Universidad de Costa Rica, San José, Costa Rica Abstract. Dengue virus (DENV) infection of humans is presently the most important arthropod-borne viral global threat, for which no suitable or reliable animal model exists. Reports addressing the effect of DENV on vascular components other than endothelial cells are lacking. Dengue virus infection of vascular smooth muscle cells, which play a physiological compensatory response to hypotension in arteries and arterioles, has not been characterized, thus precluding our un- derstanding of the role of these vascular components in dengue pathogenesis. Therefore, we studied the permissiveness of primaryhumanumbilical artery smoothmusclecells (HUASMC) toDENV1–4 infectionandcomparedwith the infection in the previously reported primary human umbilical vein endothelial cells (HUVEC) and the classically used, non-transformed, and highly permissive Lilly Laboratories Cell-Monkey Kidney 2 cells. Our results show that HUASMC are susceptible and productive to infectionwith the fourDENVserotypes, although toa lesser extentwhencomparedwith theother cell lines. This is the first report of DENV permissiveness in human smooth muscle cells, which might represent an unexplored patho- physiological contributor to the vascular collapse observed in severe human dengue infection. INTRODUCTION Dengue virus (DENV) is a member of the genus Flavivirus within the Flaviviridae family, for which five serotypes have been described (DENV 1–5).1 The virion comprises an envel- oped spherical particle that harbors a positive single-stranded RNA genome.2 Dengue virus is transmitted by mosquito vectors, mainly Aedes aegypti, and is considered the most important arthropod-borne viral disease worldwide.3 Denguevirus represents aglobal threat, forwhich there is no specific treatment available. Although a tetravalent, live- attenuated, dengue vaccine was recently approved,4 safety concerns5 have highlighted the urgent need for antiviral drugs to treat DENV infections. However, the development of an antiviral drug targeting viral factors of all DENV serotypes has been problematic. New promising approaches rely on the targeting of host factors to achieve antiviral activity.6 Never- theless, this strategy requires an in-depth understanding of the pathogenesis of dengue disease, including the identifi- cation of the key cellular targets involved in severe infections. The determinants of dengue disease severity are complex and multifactorial, and although several models have been developed over the years to bridge translation from in vitro to human observational studies, no laboratory animals (wild- type or genetically modified) develop all of the clinical mani- festations of severe dengue disease in humans.7 Dengue virus infects many animal cell lines such as the highly per- missive baby hamster kidney cells (BHK-21), C6/36, Vero and LLC-MK2 (macaque kidney cells), and human cell lines such as HepG2, U937, and HEK-293, with varying degrees of permissiveness.8–10 However, the question remains as to whether those cells types represent relevant targets of DENV infection in vivo. Therefore, most conclusions regarding the in vivo situation in humans rely on postmortem studies or on the in vitro permissiveness of primary cells such as human umbilical vein endothelial cells (HUVEC) and peripheral blood mononuclear cells to DENV.8,11 Postmortem studies depend on the detection of DENV an- tigens in tissues. The most specific marker of DENV infection in vivo is the nonstructural protein 3 (NS3) because it does not enter the secretory pathway, and thus demonstrating exclu- sively intracellular localization.11,12 However, localization of NS3protein in tissues variesdependingonwhich host species is analyzed. In mice, NS3 was detected in phagocytes of the spleen and lymph nodes, as well as in hepatocytes and my- eloid cells in the bone marrow.11 In human postmortem tis- sues, the NS3 protein was detected in phagocytes of spleen and lymph nodes, hepatocytes and endothelial cells in spleen, perivascular cells in brain, and alveolar macrophages in lungs of DENV severe cases.11 Others reported that skeletal and cardiacmuscle cells are also infected in vivo.13,14 In addition, it has been shown that myotubes can also be infected in vitro.14 Endothelial cells have long been implicated in the physio- pathology of DENV infection. Microvascular and endothelial dysfunctions are associated with the severity of dengue, and this occurs before the appearance of severe clinical mani- festations.15 Indeed, there are tests and treatments to identify and handle various forms of vascular dysfunction that could be applied for the clinical management of patients with severe dengue.3 It has been reported that endothelial cells are per- missive to DENV infection in vitro although they produce low viral titers.16 Nevertheless, DENV infection of ECV304 human endothelial cells leads to chemokine production and comple- ment activation, suggesting an important role in microvascular dysfunctionduringDENVphysiopathology.17Moreover, others have shown the effect of DENV infection on gene expression in HUVEC cells and identified potentially novel mechanisms involved in dengue disease manifestations such as hemo- static disturbances.18 However, only a small percentage of endothelial cells were productively infected in vitro using the DENV-2 16681 strain.19 Despite these observations, the impor- tance of endothelial cells as targets of DENV infection in vivo remains a subject of debate. Reports addressing the effect of DENV on other vascular components such as smooth muscle cells, which play a physiologically relevant role in arteries and arterioles, are lacking. Dengue virus infection of vascular smooth muscle cells has not been characterized, thus precluding our un- derstanding of the role of these vascular components in * Address correspondence to Rodrigo Mora-Rodrı́guez, Facultad de Microbiologı́a, Centro de Investigación en Enfermedades Tropicales, Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, San Pedro de Montes de Oca, San José 11501-2060, Costa Rica. E-mail: rodrigo.morarodriguez@ucr.ac.cr 1451 mailto:rodrigo.morarodriguez@ucr.ac.cr dengue pathogenesis. To address this issue, here we worked with human umbilical artery smooth muscle cells (HUASMC), which are primary smooth muscle cells isolated from normal healthy human umbilical arteries. Human umbilical artery smooth muscle cells have been used along HUVEC to study the dynamics, maturation, and effects of toxic stimulus on blood vessels, and constitutes a suitable and well-validated model that could be applied on DENV research.20,21 This work describes