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J. Virol. doi:10.1128/JVI.01017-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Selection and analysis of mutations in an EMCV IRES that improve the efficiency of a bicistronic flavivirus construct

Clinical Institute of Virology, Medical University of Vienna, Vienna, AUSTRIA

^* To whom correspondence should be addressed. Email: christian.mandl{at}meduniwien.ac.at.

	Abstract

Flaviviruses have a positive-stranded RNA genome, which simultaneously serves as a mRNA for translation of the viral proteins. All of the structural and nonstructral proteins are translated from a cap-dependent cistron as a single polyprotein precursor. In an earlier study (Orlinger K.K., V.M. Hoenninger, R.M. Kofler, and C.W. Mandl, J. Virol 80:12197-12208), it was demonstrated that an artificial bicistronic flavivirus genome, TBEV-bc, in which the region coding for the viral surface glycoproteins prM and E from tick-borne encephalitis virus (TBEV) had been removed from its natural context and inserted into the 3'- noncoding region under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) produces viable, infectious virus when cells are transfected with this RNA. The rates of RNA replication and infectious particle formation were significantly lower with TBEV-bc, however, than with wild-type TBEV. In this study, we have identified two types of mutations, selected by passage in BHK-21 cells, that enhance the growth properties of TBEV-bc. The first type occurred in the E protein, and these most likely increase the affinity of the virus for heparan sulfate on the cell surface. The second type occurred in the inserted EMCV IRES, in the oligoA loop of the J-K stem-loop structure, a binding site for the translation factor eIF4G. These included single nucleotide substitutions as well as insertions of additional adenines in this loop. An A-to-C substitution in the oligoA loop decreased the efficiency of the IRES itself but nevertheless resulted in improved rates of virus particle formation and overall replication efficiency. These results demonstrate the need for proper balance in the competition for free template RNA between the viral RNA replication machinery and the cellular translation machinery at the two different start sites and also identify specific target sites for the improvement of bicistronic flavivirus expression vectors.

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