New chemical method of viral inactivation for vaccine development based on membrane fusion inhibition

Stauffer, Fausto; Miranda, Joari De; Schechter, Marcos C.; Queiroz, Fernando Ávila; Santos, Nathalia O.; Alves, Ada M. B.; Poian, Andrea T. Da

doi:10.1016/j.vaccine.2007.09.025

Cited by 7 publications

(7 citation statements)

References 22 publications

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“…In flaviviruses, a conserved cluster of histidine side chains acts as a “pH sensor”, which triggers the fusogenic conformational change in response to the reduced pH of the endosome [16], [17]. The histidine modifying agent diethylpyrocarbonate (DEPC) inactivates VSV [46] and dengue virus [22] by inhibiting the fusogenic conformational change. We found that DEPC also blocked fusion of R18-labeled YFV with liposomes at pH 5.5 (Figure 6B).…”

Section: Resultsmentioning

confidence: 99%

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

Nour

Wolenski

et al. 2013

PLoS Pathog

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Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion.

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Section: Resultsmentioning

confidence: 99%

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

Nour

Wolenski

et al. 2013

PLoS Pathog

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“…DEPC treatment of viral particles is known to covalently modify histidines on viral glycoproteins, thereby abolishing viral fusion without changing the protein structure (42,43). DiD-labeled CHIKV was treated with 2 mM DEPC for 30 min at room temperature.…”

Section: Characteristics Of Did-labeled Chikungunya Virusmentioning

confidence: 99%

Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells

Hoornweg

Duijl-Richter

Nuñez

et al. 2016

J Virol

121

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Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in Africa, Asia, and the Americas. The cell entry pathway hijacked by CHIKV to infect a cell has been studied previously using inhibitory compounds. There has been some debate on the mechanism by which CHIKV enters the cell: several studies suggest that CHIKV enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. Here we applied live-cell microscopy and monitored the cell entry behavior of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain detailed insight into the dynamic events that occur during CHIKV entry. We observed that almost all particles fused within 20 min after addition to the cells. Of the particles that fused, the vast majority first colocalized with clathrin. The average time from initial colocalization with clathrin to the moment of membrane fusion was 1.7 min, highlighting the rapidity of the cell entry process of CHIKV. Furthermore, these results show that the virus spends a relatively long time searching for a receptor. Membrane fusion was observed predominantly from within Rab5-positive endosomes and often occurred within 40 s after delivery to endosomes. Furthermore, we confirmed that a valine at position 226 of the E1 protein enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV enters cells via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes. IMPORTANCESince its reemergence in 2004, chikungunya virus (CHIKV) has spread rapidly around the world, leading to millions of infections. CHIKV often causes chikungunya fever, a self-limiting febrile illness with severe arthralgia. Currently, no vaccine or specific antiviral treatment against CHIKV is available. A potential antiviral strategy is to interfere with the cell entry process of the virus. However, conflicting results with regard to the cell entry pathway used by CHIKV have been published. Here we applied a novel technology to visualize the entry behavior of single CHIKV particles in living cells. Our results show that CHIKV cell entry is extremely rapid and occurs via clathrin-mediated endocytosis. Membrane fusion from within acidic early endosomes is observed. Furthermore, the membrane fusion capacity of CHIKV is strongly promoted by cholesterol in the target membrane. Taking these findings together, this study provides detailed insight into the cell entry process of CHIKV.

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“…Indeed, VSV treatment with DEPC completely inactivated the virus, and, more importantly, the inactivation procedures did not affect the conformational integrity of its surface proteins [64]. Additionally, pathogenicity and viral replication in an animal model were abolished by viral treatment with DEPC and the antibodies elicited in mice after intraperitoneal immunization with the inactivated virus mixed with adjuvants were able to recognize and neutralize the native virus and efficiently protected animals against the challenge with lethal doses of VSV [65].…”

Section: Vaccine Developmentmentioning

confidence: 99%

“…The results obtained with the use of DEPC for VSV inactivation opened new possibilities for the development of safe vaccines by multiples reasons: (a) the inactivation by this compound is stable, since it covalently modifies His residues in viral proteins; (b) although DEPC is a cytotoxic agent, it is important to point out that free DEPC is very unstable in aqueous solution, being rapidly hydrolyzed, what assures that no free DEPC would be present in the inactivated virus preparation; (c) DEPC treatment did not altered virus structure (in marked contrast to virus inactivated by heat, DEPC treated virus was recognized by antibodies against the unmodified VSV with the same avidity); and (d) the antibodies elicited upon immunization with the inactivated virus recognized and neutralized the native virus and protected animals against a challenge [64,65].…”

Section: Vaccine Developmentmentioning

confidence: 99%

Viral Inactivation Based on Inhibition of Membrane Fusion: Understanding the Role of Histidine Protonation to Develop New Viral Vaccines

Poian¹,

Carneiro

Stauffer

2009

PPL

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Membrane fusion is an essential step in the entry of enveloped viruses into their host cells, what makes it a potentially attractive target for viral inactivation approaches. Fusion is mediated by viral surface glycoproteins that undergo conformational changes triggered by interaction with specific cellular receptors or by the exposition to low pH of endossomal medium. Here we review how several studies on the structural rearrangements of vesicular stomatitis virus (VSV) glycoprotein G during cellular recognition and fusion led us to propose a crucial role of the protonation of His residues for G protein activity. Moreover, we demonstrated that using diethylpyrocarbonate (DEPC), a histidine-modifying compound, it was possible to abolish viral infectivity and pathogenicity in mice, and to elicit neutralizing antibodies that confer protection in these animals against challenge using lethal doses of the virus. The presence of conserved His residues in a wide range of viral fusion proteins and the use of DEPC as a more general means for vaccine development will be also discussed.

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New chemical method of viral inactivation for vaccine development based on membrane fusion inhibition

Cited by 7 publications

References 22 publications

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells

Viral Inactivation Based on Inhibition of Membrane Fusion: Understanding the Role of Histidine Protonation to Develop New Viral Vaccines

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