for the first time a DENV-permissive infection of primary arterial smooth muscle cells in vitro, which might represent an unexploredpathophysiological contributor to the reduced vascular reactivity to hypotension observed during dengue shock syndrome and dengue hemorrhagic fever. MATERIALS AND METHODS Viruses. Dengue virus-1 Angola (D1/AO/XX/1988) and DENV-4 Dominica (D4/DM/814669/1981) strains were sup- plied by the Instituto de Medicina Tropical Pedro Kourı́, Ha- vana, Cuba. The clinical isolates from Costa Rican patients DENV-2 10066 (D2/CR/10066/2007) and DENV-3 14531 (D3/ CR/14531/2007) were provided by the Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud, Cartago, Costa Rica.22 Viruses were produced in C6/36 cells from Ae- des albopictus (ATCC, Manassas, VA) by inoculating cellular monolayers with DENV at a multiplicity of infection (MOI) of 0.01 and incubating for 3 days with Roswell Park Memorial Institute-1640 medium supplemented with 2% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD) at 33�C in an atmo- sphere of 5% CO2. Then, culture supernatant was collected and centrifuged at 3,000 × g for 10 minutes. Before storage at −80�C, 23% newborn calf serum (Gibco) was added.9 Culture supernatant from uninfected C6/36 cells was col- lected and used as negative control (mock control). Viruses were titrated by plaque assay in BHK-21 cells (ATCC) as previously described.23 Briefly, 10-fold serial dilutions of viruses were added to BHK-21 confluent monolayers. After 2 hours of adsorption, cells were incubated at 37�C in an at- mosphere of 5% CO2 for 5 days with minimum essential me- dium (MEM) supplemented with 2% FBS (Gibco) and 1% carboxymethylcellulose (Sigma, St. Louis, MO). Plaque num- bers were counted after staining with crystal violet. Cell lines and virus infections. Human umbilical artery smooth muscle cells and HUVEC were purchased and main- tained in smoothmuscle cell growthmediumand endothelial cell growth medium, respectively, according to the manufacturer’s instructions (Cell Applications, San Diego, CA). LLC-MK2 cells (ATCC) were grown in MEM supplemented with 10% FBS. Cell monolayers were DENV or mock infected at a MOI of 1 and allowed virus adsorption for 2 hours at 37�C. After three washes with phosphate-buffered saline (PBS), cells were incubated with 2% FBS medium at 37�C in an atmosphere of 5% CO2 for dif- ferent times. All experiments were performed with the same number of HUASMC, HUVEC, and LLC-MK2 cells. Plaque assays for virus quantification. Culture superna- tants of HUASMC were collected at 0, 24, 48, and 72 hours postinfection (p.i.) and DENV infectious particles were quan- tified by plaque assays in BHK-21 cells, as described earlier. Concomitantly, supernatants from HUVEC and LLC-MK2 cell cultures at 72 hours p.i. were titrated. Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for genome copies quantification. Culture supernatants of HUASMC cells were collected at 0, 24, 48, and72hours p.i. andDENVgenomeswerequantifiedby RT- qPCR. Briefly, viral RNA was extracted with the NucleoSpin RNA virus kit (Macherey-Nagel, Düren, Germany) and quan- tified using the Genesig RT-qPCR advanced kit for dengue virus (Primerdesign, Southampton, United Kingdom) according to themanufacturer’s instructions.The reactionswerecarriedout with a StepOne™ real-time PCR system (Applied Biosystems, Carlsbad, CA). Supernatants from HUVEC and LLC-MK2 cell cultures at 72 hours p.i. were also tested. Indirect immunofluorescence for DENV infected cells quantification.Human umbilical artery smoothmuscle cells, HUVEC, and LLC-MK2 cells were cultured on glass coverslips coated with 1% gelatin (Sigma) in 24 well plates seeded with 100,000 cells per well. At 72 hours p.i., cells were fixed with cold acetone for 10 minutes, washed with PBS, and stored at −20�C. Afterward, the slides were treated with 50mMNH4Cl for 10minutes and incubated with a 1:300 dilution of mouse anti-DENV 1, 2, 3 and 4 envelope protein monoclonal antibody (GTX29202; GeneTex, Irvine, CA) or a 1:800 dilution of rabbit anti-DENV NS3 protein polyclonal antibody (GTX124252; GeneTex) for 1 hour at 37�C. After washing, the coverslips were incubated for 30 minutes at 37�C with 1:75 diluted fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (IgG) (DAKO, Glostrup, Denmark) in 0.02%Evansblue or 1:400dilution of Alexa Fluor 647 goat anti-rabbit IgG (Invitrogen, CA) in PBS. Stained slides were mounted with Prolong Gold with 49,6-diamidino-2- phenylindole (DAPI; Invitrogen, Carlsbad, CA) and images were acquired with a Cytation 3 Cell Imaging Multi-Mode Reader (BioTeK, Winooski, VT). Image analysis of the whole coverslip was performed with the software CellProfiler 2.0 (http://www.cellprofiler.org; Broad Institute, Cambridge, MA). Statistics. Data are expressed as mean ± standard devia- tion of three independent experiments. Statistical significance of the differences between mean values was determined by using an unpaired Student’s t-test. The level of significance is denoted in each figure. RESULTS All four dengue serotypes are able to replicate in HUASMC cells. To test the permissiveness of HUASMC to DENV infection, confluent cell monolayers where infected at a MOI of 1 with each of the four DENV serotypes. Virions were quantified in culture supernatants every 24 hours for 72 hours. The supernatants of HUASMC monolayers infected with the four DENV serotypes exhibited an increasing number of plaque-forming units (PFU) after 48 hours p.i. (Figure 1A). However, there were significantly higher replication efficien- cies of the different DENV serotypes in HUVEC and LLC-MK2 cell line when compared with HUASMC at 72 hours after in- fection (Figure 1B). These results demonstrate that the HUASMC line is permissive to DENV infection by all four se- rotypes; however, virion production is lower in these cells than in the frequently used HUVEC and LLC-MK2 cells. To confirm the permissiveness of HUASMC to DENV in- fection and estimate the replicative fitness of the virus in this cell line, monolayers were infected at a MOI of 1 with the four DENV serotypes. Dengue virus RNA was quantified from culture supernatants every 24 hours for 72 hours. The super- natants of HUASMC monolayers infected with the four DENV 1452 ARIAS-ARIAS AND OTHERS http://www.cellprofiler.org serotypes showed an increase in DENV genomic RNA copies at 48 hours p.i. (Figure 2A). Nevertheless, the production of infectious virions and genomic RNA from the different DENV serotypes was significantly higher in HUVEC and LLC-MK2 cells than in HUASMC cells at 72 hours after infection (Figure 2B). Thus, the replicative fitness of DENV in HUASMC cells