cis-active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA

We have analyzed the roles of RNA structural motifs located in the 5' part of the Moloney murine leukemia virus (M-MuLV) encapsidation domain (Psi region) with regard to their effects on viral replication. Four putative stem-loop structures between the 5' splice donor site and the gag initiation codon have been examined: stem structure A, corresponding to M-MuLV viral nucleotides 211 to 224; stem-loop B, nucleotides 278 to 303; stem-loop C, nucleotides 310 to 352; and stem-loop D, nucleotides 355 to 374. By measuring infectivities, encapsidation and splicing efficiencies, and endogenous reverse transcription levels of motif A, B, C, and D deletion mutants, we identified mutations which affect replication at the encapsidation step. In particular, deletion of all four motifs in a single mutant eliminated encapsidation of viral RNA, while deletion of individual elements moderately reduced the encapsidation efficiencies. Through analysis of different deletion combinations, we found that deletion of the first two motifs (A plus B) reduced both encapsidation and reverse transcription efficiencies, while deletion of the 3' motifs (C plus D) eliminated encapsidation. Interestingly, the C and D motifs both contain a GACG loop sequence and are highly conserved among murine type C retroviruses. Our results indicate that M-MuLV motifs C and D are necessary for efficient encapsidation, and the presence of at least one of these two stem-loops is crucial to encapsidation and virus replication.