Enhancing dengue virus maturation using a stable furin over-expressing cell line

Dengue AntigensJawahar Raina
Enhancing dengue virus maturation using a stable furin over-expressing cell line
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Abstract

Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for flavivirus neutralization and structural studies.

Introduction

Flaviviruses are enveloped positive-stranded RNA viruses transmitted principally by arthropod vectors. Many viruses in this genus, such as West Nile (WNV), dengue (DENV), and Zika viruses, have a considerable impact on global health due to their potential for rapid emergence and morbidity (Fauci and Morens, 2016; Guzman and Harris, 2015; Mackenzie et al., 2004). Flavivirus virions assemble on membranes of the endoplasmic reticulum as immature virus particles that incorporate two viral structural proteins on their surface (Mackenzie and Westaway, 2001; Welsch et al., 2009; Zhang et al., 2003). The envelope (E) protein is an elongated three-domain class II fusion protein tethered to the viral membrane by a helical stem and two transmembrane helices (Zhang et al., 2013a). It is responsible for binding to cellular attachment factors (Perera-Lecoin et al., 2014) and promoting fusion of viral and cellular membranes following endocytosis and exposure to a low pH environment (Sanchez-San Martin et al., 2009). The premembrane (prM) protein associates with E shortly after synthesis (Lorenz et al., 2002) and is incorporated into virus particles as a heterotrimer. Each immature virion contains sixty heterotrimeric spikes arranged in an icosahedral fashion (Zhang et al., 2003; Zhang et al., 2007). In this configuration, E proteins cannot undergo changes in conformation required for viral fusion, rendering immature particles non-infectious (Guirakhoo et al., 1991; Zybert et al., 2008). Conversion of immature virions to an infectious form occurs while virus particles traffic through the secretory pathway. In the low pH environment of the trans-Golgi network, immature virions undergo a structural transition that exposes on prM a site recognized by host furin-like proteases (Li et al., 2008; Stadler et al., 1997; Yu et al., 2008). Cleavage of prM is the defining event in flavivirus maturation and is a required step in the virus infection cycle (Elshuber et al., 2003; Stadler et al., 1997). The products of this cleavage event are a membrane anchored nine kDa peptide (the membrane (M) protein) and a soluble “pr” fragment (~22 kDa) that disassociates from the virion upon release from the cell (Yu et al., 2009). The E proteins on mature virions are arranged as antiparallel dimers arrayed in a herringbone fashion (Kuhn et al., 2002; Zhang et al., 2013a).

Flaviviruses are secreted from cells as a heterogeneous mixture of virions due in part to inefficiency of the prM cleavage reaction (reviewed in (Pierson and Diamond, 2012)). Partially mature virions are defined herein as having structural features of both mature and immature virus particles. Structural studies suggest that the E proteins of partially mature virions are arranged as mosaics of immature virus-like prM-E heterotrimers and antiparallel E dimers, in varying proportions (Plevka et al., 2011). The fraction of virions that retain uncleaved prM in vitro and in vivo, and the distribution of uncleaved prM among individual virions, are unclear and may vary among cell types (Randolph et al., 1990). Studies of DENV revealed that ~90% of the E protein in C6/36 insect cell-derived stocks of virus can be immunoprecipitated with a prM-reactive antibody (Junjhon et al., 2010); these studies suggest prM+ virions may be common. Partially mature virions may be infectious, although the extent of cleavage required for virus infectivity remains unknown (Pierson and Diamond, 2012). Incomplete prM cleavage has a significant impact on antibody recognition of infectious viruses (Dowd et al., 2014; Mukherjee et al., 2014a; Nelson et al., 2008). Studies of several flaviviruses have identified neutralizing antibodies that bind E protein epitopes not predicted to be accessible for recognition on the mature virion (Austin et al., 2012; Cherrier et al., 2009; Cockburn et al., 2012; Lok et al., 2008; Oliphant et al., 2006; Stiasny et al., 2006). In many cases, decreasing the efficiency of prM cleavage markedly increases neutralization potency through changes in epitope accessibility. Uncleaved prM also may be bound directly by antibodies with a limited capacity to neutralize infection. In the case of DENV, occupancy by prM-reactive antibodies below the neutralization threshold allows antibody-dependent enhancement of infection (ADE) of Fc-receptor expressing cells (Dejnirattisai et al., 2010; Rodenhuis-Zybert et al., 2010), which has the potential to contribute to disease severity (Guzman et al., 2013).

