Unité de Neurovirologie et R égén ération du Système Nerveux, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France1
Author for correspondence: Florence Colbère-Garapin.Fax +33 1 45 68 87 80. e-mail fcolbere{at}pasteur.fr
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Abstract |
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Main text |
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Despite this normally very lytic nature of PV, we have shown that most PV strains can establish persistent infections in IMR-32 neuroblastoma cells and that mutant PV selected during these infections are capable of persistently infecting non-neural HEp-2c cells, unlike their corresponding parental strains (Colbère-Garapin et al., 1989 ; Pelletier et al. , 1991
). Recently, we identified viral determinants of persistence in HEp-2c cells: two on the capsid surface for the type 1 PV strain, Sabin 1 (Pelletier et al., 1998a
); and one on the inside, Leu13 (2L13), and one on the surface Asn290 (1N290) for the type 3 PV (PV-3) strain, T7-Leon (T7L) (Duncan et al., 1998
). Both PV-3 determinants are required for efficient persistence; their localization on the three-dimensional structure of the capsid indicated that the internal determinant 2L13 is located near a region critical to capsid stability (Filman et al. , 1989
), while the surface determinant 1N290 is located in neutralization antigenic site 3a (Minor et al., 1986
), outside of the putative footprint of the PVR (Colston & Racaniello, 1994
; Harber et al., 1995
; Olson et al., 1993
).
Previously, we observed several important differences in the early steps of the virus cycle between the parental lytic virus T7-Leon and the persistent double mutant T7L+2L131N290. Firstly, the persistent mutant exhibits increased adsorption and decreased elution of attached particles, suggesting an increased affinity for the PVR (Duncan et al., 1998 ). Secondly, the persistent mutant, with a sedimentation coefficient of 160S, generated a novel 147S form upon adsorption onto PVR-expressing cells, even at 0 °C.
In the current study, we have further investigated the novel receptor-mediated conformational changes and we have analysed the independent effects of each of the two determinants.
We first determined the nature of the particle(s) which elute at 37 °C in the case of the persistent mutant T7L+2L13 1N290, since when virus and cells are incubated at 37 °C for 90 min after adsorption, over 35% of the initially bound viral particles are present in the extracellular medium (Duncan et al., 1998 ). In order to know whether 147S or 135S particles are eluted, we first adsorbed [35 S]methionine-radiolabelled virus onto human HEp-2c cells at 0 °C and then removed unattached virus by repeated washes before switching the infection to 37 °C for 90 min. Cell supernatants and cell lysates were then analysed by sucrose gradient centrifugation, as previously described (Duncan et al., 1998
). In the case of T7-Leon (Fig. 1A
, top panel), the sedimentation profile revealed, as expected, the 160S virion, the 135S intermediate and the 80S empty capsid, all detectable both in cell-associated and extracellular fractions. In contrast, in the case of the persistent T7L+2L13 1N290 virus (Fig. 1A
, bottom panel), only the novel 147S form and the 80S empty capsids were visible in the cell-associated fractions. Surprisingly, however, the 135S particle was detectable in the supernatant, although the ratio of 135S particles to 160S and 80S particles appeared greatly reduced as compared to that observed for T7-Leon. Most importantly, the 147S form did not appear to be present in the extracellular fractions, suggesting that it is found solely in association with the cell.
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After adsorption for 2·5 h on ice, the two single mutants had distinctly different profiles when associated with the cell (Fig. 1B, left-hand panels). In fact, the single mutant T7L+2L13 appeared to form a peak sedimenting at the 160S position, although the peak was always slightly asymmetrical, with a longer slope towards the 147S position (Fig. 1B
, top left- hand panel). Conversely, the second single mutant, T7L+1N290 , formed a peak sedimenting at the 147S position. When the decapsidation profiles were observed after a further 90 min incubation period at 37 °C, the differences between the two viruses were even more marked. In the cell-associated fraction, the mutant T7L+2L13 (Fig. 1B
, top right-hand panel), formed a main peak at the 160S position of virions, again with a longer slope towards the top of the gradient. Very few 135S particles were eluted with this mutant, as observed for the double mutant T7L+2L 131N290. The second single mutant, T7L+1N290 (Fig. 1B
, bottom right-hand panel), formed a main peak of cell-associated particles at the 147S position, like the double persistent mutant, but had an elution profile similar to that of the parental lytic virus T7-Leon (Fig. 1A
, top panel). Taken together, these results all suggest that each of the two viral determinants, 2L13 and 1N290, exert different effects on the viral capsid during receptor-mediated uncoating.
It has been previously demonstrated that the 160S to 135S receptor- mediated conformational changes only occur at temperatures greater than 32 °C (Gomez Yafal et al., 1993 ). However, in the case of the persistent PV T7L+2L131N290, the generation of the 147S particle is observed even at 0 °C (Duncan et al., 1998
), suggesting inherent capsid instability. We therefore investigated the susceptibility of the viral capsid to heat inactivation by incubating viruses for various times at 48 °C before determining the titre by an endpoint micromethod. As can be seen in Fig. 2
, the persistent mutant T7L+2L13 1N290 was in fact extremely stable at high temperature, having over a thousandfold higher titre than the wild-type virus after 15 min incubation at 48 °C. Interestingly, the two single point-mutants were even slightly more stable than the double mutant. Altogether, these results showed that the mutant PV-3 capsids were in fact extremely heat-stable in PBS, indicating that the atypical uncoating profiles are not due to capsid instability.
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As to the nature of the 147S form, our results to date, presented here and elsewhere (Duncan et al., 1998 ) suggest that virusreceptor interactions are absolutely required for its formation. Since the 147S form appears to result from virions having an increased affinity for the PVR (Duncan et al., 1998
) coupled with remarkable capsid stability, one cannot exclude the possibility that this form corresponds to a very stable association between a viral particle and a cellular molecule, most likely the PVR, which would block PV entry. However, increasing the SDS concentration in cell extracts up to fourfold did not prevent the detection of the 147S form (data not shown). The results presented here are rather in agreement with two of our previously presented hypotheses (Duncan et al., 1998
; Pelletier et al., 1998b
), i.e. the 147S form could be either a stable, abortive, uncoating intermediate which forms immediately upon contact between certain mutant virions and the PVR, or a normally occurring but usually highly transitory uncoating intermediate, which is defective in the case of certain mutant viruses.
In conclusion, we report here the independent effects of each of two viral determinants of persistence, 2L13 and 1N290 , on virus uncoating at the molecular level, which appear to be responsible for the production of a reduced number of 135S particles and for the generation of a novel 147S form, respectively.
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Acknowledgments |
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Footnotes |
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References |
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Received 9 April 1999;
accepted 21 June 1999.
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