was significantly lower than that observed in HUVEC and LLC-MK2 cell lines, based on the genome-to-PFU ratios cal- culated at 72 hours p.i. (Figure 2C). Dengue virus antigens are detected by immunofluo- rescence in HUASMC. Human umbilical artery smooth muscle cells’ monolayers infected with the four DENV sero- types were stained by indirect immunofluorescence to quantify cellular infection. After 72 hours of infection, HUASMC, HUVEC, and LLC-MK2 cells were stained with an anti-DENV1, 2, 3 and 4envelope proteinmonoclonal antibody and fluorescence images were analyzed using the software CellProfiler 2.0. In contrast to the mock control (Figure 3A), infected HUASMC showed cytoplasmic green fluorescence staining (Figure 3B and C, white arrows), which was automatically identified by image analysis (Figure 3D, green outlines) to calculate the percentage of infected cells against the total number of identified cellular nuclei (Figure 3D, white outlines). As expected, the LLC-MK2 cell line showed higher percentages of positive cells with all four DENV serotypes compared with HUASMC cells (Figure 3E). By contrast, FIGURE 1. Humanumbilical artery smoothmuscle cells (HUASMC) are permissive to dengue virus (DENV) infection. Virionproduction and release was quantified by plaque assays of culture supernatants at different time points post-infection (p.i.) of HUASMC, human umbilical vein endothelial cells (HUVEC), and LLC-MK2 (macaque kidney cells) infectedwith each of the four DENV serotypes (multiplicity of infection:1). (A) Infection kinetics of HUASMC cells (24–72 hours p.i.) with DENV 1–4 measured by plaque assays (plaque-forming units [PFU]/mL). (B) DENV 1–4 titers in culture supernatants of HUASMC, HUVEC, and LLC-MK2 cells at 72 hours p.i. Data are expressed as themean ± standard deviation of three independent experiments. ***P < 0.001 compared with its HUVEC and LLC-MK2 cells counterparts. (D) l. = assay detection limit. DENGUE VIRUS INFECTION OF PRIMARY HUMAN SMOOTH MUSCLE CELLS 1453 HUASMC displayed a small percentage of cells (5–15%) with positive staining, indicating that this cell line has a low per- missiveness to DENV infection with all four serotypes. How- ever, only DENV-2 and DENV-3 led to a higher antigen production in HUVEC cell line compared with HUASMC cells (Figure 3E). Finally, no difference was observed in the identifi- cation of HUASMC-infected cells by immunostaining with an anti-DENV 1-4 envelope protein-specific monoclonal antibody and an anti-NS3 polyclonal antibody (Figure 3F), which indi- cates that the detected antigens are produced de novo during the infection. These results demonstrate that DENV antigens canbedetected inHUASMCdespite the lowpermissivenessas shown by the low percentage of infected cells compared with HUVEC and LLC-MK2 cells. DISCUSSION ResearchonDENVpathophysiologyhasbeenhamperedby the lack of competent animal models for reproducing the in vivo human infection.7 Therefore, most conclusions re- garding the pathophysiological mechanisms of this disease in humans rely onpostmortemstudies or are extrapolations from the in vitro permissiveness of primary cells to DENV.8,11 A key remaining question regarding DENV pathophysiology is the role of alterations in the different cellular components of the blood vessel. This has been addressed for endothelial cells because they are the major component of capillary blood vessels, and the microvascular dysfunction is closely asso- ciated with the severity of dengue.15 Our findings confirm that DENV do infect the endothelial cell line HUVEC, as shown previously.16 Indeed, future work is necessary to assess whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage, which has been previously observed in patients with dengue hemor- rhagic fever and dengue shock syndrome.19 A neglected component of the tissue response to this exten- sive vascular leakage has to do with the physiological com- pensatory mechanisms associated with the response of arteriolesandparticularlywith the regulationof vasculardiameter by smooth muscle cells present in the arteriolar wall. Significant vascular leakage and the resulting hypovolemia trigger vaso- constriction of arterioles to compensate for hypotension.24 During hemorrhagic shock, the vascular hyporeactivity is related to a desensitization to calcium andmitochondrial dysfunction in smoothmusclecells inbloodvessels.25,26 Inaddition,damage to lymphatic smooth muscle cells in collecting lymphatic vessels leads to an impairment in lymph formation and interstitial fluid balance, generating edema and thereby perturbing blood vol- ume recovery.21 Therefore, the observed effect of DENV in- fection in smooth muscle cells could play an important role in precipitating the outcome of severe shock due to a deficient compensatory response tohypotension.Ourobservations incell culture conditions may thus reveal a hitherto unexplored mechanism of vascular pathology in DENV infection. In the present work, we compared the permissiveness of primary HUASMC cells, primary HUVEC cells, and the model cell line LLC-MK2withDENVstrains of the four serotypes. The results demonstrate that HUASMC cells are permissive to DENV infection by all four serotypes. However, virus pro- duction and replicative fitness are significantly lower in this cell line than in HUVEC and LLC-MK2 cells. Indeed, on infection, HUASMC cells displayed a small percentage of DENV FIGURE 2. Human umbilical artery smoothmuscle cells (HUASMC) release dengue virus (DENV) genomes on infection. Dengue virus genomic RNAwas quantified by real-time qRT-PCR from cell culture supernatants of HUASMC, human umbilical vein endothelial cells (HUVEC), and LLC-MK2 (macaque kidney cells) infected with DENV (multiplicity of infection:1). (A) Dengue virus 1–4 infection kinetics (24–72 hours post-infection [p.i.]) of HUASMC cells measured by real-time genomic qRT-PCR (copies/mL). (B) Genome copies pre- sent in culture supernatants of HUASMC, HUVEC, and LLC-MK2 cells at 72 hours p.i. with the four DENV serotypes. (C) Calculated genome-to-plaque-forming unit (PFU) ratios of HUASMC, HUVEC, and LLC-MK2 cells supernatants at 72 hours p.i. with each DENV serotype. Data are expressed as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.005, and ***P < 0.001 calculated to its HUASMC counterpart. 