The structural heterogeneity of flaviviruses complicates detailed characterization of structure-function relationships, insights into antibody recognition and neutralization potency, and the production of uniform stocks of live-attenuated vaccine candidates. In this study, we describe the creation and utility of a Vero cell line modified to ectopically express high levels of human furin. Flaviviruses produced in these cells are considerably less heterogeneous than stocks prepared in the parental Vero cell line. These virus preparations are characterized by increased specific infectivity and decreased sensitivity to neutralization by maturation state-dependent neutralizing antibodies, thus confirming the more efficient maturation of infectious virus particles. That both laboratory-adapted and primary DENV strains grow efficiently on this cellular substrate provides a powerful new tool for the study of flavivirus structure and humoral immunity. Because primary myeloid cells appear to produce more mature virions than cell culture adapted lines (Dejnirattisai et al., 2015), virions derived from Vero-furin cells may have greater relevance for studies of the antigenic relatedness of viruses (Katzelnick et al., 2015), and facilitate efforts to identify in vitro assays that predict protection following vaccination.

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Materials and Methods

Cells

HEK-293T and Vero cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing Glutamax supplemented with 100 U/ml penicillin-streptomycin (PS)(Invitrogen) and 7% fetal bovine serum (FBS)(HyClone). Vero and HEK-293T cell lines that over-express human furin (Vero-furin and 293T-furin, respectively) were cultured in the complete DMEM formulation detailed above supplemented with 10 μg/ml or 5 μg/ml of blasticidin, respectively (Invitrogen). Raji cells that stably express DC-SIGNR (Raji-DCSIGNR) were maintained in RPMI-1640 medium supplemented with 7% FBS and 100 U/ml PS. All mammalian cell culture was performed at 37°C in the presence of 7% CO2. The insect cell line C6/36 was cultured in complete DMEM supplemented with 1X non-essential amino acids (Invitrogen), and maintained at 28°C in the presence of 7% CO2.

Stable cell line production

A bicistronic vector (pFIRB) encoding human furin and the blasticidin resistance gene (blastR) was constructed using standard molecular cloning techniques. Briefly, pFIRB was constructed by introducing a 966-bp PCR amplicon encoding the foot-and-mouth disease virus internal ribosome entry site (IRES) and blastR from a previously described WNV replicon plasmid (Pierson et al., 2006) into a human furin-expressing pcDNA3.1 vector (Davis et al., 2006). pFIRB was transfected into Vero cells and cultured in the presence of 10 μg/ml of blasticidin. Furin-expressing clones were isolated by limiting dilutions and screened for expression by Western blot, as detailed below, and flow cytometry using a furin-specific polyclonal antibody and AlexaFluor 647 labeled secondary antibody (Invitrogen).

Viruses

An infectious clone of WNV lineage I (strain NY1999) that expresses GFP following infection has been described previously. Virus production using this clone was performed as described previously (Lin et al., 2012). Infectious clones of DENV1 (Western Pacific strain), DENV2 (New Guinea C strain), DENV3 (Sleman strain) and DENV4 (18604 strain) were obtained from Dr. Stephen Whitehead (NIH, Bethesda, MD). DENV was produced by in vitro transcription and capped using AmpliCap SP6 Message Maker technology (Epicentre Technologies) according to manufacturer’s instructions. Purified viral RNA was transfected into C6/36 cells using the DOTAP liposomal transfection reagent (Roche) in HEPES-buffered saline (pH 7.6). DENV was harvested 5 d post-transfection, passed through a 0.22 μm filter, aliquotted, and frozen at −80°C until use. Three independent working stocks of each virus were produced by passage in C6/36 cells for use in experiments described within. Clinical Nicaraguan DENV2 and DENV4 viruses were a kind gift from Dr. Eva Harris, University of California, Berkeley. Propagation of the DENV2 16681 for structural studies is detailed below. To reduce the efficiency of prM cleavage in virus preparations, cells were cultured in the presence of 50 μM furin inhibitor Dec-RVKR-CMK (Enzo Life Sciences), as described previously (Mukherjee et al., 2014a; Mukherjee et al., 2011).

