Rabies Unit, Virology Department, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris Cedex 15, France1
Max-von-Pettenkofer Institute, Munich, Germany2
Veterans Administration Medical Center, East Orange, Newark, NJ, USA3
Author for correspondence: Pierre-Emmanuel Ceccaldi. Fax +33 1 40 61 30 15. e-mail ceccaldi{at}pasteur.fr
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Abstract |
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The mechanisms of transsynaptic spread of RV remain unclear. Although it has been shown that RV G is responsible for interaction of RV with cells, some authors have hypothesized that RV G would not be necessary for transsynaptic spread of RV nucleocapsid from one neuron to another. On the basis of sequential electron microscope studies, it has been suggested that, in the early phase of infection, before the assembly of full virus particles, the bare viral nucleocapsid could be transported along the axon and subsequently transferred through the synapse into the post-synaptic neuron, without acquiring a G-containing envelope (Gosztonyi, 1994 ; Gosztonyi et al., 1993
). This hypothesis of transsynaptic transfer of bare RV nucleocapsids is based on the fact that full virus particles could never be detected at the sites of synapses in these studies. These conclusions, however, are in contrast with the previously reported role of G in RV spread. Of special interest is the fact that point mutations at position 333 are able to modify virus spread within the brain (Coulon et al., 1989
; Lafay et al., 1991
).
Previously, Mebatsion et al. (1996a ) have been able to obtain a recombinant RV mutant deficient for the entire G gene (SAD
G) by a reverse genetic approach. This mutant was able to bud spikeless virions from the cell surface of fibroblast cell lines, although with a 30-fold lower efficiency than the parental strain SAD L16, but was unable to produce infectious virus particles. However, this study did not involve neuronal cells, the natural host cells for RV, and the possibility of transsynaptic spread of nucleocapsids was not addressed. In the present study, we investigated the behaviour of the G-deficient mutant on cultured neurons (cell lines or primary cultures) and characterized its pathogenic properties in murine models that allow the study of transsynaptic spread (Gillet et al., 1986
; Ceccaldi et al., 1989
).
The recombinant G-deficient RV mutant (SAD G) was generated previously (Mebatsion et al., 1996a
) by deletion of the entire G gene from a full-length infectious RV clone, pSAD L16 (Schnell et al., 1994
), which was derived from the attenuated RV strain SAD B19 (Conzelmann et al., 1990
; Schneider & Cox, 1983
). Stocks of SAD
G virus able to perform the first cycle of infection in cell cultures and animals (SAD
G+G) were generated in cells that express G protein corresponding to that of SAD L16 from transfected plasmids, as described previously (Mebatsion et al., 1996a
). The course of infection and spread of SAD
G infection were compared in this study with the parental SAD L16 RV.
The infection characteristics of G-RV were investigated in neuronal cell cultures (cell lines and primary cultures) in comparison with the reference RV SAD L16. Murine neuronal cell lines (NIE-115, Ceccaldi et al., 1997
) were infected with either SAD
G or SAD L16 at an m.o.i. of 0·01. The number of infected cells was assessed by immunofluorescence with a polyclonal anti-nucleocapsid conjugate (Sanofi-Diagnostics-Pasteur) as described previously (Atanasiu et al., 1974
). The number of infected cells did not increase between days 1 and 4 post-infection (p.i.) when infected with SAD
G strain (see Fig. 1
AC), whereas with SAD L16, the entire cell layer displayed infection as soon as day 2 p.i. (Fig. 1D
F
). These results were confirmed by counting infected cells from two different culture preparations: whereas with SAD
G, the percentage of infected cells versus total cells remained below 1% on days 1, 2, 3 and 4 p.i., a value of 100% infected cells was reached with SAD L16 from day 2 p.i. onwards. Similar kinetics were observed with another murine neuroblastoma cell line, Neuro2a (data not shown). Titration of infectious virus yield on these cultures by the fluorescence forming unit (f.f.u.) method (Bourhy & Sureau, 1990
) revealed that cells infected with SAD
G (m.o.i.=1) were unable to release infectious virus on days 2, 3 and 4 p.i. Only on day 1 p.i. could a very low titre (20 f.f.u./ml) be found, probably due to input virus (remaining in the culture medium despite careful removal of the inoculum after infection, or detached from the cell membrane). In contrast, in SAD L16-infected cells, the production of infectious particles increased with time from a value of 103 f.f.u./ml as early as day 1 p.i. to nearly 107 f.f.u./ml by day 3 p.i. and later.
