Functional characterization of the dimer linkage structure RNA of Moloney murine sarcoma virus

Several determinants that appear to promote the dimerization of murine retr oviral genomic RNA have been identified. The interaction between these dete rminants has not been extensively examined. Previously, we proposed that di merization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is ini tiated by separate G-rich stretches (H. Ly, D, P. Nierlich, J, C. Olsen, an d A. H, Kaplan, J. Virol, 73:7255-7261, 1999), The cooperative action of th ese two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementa ry to the palindrome; molecules lacking the palindrome could not dimerize i n the presence of oligomers that hybridize to two G-rich sequences. The res ults of spontaneous dimerization experiments also demonstrated that RNA mol ecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage struc ture (DLS). However, the addition of an oligonucleotide complementary to th e remaining stretch of guanines restored the kinetics of dimerization to wi ld-type levels, The ability of this oligomer to rescue the kinetics of dime rization was dependent on the presence of the palindrome, suggesting that i nteractions within the G-rich regions produce changes in the palindrome tha t allow dimerization to proceed with maximum efficiency. Further, unsuccess ful attempts to produce heterodimers between constructs lacking various com binations of these elements indicate that the G-rich regions and the palind rome do not interact directly. Finally, we demonstrate that both of these e lements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation, Overall, ou r data support a dynamic model of retroviral RNA dimerization in which disc rete dimerization elements act in a concerted fashion.