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FIG. 7. Strategy for selecting adapted replicons for noncytopathic gene expression in mammalian cells. A SIN replicon expressing the PAC gene is used to transfect BHK cells followed by puromycin selection. At low frequency, surviving puromycin-resistant (purR) foci appear and are cloned. RNA from these cell lines is screened for adaptative mutations in the SIN replicon by transformation of naive BHK cells to puromycin resistance at high frequency. Adaptive mutations are then cloned and mapped by making chimeras with the parental replicon. Modified dsSIN vectors, which include the adaptive mutations and the PAC gene, allow rapid production of BHK cell lines expressing heterologous genes without deleterious effects on the host cell.

protein products in cell culture (36). Some published examples include the hepatitis B virus pregenome RNA (59), the papillomavirus 16 capsid protein (60), the neurokinin receptor (61), the human immunodeficiency virus glycoproteins (62), and the hepatitis C virus glycoproteins (63, 64).

Given their efficient production of heterologous antigens, engineered alphavirus RNAs also have significant potential for in vivo applications (16, 51, 54, 65, 66) including vaccination against primate immunodeficiency viruses (67). Various delivery strategies are just beginning to be explored. As described above, infectious particles containing either double subgenomic RNAs or packaged RNA replicons could be used. In the case of constructs expressing alphavirus structural proteins, which have the potential to spread in vivo, safety issues related to alphavirus pathogenicity remain a major concern. Even using the best “helper-free” packaging system, packaged replicons are likely to include low levels of packaged helper RNA or recombinant wild-type virus (43, 44). Additional safeguards, such as mutations in the spike glycoproteins that require activation by in vitro proteolysis (68) or the use of packaging machinery from highly attenuated alphaviruses (51, 69, 70), may help to diminish the possibility of pathogenic consequences.

Alternatively, genetic immunization or transient gene therapy could be accomplished using DNA or RNA constructs lacking the structural proteins. In the case of DNA, a nuclear promoter can be used to drive expression of replication-competent SIN RNA replicons after transfection with DNA (14, 15). Although less stable than DNA, RNA delivery should also be considered since this would result in only transient exposure to the nucleic acid minimizing the possibility of integration and undesirable mutagenic consequences (65, 71). In addition, replicons can be engineered to express multiple subgenomic RNAs allowing coexpression of several protective antigens along with cytokines or other immunomodulators to enhance the generation of desired immune responses.

We thank our collaborators and colleagues, past and present, who have contributed to the development of Sindbis virus-based alphavirus vectors. Special thanks also go to Kaveh Ashrafi, Peter J.Bredenbeek, Joel M.Dalrymple, Jean Dubuisson, Arash Grakoui, Teryl K.Frey, Ute Geigenmuller-Gnirke, Chang S.Hahn, Young S.Hahn, Alexander A.Kolykhalov, Robin Levis, Guangpu Li, Brett D.Lindenbach, Steven D.London, Alan L.Schmaljohn, Barabara Weiss, and Cheng Xiong. Work from our laboratories has been supported by grants from the Public Health Service (AI24134, All 1377, and AI26763), the Monsanto/Washington University Biomedical Research Contract, and the Pew Memorial Trust. B.M.P. is a Visiting Professor on leave from Albert Szent-Györgyi Medical University, Department of Microbiology, Szeged, Hungary.

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