Dengue Reporter Virus Particles for Measuring Neutralizing Antibodies against Each of the Four Dengue Serotypes

Mattia, Kimberly; Puffer, Bridget A.; Williams, Katherine L.; Gonzalez, Ritela; Murray, Meredith; Sluzas, Emily; Pagano, Daniel J.; Ajith, Sandya; Bower, Megan; Berdougo, Eli; Harris, Eva; Doranz, Benjamin J.

doi:10.1371/journal.pone.0027252

Cited by 70 publications

(85 citation statements)

References 33 publications

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“…This genetic complementation results in the production of pseudoinfectious virus-like particles capable of only a single round of infection, referred to herein as reporter virus particles (RVPs). RVPs have been used extensively to study flavivirus biology (43)(44)(45)(46)(47)(48) and virus-antibody interactions (35,36,(49)(50)(51) and as tools to screen antiviral compounds (19,52,53). The mechanism of flavivirus RNA packaging is not well understood and appears to be relatively nonselective.…”

Section: Resultsmentioning

confidence: 99%

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

VanBlargan

Davis

Dowd

et al. 2015

J Virol

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The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1= position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1= position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1= position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. West Nile virus (WNV) and the four serotypes of dengue virus (DENV1 to -4) are mosquito-borne viruses of the Flavivirus genus that significantly impact public health (1, 2). Despite a clear need, neither vaccines nor therapeutics for WNV or DENV have been licensed for use in humans. The flavivirus genome is an ϳ11-kb, single-stranded, positive-sense RNA that encodes a single open reading frame flanked by 5= and 3= untranslated regions. The viral genome is translated on endoplasmic reticulum (ER)-derived membranes into a single polyprotein that undergoes co-and posttranslational cleavage by the viral protease NS2B/3 and host proteases into 10 functionally distinct proteins, including the structural proteins capsid (C), premembrane (prM), and envelope (E) that form the virus particle. During assembly, membrane-anchored prM and E glycoproteins are incorporated into virions as they bud into the ER lumen. The C protein associates with the viral genome in the cytoplasm to form an unstructured nucleocapsid that is incorporated into the budding particle via unknown mechanisms (3). The carboxy terminus (C terminus) of the C protein includes a signal sequence, flanked by protease cleavage sites, that directs the translocation of prM into the ER lumen and tethers C to the cytosolic face of the ER membrane.Cleavage at both sites is essential for virion morphogenesis and occurs in a sequenti...

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Section: Resultsmentioning

confidence: 99%

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

VanBlargan

Davis

Dowd

et al. 2015

J Virol

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“…1B). Finally, infection of target cells was measured by luciferase expression by replication-incompetent dengue reporter viruses (26) (Fig. 1C).…”

Section: Resultsmentioning

confidence: 99%

“…The BHK-DRRZ cell line, which stably propagates a DENV replicon, was produced by transduction of BHK21 cells with DENV reporter virus particles (RVPs) that encapsidate a replicon encoding both Renilla luciferase and the ShBle gene (conferring resistance to zeocin). The DENV replicon was described previously (35), and DENV RVPs have been extensively characterized for antigenic equivalence to live virus (26,35). Following infection, selection of infected cells was conferred by supplementing complete DMEM with 300 μg/mL zeocin.…”

Section: Methodsmentioning

confidence: 99%

“…RVPs produced from the mutation array were used for infectivity assays, as described previously (26), except that a Renilla luciferase reporter replaced GFP. Twenty microliters of frozen RVPs were used to infect BHK DC-SIGN cells at a density of 10,000 cells per well in DMEM with Hepes added to 10 mM, pH 8.0, in a 384-well microplate format.…”

Section: Methodsmentioning

confidence: 99%

“…Each clone in the DENV-3 mutation library was tested for (A) DENV E protein expression levels by detecting cellularly expressed prM/E protein with a mixture of diverse E protein mAbs, (B) viral particle budding levels by capturing and then detecting DENV virions released by producer cells, and (C) infectivity levels by detecting luciferase expression levels in target cells infected with DENV RVPs made with each mutated prM/E protein. RVPs are antigenically identical to live virus and have been used in numerous studies of DENV function (26,35,36). RVPs carry a reporter gene in place of structural genes and so are capable of only a single round of infection.…”

Section: Network Of Residues Proximal To the Fusion Loop Determines Itsmentioning

confidence: 99%

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Atomic-level functional model of dengue virus Envelope protein infectivity

Christian

Kahle

Mattia

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding. The locations and atomic interactions of these critical residues within different structures representing distinct fusogenic conformations help to explain how E protein (i) regulates fusion-loop exposure by shielding, tethering, and triggering its release; (ii) enables hinge movements between E domain interfaces during triggered structural transformations; and (iii) drives membrane fusion through latestage zipper contacts with stem. These results provide structural targets for drug and vaccine development and integrate the findings from structural studies and isolated mutagenesis efforts into a cohesive model that explains how specific residues in this class II viral fusion protein enable virus infectivity. D engue virus (DENV) infection of target cells proceeds through a membrane-fusion step, catalyzed by a viral fusion protein. The DENV genome, like that of other flaviviruses, encodes a single polyprotein that is processed into three structural proteins-capsid (C), precursor membrane (prM), and envelope (E)-and seven nonstructural proteins that are required for viral replication. The capsid protein and the viral RNA genome form a nucleocapsid that buds at the endoplasmic reticulum (ER) in association with 180 copies each of prM and E, as well as host-derived lipids, to form the immature virion (1). This noninfectious virion passes through the cell's secretory pathway, maturing in the Golgi where most of the prM proteins are cleaved into pr and M proteins by the host protease furin (2). The mature virions bud from the cell via exocytosis and are then available to infect new cells.DENV infects a cell by binding of E protein to one or more receptors on the cell surface, followed by clathrin-mediated endocytosis and transport to endosomes (3). The three distinct ectodomains (DI, DII, and DIII) of E protein lie over a helical stem region tethered to the virus membrane by a transmembrane anchor. In the acidic endosome, protonation of E protein histidine residues is thought to mediate conformational changes that trigger the dissociation of the E dimer into monomers and subsequent rearrangements among DI, DII, and DIII (4, 5). These conformational changes enable formation of the fusogenic E trimer and insertion of E protein into the endosomal membrane by means of a fusion loop (6, 7). Fusion between the viral and endosomal membranes is then driven by a portion of the E stem region lifting off t...

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A reporter virus particle seroneutralization assay for tick‐borne encephalitis virus overcomes ELISA limitations

Ackermann‐Gäumann,

Dentand,

Lienhard

et al. 2024

Journal of Medical Virology

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Tick‐borne encephalitis (TBE) virus is the most prevalent tick‐transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA‐based detection of specific antibodies in the patient serum. However, cross‐reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)‐based SNT in which the infectivity is measured by luminescence and that can be performed under BSL‐2 conditions. The RVP‐based SNT for TBEV exhibited a highly significant correlation with the traditional virus‐based SNT (R2 = 0.8637, p < 0.0001). The RVP‐based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%–97.4%) and specificity of 100% (95% CI: 81.6%–100%). We also tested the cross‐reactivity of serum samples in RVP‐based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV‐positive by ELISA but negative by RVP‐based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP‐based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies.

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Dengue Reporter Virus Particles for Measuring Neutralizing Antibodies against Each of the Four Dengue Serotypes

Cited by 70 publications

References 33 publications

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

Atomic-level functional model of dengue virus Envelope protein infectivity

A reporter virus particle seroneutralization assay for tick‐borne encephalitis virus overcomes ELISA limitations

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