Reporter virus particle (RVP) production

WNV RVPs were produced by complementation of a WNV subgenomic lineage II replicon with WNV structural genes as described (Mukherjee et al., 2014b; Pierson et al., 2006). The replicon used in these studies expresses a GFP reporter, allowing infection to be tracked by flow cytometry.

Large scale production and purification of DENV2 16681

Vero and Vero-furin cells were grown in 2- and 10-chambered cell stacks (Corning, New York) in DMEM as detailed above. Cells were infected at 80% confluence (~ 3 x 109 cells) with DENV2 16681 at a multiplicity of infection (MOI) of 0.1. Cells were gently agitated at room temperature for 2 h after which they were overlaid with DMEM containing 2% FBS and incubated further. Culture supernatant was harvested and replaced with fresh media on days 3, 4, 5, and 6 post-infection. Virus containing supernatants were clarified by centrifugation at 6000 rpm (JA-10, Beckman Coulter) for 30 min at 4°C, overlaid atop a 24% sucrose cushion prepared in TN buffer (20 mM Tris, 100 mM NaCl, pH 8.0), and centrifuged at 32,000 rpm (Type 50.2 Ti, Beckman Coulter) for 2 h at 4°C. The virus-containing pellet was solubilized in a small volume of TN buffer and loaded on a 10–30% potassium tartrate and 7.5–26% glycerol discontinuous gradient prepared in TN buffer and centrifuged at 32,000 rpm (SW41, Beckman Coulter) for 2 h at 4°C. The virus band was recovered from the 20% density zone by puncturing the tube using a 22-gauge needle. The virus sample was subjected to buffer exchange and was concentrated to a volume of 50–100 μl with an Amicon Ultra-4 centrifugal filter unit with an ultracell-100 membrane (EMD Millipore).

Cryo-electron microscopy, data collection and analysis

Purified DENV 16681 was placed on an ultra-thin carbon film (<3 nm) supported by a thicker holey carbon film on a 400 mesh copper grid (Ted Pella, Redding, CA). A cryoplunge 3 system (Gatan Inc.,Warrendale, PA) installed in a Class II biological safety cabinet was used to blot (~6 s) and rapidly plunge the grid into liquid ethane maintained in a liquid nitrogen bath. The grid was subsequently transferred to an FEI Titan Krios electron microscope using a cryo holder. The microscope was operated at 300 kV with a total dose of ~20 electrons per Å2 and micrographs were recorded using a 4kx4k CCD camera (Gatan Inc.). Viruses collected 5 d post-infection from both Vero and Vero-furin cells were analyzed further, as these were optimum in purity and virus concentration. Heterogeneity of each sample was analyzed by classifying ~850 particles as mature, partially mature and immature based on their smooth, mosaic or spiky appearances, respectively.

Viral RNA detection

The RNA content of DENV2 stocks was determined using quantitative real-time RT-PCR (qRT-PCR). DENV-containing supernatants were incubated with 100 U of recombinant DNase I (Roche) for 15 minutes at 37°C prior to RNA isolation using the QiaAmp Viral RNA spin column kit (Qiagen). Amplification of a portion of the 3’ untranslated region of the viral genomic RNA was accomplished using the Superscript III One-Step RT-PCR system (Invitrogen) using a previously described approach (Ansarah-Sobrinho et al., 2008).

Western blotting

Furin expression in cells was evaluated by Western blotting. Briefly, monolayers of Vero and Vero-furin cells were lysed in 1% Triton, 100 mM Tris, 2 M NaCl, and 100 mM EDTA; lysates were then clarified by centrifugation at 3,500 x g. Recombinant human furin (R&D Systems) was loaded in gels as a reference. Furin was detected using primate and human furin-reactive antibody (ThermoFisher) at 2 ug/ml. prM and E proteins in partially purified virus preparations were detected using monoclonal antibodies (mAbs) GTX128092 (Genetex) and, 4G2 respectively, at 1 μg/ml.

