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induced syncytium formation in CD4+ HeLa cells in a level comparable to that of Env-RVG. Thus, rather than the length of ≈44 amino acids, the specific sequence of the RV cytoplasmic tail is required to promote or to allow incorporation of spikes into the viral envelope. This is strongly supported by preliminary data on an additional Env mutant capable of rescuing the infectivity of SAD ΔG. The mutant has a cytoplasmic tail of 65 amino acids, the carboxyl terminus of which corresponds to the sequence of the RV G tail. As suggested by the ability of the G protein from another lyssavirus serotype to replace RV G (26) and by comparison of available lyssavirus G sequences, the critical motif might be located in the carboxyl-terminal region of the tail in which 7 of 14 residues are conserved with regard to other lyssaviruses (unpublished data).

As demonstrated recently, similar to VSV G (27), RV G protein possesses an independent exocytosis activity (7) and, in the presence of G, the yield of rhabdovirus particles is augmented. As estimated from the gradient analyses, an approximately 5-fold higher amount of particles complemented with G compared with RV(HIV) pseudotype particles was observed. Although substantial amounts of Env-RVG spikes were incorporated into the particles, as shown directly by the presence of the anchored, gp41-derived chimeric 37 kDa subunit (Fig. 5), the infectious titers differed by a factor of 20–25. Since the infectivity of viruses is largely governed by the features of their spike proteins and the interaction with receptors, the discrepancy in infectious titers may not directly reflect the efficiency by which the hybrid protein can substitute for RV G in formation of infectious virions.

It is not known so far whether oligomerization has an influence on the putative interaction of the cytoplasmic tail with internal virus proteins. Similar to the RV spike (28), HIV-Env may probably form trimers (29). The successful incorporation of Env-RVG suggests that this protein may present the tail(s) in an appropriate configuration. Incorporation of the monomeric cellular glycoprotein CD4 into VSV tsO45 has been reported previously (30). However, particles containing CD4, or a chimeric CD4 with the VSV cytoplasmic tail, were observed only at permissive temperature, suggesting that VSV G protein is required for incorporation of CD4 into VSV.

Our results verified that a recombinant rhabdovirus can be generated whose tropism is exclusively determined by a foreign surface glycoprotein incorporated specifically into the viral envelope. Due to the transient nature of the assay, the described system should provide a versatile and, most importantly, a safe tool in determining whether other glycoproteins might be able to direct rhabdovirus vectors to specific target cells.

We thank Veronika Schlatt and Karin Kegreiss for perfect technical assistance. This work was supported by Grants BEO 21/0310118A and 0311171 from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie.

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