1454 ARIAS-ARIAS AND OTHERS antigen-positive cells, indicating that this cell line has a low permissiveness to DENV infection by all four serotypes. Al- though it is evident that new infectious viral particles were produced by infectedHUASMC (Figure 1), the genome copies did not increase (for DENV1 andDENV2) or only increased one log after 48 hours p.i. (for DENV3 and DENV4), as shown in Figure 2. This observation suggests that most of the genomes detected in the supernatant are from defective particles FIGURE 3. Dengue virus (DENV) antigens are detected in human umbilical artery smooth muscle cells (HUASMC). Epifluorescence images of immunostained cells with an anti-DENV 1-4 envelope protein-specific monoclonal antibody (green), and a cytoplasmic (red) and nuclei (blue) counterstains. The images were captured at 72 hours post-infection (p.i.) with each DENV serotype at a multiplicity of infection (MOI) of 1. (A) Representative image of mock-infected HUASMC cells at ×100 magnification (scale bar = 50 μm). (B and C) Representative images of DENV- infected HUASMC (arrows) at ×400 (scale bar = 20 μm) and ×100 (scale bar = 50 μm)magnification, respectively. (D) Image analysis for quantifying infected cells (green outline) vs total cell nuclei (white outlines) with the software CellProfiler 2.0. (E) Percentages of infected cells in HUASMC, human umbilical vein endothelial cells (HUVEC), and LLC-MK2 (macaque kidney cells) cell lines. (F) Comparison of DENV labeling by immunos- tainingwith an anti-DENV 1-4 envelopeprotein-specificmonoclonal antibody (green) and an anti-NS3 polyclonal antibody (red) in HUASMCcells at 72 hours p.i. with DENV-2 andDENV-3 at aMOI of 1.Magnification of ×400 (scale bar = 20μm). Data are expressed asmean ± standard deviation of three independent experiments. **P < 0.005, ***P < 0.001 calculated to its HUASMC cells counterpart. This figure appears in color at www.ajtmh.org. DENGUE VIRUS INFECTION OF PRIMARY HUMAN SMOOTH MUSCLE CELLS 1455 http://www.ajtmh.org produced by these cells or genomes released fromdead cells, which occlude the expected elevation associated with the increased infectious particles. In the context of a viral infection with low permissiveness, a possibility to explain this phenomenon arises if the in- fected cells are able to replicate the viral genomes, but there is a problem with virion assembly or maturation in a high proportion of infected cells. These genomes would be even- tually released from the cells, explaining the high genome copies at all-time points assessed. Only the viral particles produced from a subpopulation of infected cells with rela- tively higher permissiveness would represent the PFUs, which are increasing progressively over time. This is prob- ably due to a viral morphogenesis problem in infected HUASMCcells and, therefore, the viral antigens are detected only in a small proportion of cells. Nevertheless, the immu- nofluorescence data demonstrate that new viral proteins are produced at least in the subpopulation of cells that produce viable viral particles (Figure 3). Indeed, permissiveness was very similar for HUASMC and HUVEC cells, both of which derive from the umbilical cord, where they form functional blood vessels.20 Altogether, these results support a model where DENV induces the dysfunction of smooth muscle cells, thereby contributing to the vascular hyporeactivity in vivo. The compensatory mechanisms to hypovolemia include an early sympathetic response characterized by increased heart rate andsystemic increments in vascular resistance,whichare mostly mediated by the action of catecholamines, especially noradrenaline, in cardiac muscle and in arteriolar smooth muscle cells.27,28 In addition, this compensatory vasocon- striction is mediated by thromboxane A2-triggered signaling in smooth muscle cells.29 It has been demonstrated that plasma levels of thromboxane A2 are significantly lower in dengue shock syndrome patients than in healthy populations and patients with dengue hemorrhagic fever but without shock.30 This suggests that smooth muscle cells are already hyporeactive duringDENV-induced shock. Thus, the infection of those cells by DENV would further contribute to vascular dysfunction in vivo. In support of this contention, work by Balsitis and collaborators displays a splenic artery highly positive for NS3 staining located within the muscular layer in the arterial wall,11 suggesting that the infection of smooth muscle cells might occur in vivo during the DENV infection in humans. This is the first report of DENV-permissive infection of smooth muscle cells. Despite the limitations of an in vitro model of infection, our results suggest that the infection of arteriolar, arterial, or lymphatic smooth muscle cells could have important implications for DENV-induced shock. Further work is required to demonstrate the infection and dysfunction of these cells in vivo and to design strategies to protect them for cardiovascular homeostatic mechanisms. This protection may represent a new approach in the treatment of DENV- induced hypotension. Received February 27, 2018. Accepted for publication August 14, 2018. Published online November 5, 2018. Acknowledgments:We thankCarlos Vargas Eduarte for his invaluable technical support and assistance, as well as José Marı́a Gutiérrez Gutiérrez from Insituto Clodomiro Picado (Universidad de Costa Rica) for his scientific advice and critical reading of the manuscript. We are also grateful to Christine Carrington (The University of West Indies) for English proofreading of the manuscript. Financial support: This work was supported by Universidad de Costa Rica (project VI-803-A5-025), Consejo Nacional para Investigaciones Cientı́ficas y Tecnológicas (project FI-182-10), and the Florida Ice and Farm Co. Authors’ addresses: Jorge L. Arias-Arias, Francisco Vega-Aguilar, Eugenia Corrales-Aguilar, Laya Hun, Gilbert D. Lorı́a, and Rodrigo Mora-Rodrı́guez, Facultad de Microbiologı́a, Centro de Investigación en Enfermedades Tropicales, Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, San José, Costa Rica, E-mails: jorgeluis. arias@ucr.ac.cr, francisco.vega@ucr.ac.cr, eugenia.corrales@ucr.ac. cr, ruchlia.hun@ucr.ac.cr, gilbert.loria@ucr.ac.cr, and rodrigo. morarodriguez@ucr.ac.cr. REFERENCES 1. Mustafa MS, Rasotgi V, Jain S, Gupta V, 2015. Discovery of fifth serotype of dengue virus (DENV-5): a new public health di- lemma in dengue control.Med J Armed Forces India 71: 67–70. 2. Guzman MG et al., 2010. Dengue: a continuing global threat. Nat Rev Microbiol 8 (12 Suppl): S7–S16. 3. World Health Organization and Special Programme for Research and Training in Tropical Diseases, 2009. Dengue: Guidelines for Diagnosis, Treatment, Prevention, and Control, new edition. Geneva, Switzerland: WHO and Special Programme for Re- search and Training in Tropical Diseases. 4. Scott LJ, 2016. Tetravalent dengue vaccine: a review in the pre- vention of dengue disease. Drugs 76: 1301–1312. 5. Normile D, 2017. Safety concerns derail dengue vaccination program. Science 358: 1514–1515. 