Neutralization assays

Neutralization assays were performed on Raji-DCSIGNR cells as detailed previously (Mukherjee et al., 2014b). For WNV RVPs, virus-antibody complexes were incubated for 1 h at room temperature prior to the addition of cells; these conditions have been shown previously to be sufficient to reach steady state of binding (Dowd et al., 2011). Infection was measured by flow cytometry 2 d post-infection. For infectious DENV, virus-antibody complexes were incubated at 37 °C for one hour prior to the addition of cells. Infected cells were incubated for two days. DENV infection was assayed by intracellular staining for the DENV E protein using AlexaFluor 647-labeled E protein-reactive antibody 4G2. Neutralization dose-response data was analyzed using non-linear regression on GraphPad Prism software (GraphPad Software Inc.). Comparisons of the neutralization potential of different antibodies on the two cells types were performed using a paired t test.

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Results

Creating a Vero cell line that ectopically over-expresses furin

Studies with pseudo-infectious reporter virus particles (RVPs) demonstrate that prM cleavage efficiency can be increased markedly by the over-expression of furin in virion producing cells. This approach has enabled insight into the infectious properties of partially mature flaviviruses (Davis et al., 2006), the role of furin during virus entry (Mukherjee et al., 2011), and the interplay between uncleaved prM on virions and antibody-mediated neutralization (Dowd et al., 2014; Mukherjee et al., 2014a; Nelson et al., 2008). The utility of transient furin expression for the study of infectious viruses is limited because replication competent viruses may infect non-transfected cells. To address this limitation, we and others have created HEK-293T cells that stably express furin (Dejnirattisai et al., 2015; Mukherjee et al., 2014a) . However, the utility of these reagents for the study of DENV may be limited by viral tropism. Studies by Meertens and colleagues demonstrated that HEK-293T cells have a limited capacity to support DENV entry because they lack members of the TIM/TAM family of receptors that serve as cellular attachment factors (Meertens et al., 2012). To create a new cell line that can be used to efficiently propagate flaviviruses with increased efficiency of virion maturation, we transduced Vero cells with a bicistronic plasmid that expresses human furin and a drug resistance marker. Transfected cells were grown in selective media, cloned, and characterized by flow cytometry (Figure S1A) and Western blotting (Figure S1B). Both approaches demonstrate that the resulting cell line, referred to as Vero-furin herein, expresses relatively high levels of human furin.

Dengue virus replication and growth kinetics in Vero-furin cells

To investigate the utility of Vero-furin cells for propagating DENV, we infected Vero and Vero-furin cells with strains representing the four serotypes of DENV at a multiplicity of infection (MOI) of 0.1. Virus-containing supernatants were collected from infected cells every 24 h for six days, filtered, and frozen until use. The infectious titer of each sample was assayed with an immunofocus assay on Vero cells. Analysis of the resulting growth curves from three independent experiments revealed that the growth kinetics of all four viruses were similar on both cell lines (Figure 1A–D). We next examined the ability of a clinical DENV isolate to replicate in Vero-furin cells. One-step growth curves of a Nicaraguan DENV2 clinical isolate (strain 172-06) on both Vero and Vero-furin cells were performed. Growth kinetics with this isolate were similar on both cell types, analagous to the pattern observed with the more extensively passaged viruses above (Figure 1E). Similar results were obtained with a DENV4 clinical isolate (Figure S2). By comparison, DENV2 172-06 was incapable of replicating in either parental HEK-293T cells or a HEK-293T furin over-expressing cell line previously shown to support propagation of WNV (Figure 1F)(Mukherjee et al., 2014a). Together, these data demonstrate that both laboratory strains and clinical DENV isolates are capable of efficiently replicating in Vero cells that express relatively high levels of human furin.

