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 domai n (Psi region) with regard to their effects on viral replication. Four putative stem-loop structures between the 5' splice donor site and th e gag initiation codon have been examined: stem structure A, correspon ding 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, nucl eotides 355 to 374. By measuring infectivities, encapsidation and spli cing efficiencies, and endogenous reverse transcription levels of moti f A, B, C, and D deletion mutants, we identified mutations which affec t replication at the encapsidation step. In particular, deletion of al l four motifs in a single mutant eliminated encapsidation of viral RNA , while deletion of individual elements moderately reduced the encapsi dation efficiencies. Through analysis of different deletion combinatio ns, we found that deletion of the first two moths (A plus B) reduced b oth encapsidation and reverse transcription efficiencies, while deleti on 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 t he presence of at least one of these two stem-loops is crucial to enca psidation and virus replication.