pH Dependence of Zika Membrane Fusion Kinetics Reveals an Off-Pathway State

Rawle, Robert J.; Webster, Elizabeth R.; Jelen, Marta; Kasson, Peter M.; Boxer, Steven G.

doi:10.1021/acscentsci.8b00494

Cited by 50 publications

(80 citation statements)

References 52 publications

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“…These two viral proteins were seen together for the last time in Rab7-positive structures. These results are also consistent with the data obtained with a novel a novel surrogate-receptor approach described by Rawle et al [59]. Later the ZIKV envelope appeared to be transported back to the cell surface via slow recycling endosomes.…”

Section: Discussionsupporting

confidence: 92%

Zika virus: mapping and reprogramming the entry

Owczarek

Chykunova

Jassoy³

et al. 2019

Cell Commun Signal

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Background The flaviviridae family comprises single-stranded RNA viruses that enter cells via clathrin-mediated pH-dependent endocytosis. Although the initial events of the virus entry have been already identified, data regarding intracellular virus trafficking and delivery to the replication site are limited. The purpose of this study was to map the transport route of Zika virus and to identify the fusion site within the endosomal compartment. Methods Tracking of viral particles in the cell was carried out with confocal microscopy. Immunostaining of two structural proteins of Zika virus enabled precise mapping of the route of the ribonucleocapsid and the envelope and, consequently, mapping the fusion site in the endosomal compartment. The results were verified using RNAi silencing and chemical inhibitors. Results After endocytic internalization, Zika virus is trafficked through the endosomal compartment to fuse in late endosomes. Inhibition of endosome acidification using bafilomycin A1 hampers the infection, as the fusion is inhibited; instead, the virus is transported to late compartments where it undergoes proteolytic degradation. The degradation products are ejected from the cell via slow recycling vesicles. Surprisingly, NH 4 Cl, which is also believed to block endosome acidification, shows a very different mode of action. In the presence of this basic compound, the endocytic hub is reprogrammed. Zika virus-containing vesicles never reach the late stage, but are rapidly trafficked to the plasma membrane via a fast recycling pathway after the clathrin-mediated endocytosis. Further, we also noted that, similarly as other members of the flaviviridae family, Zika virus undergoes furin- or furin-like-dependent activation during late steps of infection, while serine or cysteine proteases are not required for Zika virus maturation or entry. Conclusions Zika virus fusion occurs in late endosomes and is pH-dependent. These results broaden our understanding of Zika virus intracellular trafficking and may in future allow for development of novel treatment strategies. Further, we identified a novel mode of action for agents commonly used in studies of virus entry. Schematic representation of differences in ZIKV trafficking in the presence of Baf A1 and NH 4 Cl Electronic supplementary material The online version of this article (10.1186/s12964-019-0349-z) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 92%

Zika virus: mapping and reprogramming the entry

Owczarek

Chykunova

Jassoy³

et al. 2019

Cell Commun Signal

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“…A chemical-kinetics model formalism previously developed for Zika membrane fusion (14) was modified to incorporate three sequential steps from membrane-bound virus to hemifusion in accordance with the single-virus kinetic model used by Floyd and colleagues (11). The resulting formalism had the following kinetic parameters: B <-> I B <-> A1 -> A2 -> HF In the best-fit models, the rate constant for A1->B was zero.…”

Section: Kinetic Modelingmentioning

confidence: 99%

“…Dyes conjugated to two-tailed lipids are more difficult to load into viral particles at self-quenching concentration, but several, including Texas Red-DHPE, have been used as probes for membrane fusion in different viral systems (3,(22)(23)(24)(25). We have anecdotally found Texas Red more robust to photoinhibition effects (14,15), so we tested it more systematically. At the lowest concentrations tested, Texas Red was more robust to photoinhibition effects than R18, although photoinhibition was evident at higher concentrations and illumination intensities.…”

Section: Figure 2: Photoinhibition Of Membrane Fusion By Influenza VImentioning

confidence: 99%

“…We present a systematic study that compares the effects on influenza virus fusion using two fluorescent probes, R18 and Texas Red, widely used to monitor lipid mixing between a labeled viral particle and a target membrane. Although many other fluorescencedequenching probes exist, these two were chosen as ones that can readily be introduced into viral membranes at self-quenching concentrations and have previously been reported in singlevirus fusion measurements of multiple enveloped viruses (11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

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Detecting and controlling dye effects in single-virus fusion experiments

Rawle

Giraldo

Boxer

et al. 2019

Preprint

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Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye-and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, while Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions. Statement of SignificanceFluorescent dyes are powerful tools for labeling membranes and tracking subcellular objects, and fluorescence dequenching has further been used as a sensitive assay for membrane fusion. Here we show how incorporation of membrane dyes can perturb membrane fusion by influenza virus in a light-dependent manner. We provide a strategy to mitigate this by minimizing dye and light exposure. Finally, we show how in some cases these effects can be due to covalent reaction of some dyes with viral proteins upon illumination. These phenomena may be general and should be carefully controlled for in experiments using such labels.

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“…During ZIKV infection, AXL mediates ZIKV entry indirectly whereby the phosphatidylserine extension on ZIKV lipid membrane binds to Growth arrest-specific 6 (Gas6), one of the ligands for AXL that serves as a bridge for ZIKV and AXL interaction, resulting in clathrin-mediated virus internalization [14,15]. The acidic microenvironment within the endosome promotes fusion between the virus envelope proteins and the endosomal membrane resulting in the release of ZIKV genome into the host cell cytosolic space [15,16].…”

Section: Introductionmentioning

confidence: 99%

Endoplasmic reticulum: a focal point of Zika virus infection

et al. 2020

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Zika virus (ZIKV) belongs to the Flavivirus genus of the Flaviviridae family. It is an arbovirus that can cause congenital abnormalities and is sexually transmissible. A series of outbreaks accompanied by unexpected severe clinical complications have captured medical attention to further characterize the clinical features of congenital ZIKV syndrome and its underlying pathophysiological mechanisms. Endoplasmic reticulum (ER) and ER-related proteins are essential in ZIKV genome replication. This review highlights the subcellular localization of ZIKV to the ER and ZIKV modulation on the architecture of the ER. This review also discusses ZIKV interaction with ER proteins such as signal peptidase complex subunit 1 (SPCS1), ER membrane complex (EMC) subunits, and ER translocon for viral replication. Furthermore, the review covers several important resulting effects of ZIKV infection to the ER and cellular processes including ER stress, reticulophagy, and paraptosis-like death. Pharmacological targeting of ZIKVaffected ER-resident proteins and ER-associated components demonstrate promising signs of combating ZIKV infection and rescuing host organisms from severe neurologic sequelae.

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pH Dependence of Zika Membrane Fusion Kinetics Reveals an Off-Pathway State

Cited by 50 publications

References 52 publications

Zika virus: mapping and reprogramming the entry

Zika virus: mapping and reprogramming the entry

Detecting and controlling dye effects in single-virus fusion experiments

Endoplasmic reticulum: a focal point of Zika virus infection

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