 

 

Figure 1

Growth kinetics of DENV in human furin-expressing Vero cells. (A–D) Virus stocks of DENV strains that represent each of the four serotypes, including DENV1 strain Western Pacific, DENV2 strain New Guinea C, DENV3 strain Sleman, DENV4 strain 814, were produced in Aedes albopictus C6/36 cells. Vero and Vero-furin cells were infected at a multiplicity of infection (MOI) of 0.1. Viruses were collected from infected cells at indicated timepoints post-infection and the infectious titer determined by immunofocus assay on Vero cells in duplicate. The growth curves presented are the average titer of three or four independent onestep growth curves. Error bars indicate the standard error of the mean (SEM). (E) The growth kinetics of a clinical DENV2 isolate (strain 172-06) on Vero and Vero-furin cells was determined as above. The results presented are the average of four independent one-step growth curves. Error bars indicate the SEM. (F) Comparisons of growth kinetics of DENV2 strain 172-06 on HEK-293T and HEK-293T-furin cells were performed as detailed above. Data presented reflect the average of three independent experiments; error bars indicate the standard error. In all instances, comparisons of the titer observed at each time point between cell types were performed using a two-way ANOVA are were not found to be significant.

Furin-over expression allows the production of a more homogenous population of virions

To investigate whether furin over-expression resulted in more efficient prM cleavage in virus-producing cells, DENV2 virus (strain New Guinea C) was propagated on Vero or Vero-furin cells, partially purified, lysed, and subjected to gel electrophoresis and Western blotting with prM- and E protein-reactive antibodies. As a control, prM+ “immature-like” virus was produced in Vero cells treated with a furin inhibitor as described previously (Mukherjee et al., 2014a). Virus stocks treated with furin-inhibitor contained markedly more uncleaved prM, as compared to virus preparations from Vero or Vero-furin cells (Figure 2A). In comparison, the prM content of Vero-furin cell-derived DENV was reduced, although still detectable, when compared to standard preparations of virus derived from parental Vero cells.

 

 

Figure 2

Evaluation of the maturation state of DENV generated in Vero and Vero-furin cells. (A) The prM content of DENV2 virus preparations was evaluated by SDS-PAGE and Western blotting using E- and prM-specific antibodies. prM-specific antibody GTX128092 (Genetex, Irvine, CA) was used for detection of prM levels. (B) Electron microscopic analysis of the morphology of DENV2 stocks produced from Vero and Vero-furin cell lines. M, mature; I, immature; PM, partially mature. (C) Proportion of virions with smooth (mature, M), spiky (I), or irregular morphology (partially mature, PM) in each stock. Statistical analysis was performed using an ANOVA with Tukey’s correction for multiple comparisons. (D) The infectious titer of DENV produced in Vero or Vero-furin cells was measured by infecting Raji-DCSIGNR cells with serial two fold dilutions of virus. Infectivity was measured 48 hours post-infection by immunostaining with AlexaFluor labeled 4G2 antibody. The infectivity of the virus stock is depicted relative to its genomic RNA content, as measured by qRT-PCR.

The structural heterogeneity of flaviviruses arising from the presence of prM on virions has been documented (Cherrier et al., 2009; Pierson and Diamond, 2012; Plevka et al., 2011). Virus preparations are composed of mature virions (M) characterized by a relatively smooth appearance, spiky immature virions (I), and a heterogeneous population of virions with irregular structure (P) (Figure 2B). To investigate whether propagation of DENV in cells that ectopically-express furin increases the homogeneity of virions, DENV2 strain 16681 was grown at large scale in Vero and Vero-furin lines, purified, and analyzed by cryo-electron microscopy. Particles were classified by morphology (smooth, spiky, or other) and the distribution of each class of virions in preparations of Vero- or Vero-furin derived virions is shown in Figure 2C. These data indicate that the percentage of immature virions was markedly higher in viruses derived from Vero cells than from the Vero-furin cell line (p<0.0001). Conversely, virions with a mature morphology were enriched in the Vero-furin population relative to the parental cell line (p<0.0001). The proportion of virions with irregular structure, which includes partially mature virus particles, did not differ between virus preparations (p>0.7).