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We next studies the affects of infection in vivo. Intracerebral infection of mice (BALB/c, 20 g) with 3000 f.f.u. per animal induced 100% mortality with SAD L16 (mean time to death 9·65 days, n=20), whereas no mortality was observed in mice infected with SAD G+G (n=22). In SAD L16-infected mice, clinical signs (paralysis, cachexia and bristling) were observed from 1 to 3 days before death, whereas no changes could be observed during SAD
G infection. To assess more precisely whether SAD
G infection could have an effect on behaviour, a running-wheel activity test (Ottenweller et al., 1998
) was performed. As shown in Fig. 2
, SAD
G infection did not induce any change in activity compared to uninfected controls, whereas SAD L16-infected mice showed a dramatic decrease in running-wheel activity as soon as day 5 p.i.
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The present data indicate that RV G is absolutely required for production of infectious RV particles from cultured neurons (primary cultures or cell lines). Mebatsion et al. (1996a ) have previously demonstrated that infection of fibroblast cell lines with the same G-deficient RV did not produce infectious virus, although budding of spikeless particles occurred. These data are in agreement with previous work emphasizing the role of G in virus attachment to the cell membrane (et al., 1973
; Perrin et al., 1982
) and recognition of host-cell membrane receptors (Wunner et al., 1984
). Some of these receptors have been reported, such as the nicotinic acetylcholine receptor (Lentz et al., 1982
), NCAM (Thoulouze et al., 1998
) and p75NTR (Tuffereau et al., 1998
). In addition, other membrane components such as phospholipids could play a role (Superti et al., 1984
). In spite of the obvious involvement of these structures in virus attachment to the cell membrane, it remained unclear whether G would be absolutely required in the case of transsynaptic passage of RV. This hypothesis was based mainly on the fact that RV, which uses axonal transport to spread within the CNS (Kucera et al., 1985
; Gillet et al., 1986
; Ceccaldi et al., 1989
), has never been visualized as full virus particles at the site of synaptic junctions in sequential electron microscope studies (Gosztonyi et al., 1993
). Thus, it was speculated that the bare RV nucleocapsid could be transported along axons and further transferred to the subsequent neuron without the involvement of any envelope structure (Gosztonyi, 1986
, 1994
; Gosztonyi et al., 1993
). Such a mode of transmission, which is in contrast with the previously reported role of RV G in host cell entry, is problematic in terms of membrane traffic but could be explained by the phenomenon of spinule formation and the possible existence of perforated synapses (Calverley & Jones, 1990
). However, the present results, which use a model of axonal transport following virus inoculation into the striatum, clearly indicate that no transsynaptic spread of RV occurred in the absence of G. Actually, after infection of regions that are connected anatomically to the inoculation site, such as substantia nigra (pars compacta), cortex, thalamus and hippocampus (Gillet et al., 1986
; Robertson & Travers, 1975
; Veening et al., 1980
) with G-complemented SAD
G, no subsequent transfer of RV to other areas occurred. In contrast, although SAD L16 produced the same pattern of infection as SAD
G at first, this was followed by the spread of infection to other areas not connected directly to the striatum (e.g. cerebellum).
As a consequence of the absence of transsynaptic transfer, G-deficient recombinant RV shows restricted spread within the CNS. This reduced infection of the brain is not accompanied by the classical rabies clinical signs (e.g. locomotor activity changes) that occur with the parental strain. These results are in line with previous findings stating that modification of RV G is able to change the pathogenic characteristics of RV. A single amino acid change within G has been shown to reduce the neurovirulence of RV (Dietzschold et al., 1985 ; Coulon et al., 1989
). Here, we have shown that deletion of the entire G results in non-pathogenic virus restricted to a single round of infection and unable to infect secondary cells, even in the brain, where transsynaptic transmission of nucleocapsid was suspected to represent a possible means of spread. We have shown previously that recombinant pathogenic RV can be used as a vector to express foreign genes in brain neurons (Ceccaldi et al., 1998
; Etessami et al., 2000
). Elimination of virus-induced pathogenicity of RV by deletion of the G gene thus allows RV to be considered as a safe one-step replication vector not able to spread further in the brain, in addition to the previously reported neurotropic virus vectors (Goins et al., 1997
; Hermens et al., 1997
). Moreover, it has been shown that RV-derived pseudotype vectors can be targetted specifically to cells other than neurons (by envelope switching), considerably extending the range for vector applications (Mebatsion & Conzelmann, 1996
; Mebatsion et al., 1997
). Further investigations are now in progress to delineate the kinetics and mechanisms of virus clearance, which occurs with the G-deficient virus after infection of the first-order target cells, and to study the expression of foreign genes inserted into the RV genome (Mebatsion et al., 1996
b) and targetted to neurons (Ceccaldi et al., 1998
; Etessami et al., 2000
).
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References |
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Received 25 February 2000;
accepted 1 June 2000.