Mutational analysis of the rous sarcoma virus DR posttranscriptional control element

Abstract

The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttranscriptional control elements; at least one copy of this sequence is necessary for cytoplasmic accumulation of unspliced viral RNA. These sequences promote Rev-independent human immunodeficiency virus type 1 expression, suggesting they act as constitutive transport elements (CTEs). To determine which regions of this sequence are critical for CTE function, mutations in the downstream DR were generated and tested in a viral deletion construct lacking src and the upstream DR. Two single-point mutations and three different clustered mutations caused substantial reductions in reverse transcriptase activity, Gag protein levels, and unspliced viral RNA in the cytoplasm. Three conserved regions of the CTE, including nucleotides 8844 to 8847, 8862 to 8864, and 8868 to 8870, were most sensitive to inactivation by mutagenesis.

FIG. 1

Sites of substitution mutations on the DR2 RNA minimal sequence (RSV nt 8791 to 8891). Underlined sequences denote nucleotides conserved between the seven different DR sequences previously aligned (DR1 and DR2 of PrC and SR-A strains of RSV, and RAV-2, RAV-7, and RAV-0) (24). Mutations shown in boldface type resulted in large decreases in viral replication (decreases in RT activity of approximately 10-fold or more relative to that of the wild-type virus) (Table 1).

FIG. 2

Viral DR2 mutants have diminished levels of Gag proteins. DR2 mutants within pPrCΔDR′ were transfected into CEFs, and total cellular proteins were harvested 48 h after transfection. Western blots of cellular proteins were probed with rabbit antiserum raised against avian myeloblastosis virus p19^gag (MA). The larger-molecular-weight protein bands are incompletely processed Gag precursor proteins. MA, Gag matrix protein (p19).

FIG. 3

RSV DR2 mutants have reduced levels of total unspliced RNA. RNA isolated from CEFs transfected with DR2 mutants was harvested 48 h after transfection, and viral RNA was probed in an RNase protection assay with a riboprobe which spans the viral 5′ splice site. The pMyc23 construct was cotransfected with the viral constructs as a control for RNA recovery and transfection efficiency. (A) Clustered substitution mutations. (B) Single-point mutations.

FIG. 4

RSV DR2 mutants have reduced levels of unspliced RNA in the cytoplasm. Viral constructs were transfected into CEFs with the pMyc23 control plasmid. Cells were fractionated into nuclear and cytoplasmic fractions, and RNA was detected by RNase protection assays as described in the legend to Fig. 3.