6. Acosta EG, Bartenschlager R, 2016. The quest for host targets to combat dengue virus infections. Curr Opin Virol 20: 47–54. 7. Chan KW, Watanabe S, Kavishna R, Alonso S, Vasudevan SG, 2015. Animal models for studying dengue pathogenesis and therapy. Antiviral Res 123: 5–14. 8. DiamondMS, Edgil D, Roberts TG, Lu B, Harris E, 2000. Infection of human cells by dengue virus is modulated by different cell types and viral strains. J Virol 74: 7814–7823. 9. Medina F, Medina JF, Colon C, Vergne E, Santiago GA, Munoz- Jordan JL, 2012. Dengue virus: isolation, propagation, quanti- fication, and storage. Curr Protoc Microbiol 15D: 2.1–2.24. 10. Barr KL, Anderson BD, 2013. Dengue viruses exhibit strain- specific infectivity and entry requirements in vitro. Virus Adapt Treat 5: 1–9. 11. Balsitis SJ,ColomaJ,CastroG,AlavaA, FloresD,BeattyR,Harris E, 2008. Tropism of replicating dengue virus in mice and hu- mans defined by viral nonstructural protein 3-specific immu- nohistochemistry. Am J Trop Med Hyg 79: 38. 12. LindenbachBD, Rice CM, 2007. Flaviviridae: the viruses and their replication. Fields Virol 2007: 1101–1151. 13. Paliwal VK, Garg RK, Juyal R, Husain N, Verma R, Sharma PK, Verma R, Singh MK, 2011. Acute dengue virus myositis: a re- port of seven patients of varying clinical severity including two cases with severe fulminant myositis. J Neurol Sci 300: 14–18. 14. Salgado DM et al., 2010. Heart and skeletal muscle are targets of dengue virus infection. Pediatr Infect Dis J 29: 238–242. 15. Yacoub S, Wertheim H, Simmons CP, Screaton G, Wills B, 2015. Microvascular and endothelial function for risk predic- tion in dengue: an observational study. Lancet 385 (Suppl 1): S102. 16. Huang YH, Lei HY, Liu HS, Lin YS, Liu CC, Yeh TM, 2000. Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production. Am J Trop Med Hyg 63: 71–75. 17. Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M, 1998. Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J Immunol 161: 6338–6346. 18. Warke RV et al., 2003. Dengue virus induces novel changes in gene expression of human umbilical vein endothelial cells. J Virol 77: 11822–11832. 19. Malavige GN, Fernando S, Fernando DJ, Seneviratne SL, 2004. Dengue viral infections. Postgrad Med J 80: 588–601. 1456 ARIAS-ARIAS AND OTHERS mailto:jorgeluis.arias@ucr.ac.cr mailto:jorgeluis.arias@ucr.ac.cr mailto:francisco.vega@ucr.ac.cr mailto:eugenia.corrales@ucr.ac.cr mailto:eugenia.corrales@ucr.ac.cr mailto:ruchlia.hun@ucr.ac.cr mailto:gilbert.loria@ucr.ac.cr mailto:rodrigo.morarodriguez@ucr.ac.cr mailto:rodrigo.morarodriguez@ucr.ac.cr 20. Korff T, Kimmina S, Martiny-Baron G, Augustin HG, 2001. Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF respon- siveness. FASEB J 15: 447–457. 21. Mora J, Mora R, Lomonte B, Gutiérrez JM, 2008. Effects of bothrops asper snake venom on lymphatic vessels: insights into a hidden aspect of envenomation. PLoS Negl Trop Dis 2: e318. 22. Soto-Garita C, Somogyi T, Vicente-Santos A, Corrales-Aguilar E, 2016. Molecular characterization of two major dengue out- breaks in Costa Rica. Am J Trop Med Hyg 95: 201–205. 23. Morens DM, Halstead SB, Repik PM, Putvatana R, Raybourne N, 1985. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: com- parison of the BHK suspension test with standard plaque re- duction neutralization. J Clin Microbiol 22: 250–254. 24. GutierrezG, Reines HD,Wulf-GutierrezME, 2004. Clinical review: hemorrhagic shock. Crit Care 8: 373–381. 25. Li T, Liu L, Xu J, Yang G, Ming J, 2006. Changes of Rho kinase activity after hemorrhagic shock and its role in shock-induced biphasic responseof vascular reactivity and calciumsensitivity. Shock 26: 504–509. 26. SongR, BianH,WangX,HuangX, ZhaoK-S, 2011.Mitochondrial injury underlies hyporeactivity of arterial smooth muscle in se- vere shock. Am J Hypertens 24: 45–51. 27. Flint LM, Cryer HM, Simpson CJ, Harris PD, 1984. Microcircula- tory norepinephrine constrictor response in hemorrhagic shock. Surgery 96: 240–247. 28. Scully CG et al., 2016. Effect of hemorrhage rate on early hemo- dynamic responses in conscious sheep. Physiol Rep 4: 1–15. 29. Dorn GW, Becker MW, 1993. Thromboxane A2 stimulated signal transduction in vascular smoothmuscle. J Pharmacol Exp Ther 265: 447–456. 30. Preeyasombat C, Treepongkaruna S, Sriphrapradang ACL, 1999. The role of prostacyclin (PGI2) and thromboxane A2 (TXA2) in pathogenesis of dengue hemorrhagic fever (DHF). J Med Assoc Thai 82 (Suppl 1): S16–S21. DENGUE VIRUS INFECTION OF PRIMARY HUMAN SMOOTH MUSCLE CELLS 1457 25 Protocol. Rapid IFA for labeling DENV/ZIKV structural and nonstructural proteins. 1. Seed and infect the model cells with the DENV/ZIKV strain of interest at the desired multiplicity of infection (MOI), on a µClear black 96-well plate (Greiner Bio-One 655090). 2. Remove the culture media and wash once with 100 µL/well of phosphate-buffered saline (PBS, Gibco 10010023) to remove detached cells and cellular debris. 3. Fix the cell monolayers with 50 µL/well of a 3.5% paraformaldehyde (Sigma 158127) solution in PBS for 15 min at room temperature. Remove the fixative and wash once with 100 µL/well of PBS. 4. Permeabilize cells with 50 µL/well of 70% ethanol in water for 15 min at room temperature. Remove the ethanol and wash once with 100 µL/well of PBS. 5. Incubate for 1 h at 37 °C with 50 µL/well of one of the following primary antibodies diluted in a 0.001% Triton X-100 (Sigma 10789704001) solution in PBS: - 1:400 dilution of mouse anti-DENV 1-4 E protein monoclonal antibody (GeneTex GTX29202). - 1:400 dilution of mouse anti-ZIKV E protein monoclonal antibody (GeneTex GTX634157). - 1:800 dilution of rabbit anti-DENV NS3 protein polyclonal antibody (GeneTex GTX124252). - 1:400 dilution of rabbit anti-ZIKV NS3 protein polyclonal antibody (GeneTex GTX133309). 6. Wash twice with 100 µL/well of PBS and once with 100 µL/well of 0.001% Triton X-100 solution in PBS. 7. Incubate for 30 min at 37 °C with 50 µL/well of one of the following secondary antibodies (accordingly) diluted in a solution of 0.001% Triton X-100, 0.02% Evans blue (Sigma E2129), and 1 µg/mL Hoechst 33342 (Invitrogen H3570, 1:10 000 dilution) in PBS: - 1:400 dilution of Alexa Fluor 488 goat anti-mouse IgG, IgM (Invitrogen A-10684), Alexa Fluor 568 goat anti-mouse IgG (Invitrogen A-11031), or Alexa Fluor 647 goat anti-mouse IgG (Invitrogen A-21237). - 1:400 dilution of Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen A-11034), Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen A-11037), or Alexa Fluor 647 goat anti-rabbit IgG (Invitrogen A-21245). 