An attenuated replication-competent chikungunya virus with a fluorescently tagged envelope

Jin, Jing; Sherman, Michael B; Chafets, Daniel M.; Dinglasan, Nuntana; Lü, Kai; Lee, Tzong Hae; Carlson, Lars A.; Muench, Marcus O.; Simmons, Graham

doi:10.1371/journal.pntd.0006693

Cited by 9 publications

(11 citation statements)

References 30 publications

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“…We have previously described CHIKV constructs with mCherry fluorescent protein inserted immediately downstream of the furin cleavage site between E3 and E2. This results in functional envelope with the reporter protein fused to the N-terminus of E2 protein [ 39 ]. However, there is some attenuation, and if mCherry is used in combination with an already attenuated vaccine strain like CHIKV 181/25, there is a lack of replication [ 39 ].…”

Section: Resultsmentioning

confidence: 99%

“…This results in functional envelope with the reporter protein fused to the N-terminus of E2 protein [ 39 ]. However, there is some attenuation, and if mCherry is used in combination with an already attenuated vaccine strain like CHIKV 181/25, there is a lack of replication [ 39 ]. Therefore, to make a BSL-2 safe reporter virus based on CHIKV 181/25, we utilized the smaller reporter protein, nano-luciferase (nLuc).…”

Section: Resultsmentioning

confidence: 99%

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A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release

Ramjag

Cutrone

Lü

et al. 2022

Virol J

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Background Several studies have demonstrated neutralizing antibodies to be highly effective against alphavirus infection in animal models, both prophylactically and remedially. In most studies, neutralizing antibodies have been evaluated for their ability to block viral entry in vitro but recent evidence suggests that antibody inhibition through other mechanisms, including viral budding/release, significantly contributes to viral control in vivo for a number of alphaviruses. Results We describe a BSL-2, cell-based, high-throughput screening system that specifically screens for inhibitors of alphavirus egress using chikungunya virus (CHIKV) and Mayaro virus (MAYV) novel replication competent nano-luciferase (nLuc) reporter viruses. Screening of both polyclonal sera and memory B-cell clones from CHIKV immune individuals using the optimized assay detected several antibodies that display potent anti-budding activity. Conclusions We describe an “anti-budding assay” to specifically screen for inhibitors of viral egress using novel CHIKV and MAYV nLuc reporter viruses. This BSL-2 safe, high-throughput system can be utilized to explore neutralizing “anti-budding” antibodies to yield potent candidates for CHIKV and MAYV therapeutics and prophylaxis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release

Ramjag

Cutrone

Lü

et al. 2022

Virol J

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“…The chikungunya virus strains; East Central South African (ECSA) LR2006-OPY1 strain (3′GFP-CHIKV) [ 50 ] and West African (WA) 37997 strain (5′GFP-CHIKV) [ 51 ], both encoding green fluorescent protein (GFP) gene, and Asian (181/25) vaccine strain [ 52 ] encoding either mCherry-fluorescent protein (mc-CHIKV) or nano-luciferase gene (nLuc-CHIKV) fused to the N-terminus of E2 glycoprotein ( Supplementary Figure S1 ). The mCherry or nano-luciferase proteins are expressed on the viral envelope as previously described [ 53 ]. The plasmids used for the production of CHIKV were kindly provided by Graham Simmons, San Francisco, CA, USA.…”

Section: Methodsmentioning

confidence: 99%

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Kirui

Abidine

Lenman

et al. 2021

Cells

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Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

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“…Semliki Forest virus (SFV) as well as the engineered Chikungunya virus (CHIKV) encoding GFP or Nano-luciferase have been described previously (27)(28)(29)(30)(31). UUKV S23, TOSV ISS, and SFV were produced and titrated in BHK-21 cells (12).…”

Section: Virusesmentioning

confidence: 99%

Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses

Uckeley

Moeller

Kühn

et al. 2019

Molecular & Cellular Proteomics

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Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immuno-precipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin Aresistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae. In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both occurs in the nucleus. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.

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An attenuated replication-competent chikungunya virus with a fluorescently tagged envelope

Cited by 9 publications

References 30 publications

A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release

A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses

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