ALTERATION OF LOCATION OF DIMER LINKAGE SEQUENCE IN RETROVIRAL RNA - LITTLE EFFECT ON REPLICATION OR HOMOLOGOUS RECOMBINATION

Retrovirus particles contain a dimer of retroviral genomic RNA. A defi ned region of the retrovirus genome has previously been shown to be im portant for both dimerization and encapsidation. To study the importan ce of the position of this encapsidation and dimerization signal for r etroviral replication and homologous recombination, we used a previous ly described spleen necrosis virus-based helper cell system. This syst em allows retroviral vectors with multiple genetic markers to be studi ed after a single cycle of retroviral replication. The sequence respon sible for dimerization, the encapsidation/dimer linkage sequence (E/DL S), was moved from its normal location near the 5' end of the retrovir al genome to a location near the 3' end of the genome. We characterize d four pairs of retroviral vectors: (i) with both E/DLSs at the 5' end s of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at t he 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reducti on in virus titer in a single cycle of retroviral replication. Further more, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombina tion, or the number of internal template switches in recombinant provi ruses. These results indicate that any alignment or conformation neces sary for retroviral replication or recombination is not the result of the position of the E/DLS.