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First published online July 10, 2003
doi: 10.1242/10.1242/jcs.00613

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Targeting of incoming retroviral Gag to the centrosome involves a direct interaction with the dynein light chain 8

Coralie Petit1, Marie-Lou Giron1, Joelle Tobaly-Tapiero1, Patricia Bittoun1, Eléonore Real2, Yves Jacob2, Noël Tordo2, Hugues de Thé1 and Ali Saïb1,*

1 CNRS UPR9051, Hôpital Saint-Louis, Conventionné par l'Université Paris 7, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France
2 Laboratoire des Lyssavirus, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France



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  		Fig. 1. MTOC targeting of incoming HFV in the early step of infection. Five hours after infection, numerous free incoming viral capsids are detected around the centrosome at a low (top) and a high (bottom) m.o.i. by electron microscopy. Viral capsids in close vicinity to microtubules are easily observed in the higher resolution picture of the centrosomal region. Scale bar, 450 nm (upper and lower insets). N, nucleus.

 


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  		Fig. 2. The structural Gag protein targets the MTOC in transfected Cos6 cells. Following transfection of the full-length Gag expression vector, a confocal section reveals that HFV Gag proteins are mainly detected around the MTOC, which is revealed by the CTR910 antibody (left). In Gag-expressing cells, the vimentin network is not affected as observed by fluorescence microscopy (right). Nuclei are stained with DAPI.

 


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  		Fig. 3. (A) Minimal Gag sequence requirement for MTOC targeting. Schematic representation of the GFP-Gag fusion proteins. The locations of the three glycine/arginine rich motifs (GR I, II, III), the Gag-Gag interaction domain (ID) and the cytoplasmic targeting and retention signal (CTRS) are indicated by black boxes. The numbers indicate the percentage of GFP-positive cells harboring a centrosomal staining; values are the average of three independent experiments. (B) Western-blot analysis of the different GFP fusions revealed by rabbit anti-GFP antibodies. (C) Subcellular localization of GFP-Gag fusions as observed by fluorescence microscopy and prediction of coiled-coil motifs in the first 270 amino acids of HFV Gag as revealed by the COILS program (matrix: MTIDK, weight of 2.5 for position N).

 


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  		Fig. 4. (A) Involvement of the dynein-dynactin complex in MTOC targeting of Gag. In cells overexpressing CC1, the central coiled-coil domain of p150Glued, GFP-Gag is no longer able to reach the centrosome (top). By contrast, expression of CC1 does not disrupt the MT network (bottom). (B) Following nocodazole treatment, GFP-Gag is no longer linked to the pericentrosomal region but dispersed within the cytoplasm.

 


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  		Fig. 5. Interaction between the structural Gag and the cytoplasmic light chain of dynein LC8. (A) Cos6 cells were co-transfected with pSG-Gag and pGFP-LC8, and analyzed 20 hours later. As shown in this confocal section, 97% of Gag co-localizes with GFP-LC8 at the MTOC [measured using Lasersharp software (BioRad)], whereas GFP-LC8 is evenly distributed in the cytoplasm when expressed alone. Nuclei are stained with DAPI. (B) A direct interaction between LC8 and Gag was observed by immunoprecipitation experiments followed by western blotting when 293T cells were co-transfected with the expression vectors, whereas the L171G mutation abolishes this interaction. Anti-Gag and anti-Flag antibodies do not precipitate LC8 or HFV Gag, respectively, when these proteins are expressed alone. A similar interaction was observed with the M2 mutant (GFP-Gag150-250) but not with the M9 mutant, which lacks the domain involved in MTOC targeting.

 


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  		Fig. 6. Biological role of the coiled-coil motif. Western-blot analysis of cell extracts and virus pellets from cells transfected with the infectious clone pHFV13, the corresponding clone harboring the GagL171G mutation (pHFVGagL171G), pSG-Gag and pCgp9, detected by polyclonal anti-Gag antibodies. The virus titer from cells transfected with pHFV13, corresponding to 9x104 FCFU ml–1, was arbitrarily set at 100%. The L171G point mutation does not affect particle release but dramatically reduces infectivity of the corresponding virus by more than 90% (6.2x103 FCFU ml–1).

 

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© The Company of Biologists Ltd 2003