Antigenic properties of DENV released from Vero-furin cells. Because biochemical and structural approaches do not distinguish between infectious and non-infectious virus particles, we performed experiments to determine the relative prM content of infectious virions derived from furin over-expressing and parental Vero cells. To provide an estimate of the specific infectivity of DENV preparations we first measured the RNA content of two independent virus stocks obtained from Vero and Vero-furin cells. RNA copy number was then used to normalize graphs depicting the number of Raji-DCSIGNR cells infected by serially diluted stocks of viruses. These studies revealed that production of DENV in Vero-furin cells resulted in virus preparations that were more infectious per copy of RNA than Vero cell-derived virus stocks analyzed in parallel (Figure 2D). Similar patterns were observed previously with RVPs produced in cells transiently transfected with furin-expressing plasmids (Nelson et al., 2008). These studies suggest that the expression of furin at high levels results in the production of infectious viruses with reduced prM content and greater specific infectivity.

Previous studies have shown that uncleaved prM governs the sensitivity of virions to neutralization by a subset of mAbs and polyclonal sera (Dejnirattisai et al., 2015; Dowd et al., 2014; Mukherjee et al., 2014a; Nelson et al., 2008). For example, studies of the WNV E protein specific mAb E53 revealed maturation state-dependent patterns of neutralization that can be explained structurally by the limited accessibility of the domain II fusion loop (DII-FL) epitope on mature but not partially mature prM+ virions (Cherrier et al., 2009). In contrast, mAb E16 binds an E protein domain III (DIII) epitope that is accessible on both immature and mature virions, allowing potent neutralization of infection irrespective of the prM content of the virion (Kaufmann et al., 2006; Nybakken et al., 2005). To further confirm that Vero-furin cells produce infectious virions with reduced prM content, we first performed control neutralization studies with WNV and this pair of extensively characterized antibodies. WNV RVPs were produced by transfection of Vero or Vero-furin cells and used in neutralization studies. The sensitivity of WNV RVPs produced in both cell types to neutralization by E16 was similar (~2-fold difference in EC50, n=3, Figure 3A), in agreement with prior studies (Dowd et al., 2014; Mukherjee et al., 2011; Nelson et al., 2008). In contrast, RVPs produced in Vero-furin cells were much less sensitive to neutralization by mAb E53, as reflected by significant differences in the size of the population of virions sensitive to neutralization (Figure 3B).

 

Figure 3

Impact of furin over-expression on maturation state-sensitive patterns of antibody-mediated neutralization. Neutralization of WNV RVPs produced in Vero and Vero-furin cells by (A) mAb E16 and (B) mAb E53 was measured as detailed previously (Mukherjee et al., 2014b). Serial dilutions of antibody were mixed with virus particles to allow for steady state binding and then added to Raji-DC-SIGNR cells. Infection was measured by flow cytometry two days post infection. Data was analyzed by non-linear regression using GraphPad Prism. Error bars represent the range of two technical replicates. Data are representative of at least three independent antibody-dose response curves. (C-E) Neutralization of infectious DENV2 derived from Vero and Vero-furin cell lines by mAbs DV-96 (C), E60 (D), and prM22 (E) was measured as described previously (Mukherjee et al., 2014b). Virus-antibody mixtures were incubated for 1 h at 37 ºC prior to the addition of Raji-DC-SIGNR cells. Cells were incubated at 37ºC for 48 hours prior to being fixed and stained for intracellular E protein expression. Infection was measured by flow cytometry. Data were analyzed by non-linear regression using GraphPad Prism. Error bars represent the range of two technical replicates. Data are representative of at least three independent antibody-dose response curves.

We next performed neutralization studies with DENV2 produced using Vero and Vero-furin cell lines and antibodies specific for E or prM. Neutralization studies with the DENV2 E-DIII-specific mAb DV-96 revealed similar neutralization sensitivity for stocks derived from both Vero and Vero-furin cells, analogous to the pattern observed with mAb E16 against WNV (Figure 3C). In contrast, virions produced in Vero-furin cells were considerably less sensitive to neutralization by the DII-FL-specific mAb E60 (Figure 3D). Because exposure of the E60 epitope on DENV is sensitive to both the maturation state of the virion and the conformational flexibility of the virion (Dowd et al., 2014), additional studies were performed using prM22, a murine mAb specific for DENV prM (Mukherjee, Diamond, and Pierson, in preparation). Stocks of DENV produced in Vero-furin were refractory to neutralization by this antibody, as evidenced by the significant difference in the size of the resistant population at the highest concentration of antibody tested (n=3, p = 0.005, Figure 3E).