26 8. Wash three times with 100 µL/well of PBS, add 100 µL/well of FluoroBrite Dulbecco’s modified Eagle’s medium (DMEM, Gibco A1896701), and acquire images at the desired magnification with an automated fluorescence microscope (e.g., BioTek Lionheart FX). 9. Analyze the images using an image analysis software (e.g., CellProfiler 3.0, Broad Institute https://www.cellprofiler.org/) to quantify the percentages of infected cells. Notes: Cells could be also seeded and infected on round glass coverslips into a 24-well plate, stained and mounted in slides to analyze the results with a conventional fluorescence microscope. We have also labeled all the above-mentioned primary antibodies using commercial labeling kits for Alexa Fluor 488 (Invitrogen A20181), Alexa Fluor 568 (Invitrogen A20184), and Alexa Fluor 647 (Invitrogen A20186), facilitating a much faster direct IFA protocol applying dilutions in the range 1:75-1:150, as well as flow cytometry assays with dilutions 1:300-1:400. https://www.cellprofiler.org/ 27 Chapter 2 Development and validation of cell-based molecular reporters and cytopathic effect fluorescent labeling approaches for the study of flavivirus infection kinetics in single cells and viral plaques by live-cell imaging Summary The identification of therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with wild-type viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. This chapter describes the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus internal NS3 cleavage site linker (AAQRRGRIG) presented the highest fluorescence activation in stably transduced mammalian cells upon DENV- 2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco’s plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with DNA fluorescent dyes for the detection of virus- induced chromatin condensation and cell death (CPE labeling), enabling studies of viral plaque formation with single-cell resolution. Finally, the application of the multireporter platform also enabled the study of cell-population kinetics of infection and CPE induction by DENV-2, ZIKV, and YFV. Such approaches constitute valuable tools for both basic and applied research in flavivirology. 28 Published articles Arias-Arias, J.L., MacPherson, D.J., Hill, M.E., Hardy, J.A., Mora-Rodríguez, R., 2020. A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques. J. Biol. Chem. 295, 2212–2226. https://doi.org/10.1074/jbc.RA119.011319 Arias-Arias, J.L., Corrales-Aguilar, E., Mora-Rodríguez, R.A., 2021. A fluorescent real-time plaque assay enables single-cell analysis of virus-induced cytopathic effect by live-cell imaging. Viruses 13, 1193. https://doi.org/10.3390/v13071193 https://doi.org/10.1074/jbc.RA119.011319 https://doi.org/10.3390/v13071193 A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques Received for publication, October 2, 2019, and in revised form, January 2, 2020 Published, Papers in Press, January 9, 2020, DOI 10.1074/jbc.RA119.011319 Jorge L. Arias-Arias‡, Derek J. MacPherson§, Maureen E. Hill§, Jeanne A. Hardy§, and X Rodrigo Mora-Rodríguez‡1 From the ‡Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José 11501-2060, Costa Rica and the §Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003 Edited by Craig E. Cameron The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for thera- peutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus–host interactions during infectionswithnative viral strains.However, this is precludedby limitations of current cell-based systems for monitoring flavivi- rus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirusNS2B–NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluo- rescent upon cleavage by recombinantDENV-2/ZIKVproteases in vitro. A version of this sensor containing the flavivirus inter- nal NS3 cleavage site linker reported the highest fluorescence activation in stably transducedmammalian cells uponDENV-2/ ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensorhad thebest signal-to-noise ratio in a fluorescentDulbec- co’s plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus–host interactions and facilitate future applications in antiviral drug research to manage flavivi- rus infections. The genus Flavivirus in the family Flaviviridae comprises more than 70 species of arthropod-borne viruses (arboviruses) that are transmitted to vertebrates by infected mosquitoes or ticks, producing diseases in animals and humans, including many medically important viruses like West Nile virus (WNV),2 yellow fever virus (YFV), St. Louis encephalitis virus, dengue virus (DENV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) (1). The genome of flaviviruses is a positive sense RNA of�11 kb that encodes three structural proteins, i.e. capsid (C), mem- brane precursor (prM), and envelope (E), and seven nonstruc- tural proteins, i.e. NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. These proteins initially form a precursor polyprotein (NH2- C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH) that is cleaved by both cellular and viral proteases to release the mature viral proteins (2). The flavivirus serine protease NS2B– NS3 consists of theN-terminal domain of theNS3 protein asso- ciated with the membrane-resident NS2B cofactor to form an active complex. This viral protease cleaves the precursor poly- protein at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, andNS4B/ NS5 junctions, as well as at internal sites within C, NS2A, NS3, and NS4A (3–5). Flaviviruses have continued to emerge in recent years, and together represent a global threat responsible for pandemics associated with encephalitis and hemorrhagic fever diseases for which there are no specific treatments available other than sup- portive care upon hospitalization (2). Moreover, the develop- ment of successful human vaccines seems to be challenging for some flaviviruses. Although YFV, JEV, and TBEV vaccines are highly effective, the development of vaccines for other flavivi- ruses like WNV and DENV have presented some drawbacks and safety concerns (6–8) This situation partially arises from the limitations of clinical studies, and although there are estab- lished animal models for flaviviruses, they do not faithfully reproduce all the clinicalmanifestations observed in the human host (9, 10). Therefore, post-mortem studies and cell culture models are still an important approach to study flavivirus dis- eases (11–13), especially for the quest of novel therapeutic tar- gets to combat these infections, either on the virus or on the host (14, 15). Currently, the identification of flavivirus-infected cells relies on either immunostaining of viral proteins (12), the application This work was supported by Universidad de Costa Rica Project VI-803-B9 –505 (to J. L. A.