Mutational Analysis of Stem-Loops in the RNA Packaging Signal of the Moloney Murine Leukemia Virus

Abstract

The retroviral RNA genome is targeted for incorporation into the nascent virion particle by the psi region, a specific block of RNA sequences near the 5' end. A number of deletions and linker insertion mutations were introduced into the psi region of cloned DNA of the Moloney murine leukemia virus, and the mutants were introduced into cells in culture and tested for their ability to direct the assembly of virions and the packaging of viral RNA. Only a small portion of the psi region was important for packaging, containing the so-called stem-loops C and D. Additional mutants were used to demonstrate that the base pairing of stem D, and the sequence of loop D, were essential for normal packaging of the RNA. Two mutants with alterations near the 5' splice donor were also replication-defective, probably due to effects on gene expression. The results allow a high-resolution definition of the RNA structures required during virus replication in culture.

RNA Sequences in the Moloney Murine Leukemia Virus Genome Bound by the Gag Precursor Protein in the Yeast Three-Hybrid System

“…The SL355-4 mutation disrupts the base and top of the SL-D stem. This mutation reduced RNA incorporation into virions by more than 100-fold and severely disrupted replication ability, almost abolishing viral spread as detected by reverse transcriptase assay (17). A second SL-D mutation, G364A, changed the first G in the SL-D tetraloop to an A.…”

“…Therefore, it was of interest to test how mutations previously shown to impair RNA encapsidation in vivo would affect the interaction with MMLV Gag in the yeast three-hybrid system. A report from Fisher and Goff (17) described an analysis of the effects of MMLV ⌿ mutations on in vivo RNA encapsidation and viral spread. In this study, SL-C and SL-D mutations were shown to have dramatic effects on viral RNA packaging.…”

“…4A]), caused an in vivo deficiency in RNA packaging (Ͼ10-fold by RNase protection assay of purified virions) and a delay in viral spread (approximately 2 or 3 days by reverse transcriptase assay) (17). To assay the effects of this mutation in the yeast three-hybrid system, this mutation was incorporated into progressively smaller regions of ⌿ containing SL-C RNA baits, and these RNAs were assayed for interaction with GAD-MoGag.…”

“…Many stem-loops (SL) and tertiary contacts are predicted in this region, with the major structural features designated SL-A through SL-D. Most functional mapping studies of the MMLV packaging signal have involved mutagenesis of the region and characterization of the effects of these mutations on the viral life cycle (17,29,30). These studies have shown a critical role for SL-C and -D, with contributions from upstream elements.…”

A unique, thermostable dimer linkage structure of RD114 retroviral RNA

“…Psl-3 and Psl-4 are positioned at the same distance from the primer binding site in RD114, as are two psls DIS-1 and DIS-2 of the MLVs. Moreover, two GACGhairpins downstream from DIS-2 are implicated in the dimerization and packaging of MLV-RNA (Mougel and Barklis 1997;De Tapia et al 1998;Fisher and Goff 1998;Kim and Tinoco 2000;Badorrek et al 2006). Such GACGhairpin motifs are also found in RD114 RNA (as well as in BaEV and PcEV), with GACG-sequences at nucleotide positions 336-339 and 371-375, respectively (see Fig.…”