Background: The retrovirus genome is a dimer of 2 identical positive-strand RNAs. These RNA dimers from mature (protease active) and immature (protease inactive) virus, have different RNA conformations. The most stable junction in mature dimers, termed the Dimer Linkage Site (DLS), is near the 5’-termini. The location of the dimer linkages in immature dimers as well as the precise location of the DLS in mature dimers is unknown. The 5’-end of the Murine Leukemia Virus (MLV) genome contains stem-loops implicated in in vitro dimerization (SL-B’, SL-B), and in vivo packaging (SL-C and SL-D). Here, we study the contribution of these 5’-end sequences to dimerization, in vivo.

Methods: We used both a physical and a genetic approach to study the contribution of the 5’-termini to in vivo dimerization. We mapped the linkages in genomic dimers using oligonucleotide-directed Rnase H cleavage of viral RNA, followed by native Northern analysis. To ask if sequences in the 5’-termini were sufficient to produce virus-like dimer linkages outside the context of a full-length genome, non-viral mRNAs, containing retroviral 5’-end sequences, were packaged into either mature or immature particles, isolated and analyzed. Using mutant constructs of the DLS region, we tested the effect of the MLV SL-B and surrounding sequences on dimer stability in vivo.

Results: The physical mapping results show that the strongest link in immature dimers, as in mature dimers, is near the 5’-end of the genome. Weaker, tethering interactions, joining the 5’- and 3’-ends, in both immature and mature dimers were also identified. Studies of the chimeric mRNAs show that the retroviral 5’-termini were sufficient to produce dimers with thermostability and maturation profiles identical to the profiles of genomic dimers from which the sequences were taken. Sequences outside the MLV SL-B affected the dimer stability, and deletion of SL-B greatly reduced the thermostability of immature, but not mature dimers.

Conclusions: These results confirm the hypothesis that the most stable junction in both immature and mature dimers of genomic RNAs is in the psi region, further supporting a close relationship between dimerization and packaging. These results provide the first direct in vivo evidence that the MLV SL-B participates in the immature dimer linkage but may not be part of the mature dimer linkage, suggesting other sequences must be involved.