FUNCTIONAL-ANALYSIS OF THE CORE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PACKAGING SIGNAL IN A PERMISSIVE CELL-LINE

Packaging of type C retrovirus genomic RNAs into budding virions requi res a highly specific interaction between the viral Gag precursor and unique cis-acting packaging signals on the full-length RNA genome, all owing the selection of this RNA species from among a pool of spliced v iral RNAs and similar cellular RNAs. This process is thought to involv e RNA secondary and tertiary structural motifs since there is little c onservation of the primary sequence of this region between retroviruse s. To confirm RNA secondary structures, which we and others have predi cted for this region, disruptive, compensatory, and deletion mutations were introduced into proviral constructs, which were then assayed in a permissive cell line. Disruption of either of two predicted stem-loo ps was found to greatly reduce RNA encapsidation and replication, wher eas compensatory mutations restoring base pairing to these stem-loops had a wild-type phenotype. A GGNGR motif was identified in the loops o f three hairpins in this region. Results were consistent with the hypo thesis that the process of efficient RNA encapsidation is linked to di merization. Replication and encapsidation were shown to occur at a red uced rate in the absence of the previously described kissing hairpin m otif.