-A. and R. M.-R.), National Science Foundation Grant NSF CBET1511367 (to J. A. H.), and International Centre for Genetic Engineering and Biotechnology Grant CRP/CRI18-02 (to R. M.-R). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Table S1 and Figs. S1–S5. 1 To whom correspondence should be addressed: Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, San Pedro de Montes de Oca, San José 11501-2060, Costa Rica. Tel.: 506-2511-8635; E-mail: rodrigo.morarodriguez@ucr.ac.cr. 2 The abbreviations used are: WNV, West Nile virus; DENV, dengue virus; ZIKV, Zika virus; YFV, yellow fever virus; JEV, Japanese encephalitis virus; TBEV, tick-borne encephalitis virus; CA, caspase-activatable; FlaviA, flavivirus-ac- tivatable; MOI, multiplicity of infection; FBS, fetal bovine serum; MEM, min- imum essential medium; DMEM, Dulbecco’s modified Eagle’s medium. croARTICLE 2212 J. Biol. Chem. (2020) 295(8) 2212–2226 © 2020 Arias-Arias et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from https://orcid.org/0000-0001-7964-3575 https://www.jbc.org/cgi/content/full/RA119.011319/DC1 mailto:rodrigo.morarodriguez@ucr.ac.cr https://crossmark.crossref.org/dialog/?doi=10.1074/jbc.RA119.011319&domain=pdf&date_stamp=2020-1-9 http://www.jbc.org/ http://www.jbc.org/ http://www.jbc.org/ http://www.jbc.org/ http://www.jbc.org/ http://www.jbc.org/ http://www.jbc.org/ http://www.jbc.org/ http://www.jbc.org/ of recombinant reporter replicons or viral genomes (16–20), or the use of cell-based molecular reporters of the NS2B–NS3 activity (21–23). Antibody staining techniques require both fix- ation and permeabilization because of the lack of flavivirus expressed proteins directly on the cell surface of infected cells as a part of the viral replication cycle (2, 24, 25), which precludes their application for live-cell imaging. Reporter replicons and viral genomes allow kinetic studies in living cells but are limited to molecular clones and thus not suitable to study clinical isolates or native virus strains. In this respect, genetically encodedmolecular reportersmonitoring the flavivirusNS2B–NS3proteolytic activity upon infection are an advantageous approach that is suitable for live-cell imaging studies of native flavivirus strains. Previously, we developed a series of caspase-activatable reporters by fusing, via a linker containing the caspase-3/7 cleavage site DEVD, a hydrophobic quenching peptide to the C terminus of a fluorescent protein (26–28). This quenching pep- tide inhibits the maturation of the chromophore in the fluores- cent protein until it is proteolytically removed by an active caspase, fully restoring the fluorescence (26, 27). In the present study, we developed genetically encoded flavivirus molecular reporters by inserting a flaviviral NS2B–NS3 cleavage site into our caspase-activatable (CA) GFP (26) or CA-mNeptune (28), giving rise to the flavivirus-activatable (FlaviA) GFP and Fla- viA-mNeptune reporters, respectively. To our knowledge, this is the first fluorescence-activatable molecular reporter system for live-cell imaging of the infection by both reference and native strains of flaviviruses like DENV, ZIKV, and YFV. Results Fluorescence-activatable GFP-based reporters of flavivirus NS2B–NS3 protease activity become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro We based the design of a molecular sensor for flavivirus pro- teases on our previously reported CA-GFP sensor that com- prises GFP, a linker for caspase cleavage and a C-terminal quenching peptide (26–28). However, we encountered several limitations for the development of the new sensor, mainly with the linker sequence for the reporter function. This led us to envisage several alternative designs by changing the linker sequence. Indeed, we generated several variants of the reporter that remained uncleaved and/or nonfluorescent upon DENV-2 NS2B–NS3 protease treatment in vitro (Table S1). Therefore, we designed a linker based on previously characterized flavivi- rus polyprotein cleavage sites (29). After careful analysis and avoiding the formation of cleavage sites for other cellular proteases within the resulting protein sequence of the sensor (http://web.expasy.org/peptide_cutter/),3 we selected the cleavage sequences that define the linker. Three variants of this reporter were constructed by changing the linker sequence: ZIKVA-GFP (ZIKV polyprotein NS2B/NS3 cleavage site linker), DENV2A-GFP (DENV-2 polyprotein NS2B/NS3 cleav- age site linker), and FlaviA-GFP with the internal NS3 cleavage site present in many members of the Flavivirus genus (3, 5, 30). Using these variants of the reporter, we verified the cleavage in vitro by Coomassie Blue–stained SDS-PAGE gels (Fig. 1, A and B, and Fig. S1) and the fluorescence activation (Fig. 1C) to eval- uate at the protein level the potential of these linkers to be used within reporters of viral protease activity. Purified recombinantDENV-2NS2B–NS3 protease (Fig. 1A, left panels) or ZIKVNS2B–NS3 protease (Fig. 1A, right panels) were added to the three purified FlaviA-GFP reporter proteins. The DENV-2 NS2B–NS3 protease band was observed at 25 kDa, and the ZIKV NS2B–NS3 protease was located below 20 kDa, whereas all three full-length reporter proteins appeared above 30 kDa. To determine the location of the cleaved reporters, we gen- erated a truncated variant of the FlaviA-GFP reporter protein (tRep/control) by inserting a stop codon downstreamof the cleav- age site in the DNA sequence of the linker. The bands of the cleaved reporters appeared between the 25- and 30-kDamarkers. The cleavage kinetics of the reporters can be observed over time for the three variants tested (Fig. 1,A and B). The intensities of these bands were quantified, and a ratio of the cleaved reporter to the total amount of reporter protein for each time point was calculated. The results are displayed as time-resolved cleavage efficiency (%) to compare among the different variants of the reporter (Fig. 1B). TheDENV-2NS2B– NS3 protease has very similar cleavage kinetics for the three variants with some slight differences. Although the FlaviA-GFP reporter showed an earlier increase, the ZIKVA-GFP reporter also reached �80% of cleavage efficiency. The DENV2A-GFP reached only �50% efficiency (Fig. 1B, left panel). On the other hand, striking differences are observed for the cleavage efficiency of the three variants of the reporter by the ZIKV NS2B–NS3 protease. The ZIKVA-GFP reporter had a much earlier increase, reaching almost 100% cleavage by 10 h. In contrast, the FlaviA-GFP and the DENV2A-GFP variants reached only 40% of cleavage efficiency after 20 h (Fig. 1B, right panel). These results indicate