Identification of conserved motifs in the Westnile virus envelope essential for particle secretion

Garg, Himanshu; Lee, Raphael Tze Chuen; Tek, Ng Oon; Maurer‐Stroh, Sebastian; Joshi, Anjali

doi:10.1186/1471-2180-13-197

Cited by 11 publications

(13 citation statements)

References 71 publications

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“…reporter VLPs by using WNV C-prM-E and the Rep/GFP construct as described previously (38). Interestingly, sera obtained from ZIKV VLP/DNA-immunized mice were able to cross-neutralize WNV RVP infection in a trend that was similar to that of ZIKV RVP inhibition, although with a lower efficacy (Fig.…”

Section: Figmentioning

confidence: 67%

“…To make and test ZIKV RVPs, we used the method described previously by Garg et al (38) and adapted from the work of Pierson et al (39). 293T cells were cotransfected with the ZIKV C-prM-E construct along with the WNV replicon reporter plasmid Rep/GFP (29,39,40), which provided the WNV accessory proteins and the GFP reporter ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In this study, a construct expressing the C-prM-E polyprotein of ZIKV was used to package a GFP reporter-containing WNV replicon to generate RVPs similar to those generated with WNV (38,39). WNV-based RVPs have been used by us and others to study WNV E biology (29,38,39).…”

Section: Discussionmentioning

confidence: 99%

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Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Garg

Sedano

Plata

et al. 2017

J Virol

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Recent worldwide outbreaks of Zika virus (ZIKV) infection and the lack of an approved vaccine raise serious concerns regarding preparedness to combat this emerging virus. We used a virus-like particle (VLP)-based approach to develop a vaccine and a microneutralization assay for ZIKV. A synthetic capsid-premembrane-envelope (CprM-E) gene construct of ZIKV was used to generate reporter virus particles (RVPs) that package a green fluorescent protein (GFP) reporter-expressing West Nile virus (WNV) replicon. The assay was adapted to a 96-well format, similar to the plaque reduction neutralization test (PRNT), and showed high reproducibility with specific detection of ZIKV neutralizing antibodies. Furthermore, C-prM-E and prM-E VLPs were tested as vaccine candidates in mice and compared to DNA vaccination. While the ZIKV prM-E construct alone was sufficient for generating VLPs, efficient VLP production from the C-prM-E construct could be achieved in the presence of the WNV NS2B-3 protease, which cleaves C from prM, allowing virus release. Immunization studies in mice showed that VLPs generated higher neutralizing antibody titers than those with the DNA vaccines, with C-prM-E VLPs giving slightly higher titers than those with prM-E VLPs. The superiority of C-prM-E VLPs suggests that inclusion of capsid may have benefits for ZIKV and other flaviviral VLP vaccines. To facilitate the VLP platform, we generated a stable cell line expressing high levels of ZIKV prM-E proteins that constitutively produce VLPs as well as a cell line expressing ZIKV C-prM-E proteins for RVP production. While several vaccine platforms have been proposed for ZIKV, this study describes a safe, effective, and economical VLP-based vaccine against ZIKV.IMPORTANCE To address the growing Zika virus epidemic, we undertook this study with two objectives: first, to develop a safe, effective, and economical vaccine for ZIKV, and second, to develop a rapid and versatile assay to detect the anti-ZIKV immune response. We generated a cell line stably expressing ZIKV prM-E that produces large amounts of VLPs in the supernatant and a ZIKV C-prM-E cell line that produces reporter virus particles upon transfection with a GFP replicon plasmid. The prM-E VLPs induced a strong neutralizing antibody response in mice that was better when the capsid was included. VLP-based vaccines showed significantly better neutralizing antibody responses than those with their DNA counterparts. The RVP-based microneutralization assay worked similarly to the PRNT assay, with a rapid GFP readout in a 96-well format. Our VLP-based platform provides a source for a ZIKV vaccine and diagnosis that can rapidly be adapted to current outbreaks.KEYWORDS C-prM-E, diagnostics, microneutralization, neutralization, PRNT, reporter, reporter virus particles, vaccine, Zika, prM-E S ince the identification of Zika virus (ZIKV) from a rhesus monkey in Uganda in 1947 (1, 2) until 2010, the virus predominantly circulated between Aedes mosquitoes and nonhuman primates. Periodic episodes were inde...

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Section: Figmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

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Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Garg

Sedano

Plata

et al. 2017

J Virol

Self Cite

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“…We conducted a Renilla-luciferasebased virus assembly and release assay (Fig. 1A) as described previously (19). The gene-silenced 293T cells were co-transfected with plasmids expressing structural proteins (C, prM, and E) and a subgenomic replicon that expressed the WNV nonstructural proteins and Renilla luciferase, resulted in the production of infectious VLPs in the culture fluid.…”

Section: Rab8b Involved In Wnv Particlementioning

confidence: 99%

“…There are several reports of WNV virus-like particles (VLPs), which are produced by complementation of replicon RNA with WNV structural genes expressed in trans (17), for the screening of siRNA or compound libraries (18,19). In this study, we performed siRNA-based screen silencing of Rab proteins related to vesicle transport from the ER to the plasma membrane to elucidate the mechanisms of WNV particle release.…”

mentioning

confidence: 99%

Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes

Kobayashi

Suzuki

Kawaguchi

et al. 2016

Journal of Biological Chemistry

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West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.

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Unique Requirement for ESCRT Factors in Flavivirus Particle Formation on the Endoplasmic Reticulum

et al. 2016

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Flavivirus infection induces endoplasmic reticulum (ER) membrane rearrangements to generate a compartment for replication of the viral genome and assembly of viral particles. Using quantitative mass spectrometry, we identified several ESCRT (endosomal sorting complex required for transport) proteins that are recruited to sites of virus replication on the ER. Systematic small interfering RNA (siRNA) screening revealed that release of both dengue virus and Japanese encephalitis virus was dramatically decreased by single depletion of TSG101 or co-depletion of specific combinations of ESCRT-III proteins, resulting in ≥1,000-fold titer reductions. By contrast, release was unaffected by depletion of some core ESCRTs, including VPS4. Reintroduction of ESCRT proteins to siRNA-depleted cells revealed interactions among ESCRT proteins that are crucial for flavivirus budding. Electron-microscopy studies revealed that the CHMP2 and CHMP4 proteins function directly in membrane deformation at the ER. Thus, a unique and specific subset of ESCRT contributes to ER membrane biogenesis during flavivirus infection.

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Identification of conserved motifs in the Westnile virus envelope essential for particle secretion

Cited by 11 publications

References 71 publications

Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes

Unique Requirement for ESCRT Factors in Flavivirus Particle Formation on the Endoplasmic Reticulum

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