Near-Atomic Resolution Cryo-Electron Microscopic Structure of Dengue Serotype 4 Virus

Kostyuchenko, V.A.; Chew, Pau Ling; Ng, Thiam-Seng; Lok, Shee-Mei

doi:10.1128/jvi.02641-13

Cited by 86 publications

(75 citation statements)

References 34 publications

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“…ible conformational changes that have the potential to increase epitope accessibility. Of note, DENV serotypes 1 and 4 were not observed to adopt a "bumpy" conformation at 37°C, indicating variability among strains (54). Furthermore, although cryo-EM constructions of immature DENV incubated at room temperature and 37°C are similar (53), our studies demonstrate that preparations of virions that retain significant levels of uncleaved prM also sample an ensemble of conformations with the potential to alter neutralization sensitivity.…”

Section: Fig 5 Neutralization Potency Against Wnv-prmmentioning

confidence: 83%

“…The use of neutralization assays that report the biology of only infectious virions provides a functional probe for the way in which the antigenic surface of infectious virions changes with time. These approaches should be of significant utility in studies designed to probe the viral determinants that govern the dynamics of the virus particle, which appear to vary among virus types (28,54) and even viral strains (22). One intriguing hypothesis is that the contribution of interactions that form the mature virion (e.g., the dimer interface) may differ among viruses (55) or may be impacted by variation within a virus population.…”

Section: Fig 5 Neutralization Potency Against Wnv-prmmentioning

confidence: 99%

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Combined Effects of the Structural Heterogeneity and Dynamics of Flaviviruses on Antibody Recognition

Dowd

Mukherjee

Kühn

et al. 2014

J Virol

101

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Flaviviruses are thought to sample an ensemble of structures at equilibrium. One consequence of a structurally dynamic virion is the observed time-dependent increases in neutralization sensitivity that can occur after prolonged incubation with antibody. Differences in how virus strains "breathe" may affect epitope exposure and contribute to the underlying mechanisms of straindependent neutralization sensitivity. Beyond the contribution of structural dynamics, flaviviruses exist as a structurally heterogeneous population due to an inefficient virion maturation process. Here, we investigate the interplay between virion maturation and structural dynamics that contributes to antibody-mediated neutralization. Using West Nile (WNV) and dengue (DENV) viruses produced under conditions that modify the extent of virion maturation, we investigated time-dependent changes in neutralization sensitivity associated with structural dynamics. Our results identify distinct patterns of neutralization against viruses that vary markedly with respect to the extent of virion maturation. Reducing the efficiency of virion maturation resulted in greater time-dependent changes in neutralization potency and a marked reduction in the stability of the particle at 37°C compared to more mature virus. The fact that the neutralization sensitivity of WNV and DENV did not increase after prolonged incubation in the absence of antibody, regardless of virion maturation, suggests that the dynamic processes that govern epitope accessibility on infectious viruses are reversible. Against the backdrop of heterogeneous flavivirus structures, differences in the pathways by which viruses "breathe" represent an additional layer of complexity in understanding maturation state-dependent patterns of antibody recognition. IMPORTANCEFlaviviruses exist as a group of related structures at equilibrium that arise from the dynamic motion of E proteins that comprise the antigenic surface of the mature virion. This process has been characterized for numerous viruses and is referred to as viral "breathing." Additionally, flaviviruses are structurally heterogeneous due to an inefficient maturation process responsible for cleaving prM on the virion surface. Both of these mechanisms vary the exposure of antigenic sites available for antibody binding and impact the ability of antibodies to neutralize infection. We demonstrate that virions with inefficient prM cleavage "breathe" differently than their more mature counterparts, resulting in distinct patterns of neutralization sensitivity. Additionally, the maturation state was found to impact virus stability in solution. Our findings provide insight into the complex flavivirus structures that contribute to infection with the potential to impact antibody recognition. F laviviruses are small, enveloped, single-stranded RNA viruses that cause significant morbidity and mortality worldwide. West Nile (WNV) and dengue (DENV) viruses are members of this genus that are transmitted to humans through the bite of an infected mosqui...

