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Moloney murine sarcoma virus genomic RNAs dimerize via a two-step process: a concentration-dependent kissing-loop interaction is driven by initial contact between consecutive guanines.

ABSTRACT: Retroviruses contain two plus-strand genomic RNAs, which are stably but noncovalently joined in their 5' regions by a dimer linkage structure (DLS). Two models have been put forward to explain the mechanisms by which the RNAs dimerize; each model emphasizes the role of specific molecular determinants. The kissing-loop model implicates interactions between palindromic sequences in the DLS region. The second model proposes that purine-rich stretches in the region form purine quartet structures. Here, we present an examination of the in vitro dimerization of Moloney murine sarcoma virus (MuSV) RNA in the context of these two models. Dimers were found to form spontaneously in a temperature-, time-, concentration-, and salt-dependent manner. In contrast to earlier reports, we found that deletion of neither the palindrome nor the consensus purine motifs (PuGGAPuA) affected the level of dimer formation at low concentrations of RNA. Rather, different purine-rich sequences, i.e., consecutive stretches of guanines, were found to enhance both in vitro RNA dimerization and in vivo viral replication. Biochemical evidence further suggests that these guanine-rich (G-rich) stretches form guanine quartet structures. We also found that the palindromic sequences could support dimerization at significantly higher RNA concentrations. In addition, the G-rich stretches were as important as the palindromic sequence for maintaining efficient viral replication. Overall, our data support a model that entails contributions from both of the previously proposed mechanisms of retroviral RNA dimerization.

SUBMITTER: Ly H

PROVIDER: S-EPMC104250 | biostudies-literature | 1999 Sep

REPOSITORIES: biostudies-literature

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Project description:Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255-7261, 1999). The cooperative action of these 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 complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements 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, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.

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Moloney murine sarcoma virus genomic RNAs dimerize via a two-step process: a concentration-dependent kissing-loop interaction is driven by initial contact between consecutive guanines.

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