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Discussion

An understanding of the structural states of flaviviruses is evolving rapidly (Plevka et al., 2011). While initial studies suggested that flaviviruses exist as either non-infectious immature (prM+) structures or mature (prM-) infectious virions, it has become clear that the populations of virions released from cells are both heterogeneous and dynamic (Dowd et al., 2011; Fibriansah et al., 2013; Kuhn et al., 2002; Nelson et al., 2008; Zhang et al., 2013b; Zhang et al., 2003; Zhang et al., 2007). This heterogeneity introduces challenges for studying flavivirus biology and immunology, especially at the level of analyzing neutralization by serum antibodies that are generated after natural infection or vaccination. In this report, we describe a Vero cell line that ectopically expresses human furin that supports robust replication of both laboratory strains and clinical DENV isolates. Biochemical, structural, and functional experiments established that prM is more efficiently cleaved on infectious virions released from Vero-furin cells. This ability to produce more homogeneous stocks of virions has utility in multiple areas of flavivirus research.

The neutralizing activity of mAbs and flavivirus-immune sera may be influenced by the maturation state of the virion (Mukherjee et al., 2014a; Nelson et al., 2008). This arises in part from differences in epitope accessibility on E proteins in different orientations and arrangements on the virion. In this context, standardizing assays involving multiple virus strains or preparations is complicated by the potential for non-uniform maturation among the viruses in a given study. Because neutralization assays are a critical component of vaccine evaluation and some diagnostic procedures, steps to increase the uniformity among viruses may be of considerable value (Halstead, 2013). Increased structural homogeneity of flaviviruses also is of considerable utility for structural studies. Homogeneous preparations are critical for obtaining near atomic resolution structures of flaviviruses using single particle reconstruction. Furthermore, homogenous ‘mature’ preparations may be useful for developing inactivated vaccines against DENV to offset the risk of ADE in vaccine recipients through induction of poorly neutralizing and potentially enhancing anti-prM antibodies.

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RESEARCH HIGHLIGHTS

  • Construction of a Vero cell line that expresses high levels of human furin
  • Over-expression of furin increases maturation efficiency of infectious flaviviruses

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Supplementary Material

1

Figure S1. Vero-furin cells express high levels of human furin. (A) Furin expression in parental Vero and human furin-expressing stable Vero-furin cell lines (top and bottom, respectively) as measured by flow cytometry. Cells were stained with a primary antibody specific for non-human primate furin (clone PA1-062), or an appropriate isotype control, followed by an AlexaFluor 647-conjugated anti-rabbit secondary antibody. Mean fluorescence intensity and the number of events (normalized to the maximum) are presented on the x- and y-axis, respectively. Data are representative of four independent staining experiments. (B) Furin expression in Vero and Vero-furin cells as detected by immunoblotting. Cell lysates of Vero and Vero-furin cells were separated using SDS-PAGE and probed using furin-specific antibody (clone PA1-062: 2 μg/ml). A mass of 20 ng recombinant furin was included on the gel as a positive control.

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2

Figure S2. Growth kinetics of a clinical DENV4 isolate in human furin-expressing Vero cells. The grown kinetics of a clinical DENV4 isolate (strain Nicaragua 703-99) on Vero and Vero-furin cells was determined. Vero and Vero-furin cells were infected at a multiplicity of infection (MOI) of 0.1. Viruses were collected from infected cells at indicated time-points post-infection and the infectious titer determined in duplicate by immunofocus assay on Vero cells. The growth curves presented are the average titer of three independent one-step growth curves. Error bars indicate the SEM.

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Acknowledgments

This work was supported in part by the intramural program of the National Institute of Allergy and Infectious Diseases (TCP) an extramural grant under award numbers R01AI073755 (MSD and RJK). We are grateful to Heather Hickman for her constructive comments on the manuscript. Finally, the authors are grateful to members of their laboratories for helpful discussions.

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Footnotes

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