that the reporters are sensitive to fla- vivirus protease cleavage as designed, although with differ- ent efficiencies and kinetics. To determine whether these cleavage kinetics correlate with fluorescence activation of the reporters, wemonitored the fluo- rescence signal of each reporter as a function of time and normalized it to the background signal for each construct, obtaining thereby a time-resolved signal-to-noise ratio for the fluorescence of the reporters. All three reporters showed an increased in this signal-to-noise ratio for both protease treat- ments, indicating that the cleavage of the constructs correlates with the fluorescence increase of the GFP. The ZIKVA-GFP reporter showed the highest increase in fluorescence for both protease treatments, followed by the FlaviA-GFP and the DENV2A-GFP. These results indicate that the increase of the signal-to-noise ratio is a sensitivemarker of cleavage, especially for the ZIKVA-GFP reporter (Fig. 1C). The FlaviA-GFP sensor reports the highest fluorescence increase in stably transduced mammalian cells upon DENV-2/ ZIKV infection To validate our candidate GFP-based reporters of flavivirus NS2B–NS3 proteases, we generated three BHK-21 stable cell lines expressing each reporter. Upon DENV-2 or ZIKV infec- 3 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site. A cell-based fluorescent reporter for flavivirus infection J. Biol. Chem. (2020) 295(8) 2212–2226 2213 at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from https://www.jbc.org/cgi/content/full/RA119.011319/DC1 https://web.expasy.org/peptide_cutter/ https://www.jbc.org/cgi/content/full/RA119.011319/DC1 http://www.jbc.org/ tion at a low multiplicity of infection (MOI) of 0.25, we moni- tored the cellular fluorescence as a function of time using live imaging. A qualitative assessment of the images suggested that the cell fluorescence started to increase significantly at 48 h post-infection (Fig. 2A and Fig. S2A). To quantify this increase, we constructed an image analysis pipeline usingCellProfiler 2.0 to identify single cells based on their nuclei, recognize their cytoplasms (white outlines), classify them as live (blue dots) or dead cells (red outlines and dots) and quantify the total cell fluorescence (Fig. 2,A andB). Our results showed that the viral- Figure 1. Fluorescence-activatable GFP-based reporters for flavivirus NS2B–NS3 protease activity become fluorescent upon cleavage by DENV-2/ ZIKV recombinant proteases in vitro. The flavivirus-activatable GFP reporters contain a quenching peptide (QP) at the C terminus of GFP joined by a linker consisting of a cleavage site for the flavivirus NS2B–NS3 proteases. When the viral proteases cleave the linker, the quenching peptide is removed, and the GFP adopts a conformation promoting chromophore maturation. Three variants of this reporter were developed by changing the linker sequence: ZIKVA-GFP (ZIKV polyprotein NS2B/NS3 cleavage site linker), DENV2A-GFP (DENV-2 polyprotein NS2B/NS3 cleavage site linker), and FlaviA-GFP with the internal NS3 cleavage site linker, which is present in many members of the Flavivirus genus. A, in vitro cleavage kinetics of the flavivirus-activatable GFP reporter. Purified reporter proteins were mixed with purified DENV-2 NS2B–NS3 protease (left panels) or ZIKV NS2B–NS3 protease (right panels) at a molar ratio of 1:1 and incubated for given times. The reactions were quenched by thermal treatment in SDS loading buffer, and samples were analyzed by SDS-PAGE and staining of the gels with Coomassie Blue. tRep/control is an engineered cleaved variant of the FlaviA-GFP protein and was used as size marker of cleaved reporters. Representative cropped images from three independent experiments are shown. B, cleavage efficiency kinetics of the purified flavivirus-activatable GFP reporter proteins treated with purified DENV-2 NS2B–NS3 protease (left panel) and ZIKV NS2B–NS3 protease (right panel). C, time-resolved fluorescence signal-to-noise ratio of the purified flavivirus-activatable GFP reporter proteins treated with purified DENV-2 NS2B–NS3 protease (left panel) and ZIKV NS2B–NS3 protease (right panel). The data are expressed as means � S.D. of three independent experiments. **, p � 0.001 compared with the other two reporter variants at 20 h post-treatment. A cell-based fluorescent reporter for flavivirus infection 2214 J. Biol. Chem. (2020) 295(8) 2212–2226 at U N IV E R SID A D D E C O ST A R IC A on M arch 18, 2020 http://w w w .jbc.org/ D ow nloaded from https://www.jbc.org/cgi/content/full/RA119.011319/DC1 http://www.jbc.org/ induced cytotoxicity started �50–60 h postinfection with DENV-2 and �40–50 h post–ZIKV infection (Fig. 2B, red dots). However, the cellular fluorescence started to increase in living cells (Fig. 2B, blue dots) approximately at 48 h postinfec- tion, and we could quantify living cells with increased fluores- cence until the end of this time course (96 h). The population mean values for each condition are represented by the green continuous lines. To compare the different variants of the reporter, wemonitored the cell population kinetics of the flavi- virus-activatable GFP sensor’s fluorescence across multiple Figure 2. The FlaviA-GFP sensor reports the highest fluorescence increase in stably transduced mammalian cells upon DENV-2/ZIKV infection. We generated three BHK-21 stable cell lines expressing the flavivirus-activatable GFP reporters, each with one of the previously tested linker sequences. After cell sorting of subpopulations with homogeneous expression of each reporter, the cells were grown and infected with either infectious or UV-inactivated DENV-2 13538/ZIKV CIET-01 at a low MOI of 0.25, for the specified time periods. A, an automated image analysis protocol was constructed in CellProfiler 2.0 for the quantification of live (white outline), dead (red outline), and activated FlaviA-GFP fluorescent cells (green). A representative experiment is shown for the FlaviA-GFP stable cell line infected with DENV-2 (n � three independent experiments, magnification of 200�; scale bar, 100 �m). B, the flavivirus-activatable GFP reporter activation is represented by scatter plots showing the time-resolved fluorescence of the population of single live (blue) and dead (red) reporter cells after the exposure to infectious or UV-inactivated DENV-2 (left panels) or ZIKV (right panels). The population mean values for each condition are repre- sented by the green continuous lines. Representative scatter plots are shown (n � three independent experiments). C, the cell population kinetics of the flavivirus-activatable GFP sensors fluorescence across multiple experiments