Three dimensional morphology of rabies virus studied by cryo-electron tomography

Guichard, Paul; Krell, Tino; Chevalier, Michel; Vaysse, Carole; Adam, Olivier; Ronzon, Frédéric; Marco, Sergio

doi:10.1016/j.jsb.2011.07.003

Cited by 28 publications

(17 citation statements)

References 56 publications

(63 reference statements)

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“…These dimensions are typical for RABV and also match those of the parental VSV strain rVSV eGFP ( Fig. 1B and C) (27,28). Purified rVSV RABV G virions incorporate RABV G to an extent comparable to VSV G incorporation into particles (Fig.…”

Section: Resultssupporting

confidence: 64%

Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

Piccinotti

Kirchhausen

Whelan

2013

J Virol

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dRabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin. R abies virus (RABV) is the prototypical member of the zoonotic lyssavirus genus responsible for fatal encephalitis in animals and humans. A single-stranded negative-sense RNA virus, RABV encases its RNA genome in a bullet-shaped, enveloped particle that incorporates a single surface glycoprotein (G). RABV G mediates all internalization steps from cell binding to membrane fusion. In addition, G is a major determinant of RABV neurotropism (1). Conjugation or pseudotyping with the ectodomain of pathogenic RABV G or peptides derived from receptor-binding regions allows retargeting of biologically active molecules to the central nervous system (CNS) for drug delivery or as neurotracers (2, 3). Since a large part of RABV pathogenesis is reliant on the virus garnering access to neurons and the CNS, G is also a determining factor in RABV virulence. The pathogenicity of attenuated strains can be effectively increased by replacing the glycoprotein with one from a neurotropic, virulent strain (4).Like other rhabdoviruses, RABV gains access to the cellular interior by endocytosis and subsequent low pH-dependent fusion (5-7). Electron micrographs of viral particles in vesicles with electron-dense coats suggest that clathrin-coated pits mediate the uptake of RABV in both neuronal and nonneuronal cells (8, 9). However, static images cannot inform on the fate of such particles or the relevance of these interactions for subsequent infection. High-resolution live-imaging techniques permit tracking of viral uptake into coated pits (10-15). Fluorescence tagging of coatedpit components and quantitative analysis methods have revealed differences for the pits engaging fluoresc...

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Section: Resultssupporting

confidence: 64%

Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

Piccinotti

Kirchhausen

Whelan

2013

J Virol

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“…The virions as observed in electron microscopy or cryo-electron-tomography are characterized by the eponymous rod-or bullet-shaped morphology with one flat and one conical end, a diameter of about 75 nm, and a length of in average 180 nm ( Fig. 1) (Ge et al, 2010;Guichard et al, 2011;Matsumoto, 1962). In the virion, the N-RNA complex comprising 12 kb RNA and approximately 1300 N protomers is present in a highly condensed superhelical form and associated with approximately 650 copies of P protein dimers, which bind between two adjacent N molecules, and 50 molecules of the large RNA polymerase subunit L. The superhelix is surrounded by an envelope comprising a continuous inner M protein mesh, a lipid membrane, and trimers of the transmembrane glycoprotein G. The overall shape of the virion is imposed by the M protein layer, which determines the number of N subunits per turn, the diameter of the trunk, and the pitch of the RNP superhelix (Green et al, 2006).…”

Section: Rabies Virionsmentioning

confidence: 99%

G gene-deficient single-round rabies viruses for neuronal circuit analysis

Ghanem

Conzelmann

2016

Virus Research

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“…14 Both wild-type rabies and VSV are, in general, bullet shaped, 15,16 but, interestingly, for the rabies virus, pleomorphic forms have been reported. 17 Our ultrastructural investigation also highlights an issue with traditional recombinant lentiviral purification techniques, in that ultracentrifugation results in a high titer but impure preparation containing a mixture of complete particles, empty membranes devoid of capsids and non-enveloped capsids (Figure 2b, c and e), as well as packaging cell debris. The procedures used for viral particle concentration are common to most major lentiviral facilities, for example, 18 however, further purification methods such as size-exclusion chromatography to minimize cell debris Transduction around the injection site in the lumbar spinal cord was predominantly neuronal.…”

Section: Discussionmentioning

confidence: 78%

Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord

et al. 2015

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Lentiviral vectors have proved an effective method to deliver transgenes into the brain; however, they are often hampered by a lack of spread from the site of injection. Modifying the viral envelope with a portion of a rabies envelope glycoprotein can enhance spread in the brain by using long-range axon projections to facilitate retrograde transport. In this study, we generated two chimeric envelopes containing the extra-virion and transmembrane domain of rabies SADB19 or CVS-N2c with the intra-virion domain of vesicular stomatitis virus. Viral particles were packaged containing a green fluorescent protein reporter construct under the control of the phosphoglycerokinase promoter. Both vectors produced high-titer particles with successful integration of the glycoproteins into the particle envelope and significant transduction of neurons in vitro. Injection of the SADB19 chimeric viral vector into the lumbar spinal cord of adult mice mediated a strong preference for gene transfer to local neurons and axonal terminals, with retrograde transport to neurons in the brainstem, hypothalamus and cerebral cortex. Development of this vector provides a useful means to reliably target select populations of neurons by retrograde targeting.

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Three dimensional morphology of rabies virus studied by cryo-electron tomography

Cited by 28 publications

References 56 publications

Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

G gene-deficient single-round rabies viruses for neuronal circuit analysis

Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord

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