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Section: Fig 5 Neutralization Potency Against Wnv-prmmentioning

confidence: 83%

Section: Fig 5 Neutralization Potency Against Wnv-prmmentioning

confidence: 99%

Combined Effects of the Structural Heterogeneity and Dynamics of Flaviviruses on Antibody Recognition

Dowd

Mukherjee

Kühn

et al. 2014

J Virol

101

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“…Kostyuchenko and colleagues recently solved a near-atomic-resolution structure of DENV4 (22). DENV4 surface E molecules are tightly packed compared to those of other serotypes, which may explain the more rigid and stable nature of DENV4.…”

Section: Importancementioning

confidence: 99%

Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination

Nivarthi

Kose

Sapparapu

et al. 2017

J Virol

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The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination.IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The

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“…particle even at 40 °C, although considerable aggregation of particles occurs 3 . ZIKV seemed to be even more stable than DENV4, as the smooth surfaced particles did not aggregate at 40 °C (Fig.…”

mentioning

confidence: 97%

“…2 Centre for BioImaging Sciences, Department of Biological Sciences, National University of Singapore, Singapore 117557, Singapore. 3 CryoEM unit, Department of Biological Sciences, National University of Singapore, Singapore 117557, Singapore. *These authors contributed equally to this work.…”

mentioning

confidence: 99%

Structure of the thermally stable Zika virus

Kostyuchenko

Lim

Zhang

et al. 2016

Nature

Self Cite

448

469

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Zika virus (ZIKV), formerly a neglected pathogen, has recently been associated with microcephaly in fetuses 1 , and with Guillian-Barré syndrome in adults 2 . Here we present the 3.7 Å resolution cryoelectron microscopy structure of ZIKV, and show that the overall architecture of the virus is similar to that of other flaviviruses. Sequence and structural comparisons of the ZIKV envelope (E) protein with other flaviviruses show that parts of the E protein closely resemble the neurovirulent West Nile and Japanese encephalitis viruses, while others are similar to dengue virus (DENV). However, the contribution of the E protein to flavivirus pathobiology is currently not understood. The virus particle was observed to be structurally stable even when incubated at 40 °C, in sharp contrast to the less thermally stable DENV 3 . This is also reflected in the infectivity of ZIKV compared to DENV serotypes 2 and 4 (DENV2 and DENV4) at different temperatures. The cryoelectron microscopy structure shows a virus with a more compact surface. This structural stability of the virus may help it to survive in the harsh conditions of semen 4 , saliva 5 and urine 6 . Antibodies or drugs that destabilize the structure may help to reduce the disease outcome or limit the spread of the virus.Zika virus (ZIKV), a flavivirus, is thought to be principally transmitted to humans by the mosquito (Aedes aegypti) vector. Other flaviviriuses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DENV) and yellow fever virus (YFV). ZIKV generally causes a mild disease. However, when pregnant women are infected with ZIKV, there is an increased risk of developing microcephaly in the fetus 1 .Retrospective analysis of data collected from a ZIKV outbreak in French Polynesia in 2013-2014 showed association of the virus with microcephaly 7 . Here we present the 3.7 Å resolution structure of ZIKV strain H/PF/2013 isolated during that outbreak 8 .For cryo-electron microscopy (cryoEM) studies, ZIKV was grown in the mosquito cell line at 28 °C and purified at 4 °C by polyethylene glycol precipitation, a sucrose cushion, followed by a potassium tartrate gradient. The gel analysis of the purified sample suggested it contained mostly mature virus (Extended Data Fig. 1). The ZIKV samples were incubated at 28 °C, 37 °C and 40 °C (mimicking high fever) for 30 min, before imaging by cryoEM (Fig. 1a). At 28 °C, there were broken and shrivelled particles together with some smooth surfaced particles (about 500 Å in diameter), similar to the compact DENV mature particles. Conversely, samples incubated at 37 °C and 40 °C showed many more smooth surfaced particles. The presence of a larger fraction of shrivelled particles at 28 °C could be due to the exposure of particles to high osmolality during purification. We speculate that ZIKV particles may expand into smooth surfaced particles when incubated at higher temperatures, making the lipid envelope more fluid, and allowing the structure to revert to its normal state. Some strains of DENV2 (New G...

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Near-Atomic Resolution Cryo-Electron Microscopic Structure of Dengue Serotype 4 Virus

Cited by 86 publications

References 34 publications

Combined Effects of the Structural Heterogeneity and Dynamics of Flaviviruses on Antibody Recognition

Combined Effects of the Structural Heterogeneity and Dynamics of Flaviviruses on Antibody Recognition

Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination

Structure of the thermally stable Zika virus

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