Evidence for the Identity of Shared 5′-Terminal Sequences Between Genome RNA and Subgenomic mRNA's of B77 Avian Sarcoma Virus

Abstract

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.

Nucleotide sequence of the 5' noncoding region and part of the gag gene of Rous sarcoma virus

“…The genomes of retroviruses assume multiple roles during the viral life cycle, serving as template for the synthesis of viral DNA by reverse transcriptase, messenger for the synthesis of several viral gene products, precursor for the genesis of subgenomic mRNA's, and vehicle for the transmission of viral genes from one host cell to another (1). Each of these roles involves structural features located in the vicinity of the 5' terminus of the viral genome: the initiation site for reverse transcription (41); a nucleotide sequence (R) repeated at the 5' and 3' termini of viral RNA that is required for chain propagation by reverse transcriptase (3a); a ribosome binding site and AUG codon required to initiate translation (29, 45); a splice donor site used in the genesis of subgenomic mRNA's (5,8,9,11,25,28,30,38,46); and a nucleotide sequence of uncertain size that is apparently necessary for incorporation of viral RNA into maturing virions (23,34). To facilitate the full elucidation of these functionally important features, we have determined the sequence of 1,010 nucleotides at the 5' end of the genome of the subgroup A Schmidt-Ruppin strain of Rous sarcoma virus (RSV).…”

The leader sequence of the subgenomic mRNA's of Rous sarcoma virus is approximately 390 nucleotides

“…The mRNA's of all three size classes are capped (7,23) and polyadenylated (15,46) and contain a common leader sequence at their 5' ends; this sequence apparently is spliced from a primary transcript of the RSV genome to the subgenomic mRNA's env and src (7,19,23,37,46). Previous studies of the mRNA's showed that the leader was composed of at least 104 nucleotides (7).…”

Selective inhibition of avian sarcoma virus protein synthesis in 3-deazaadenosine-treated infected chicken embryo fibroblasts

“…Isolation of RNA. Total RNA was isolated from cells by phenol-chloroform extraction, as described previously (20). Cells were fractionated into nuclei and cytoplasm by lysis with 1% (vol/vol) Nonidet P40-1% (wt/vol) deoxycholate, and the polysomes were isolated by discontinuous sucrose gradients as previously described (8).…”

“…Cells were fractionated into nuclei and cytoplasm by lysis with 1% (vol/vol) Nonidet P40-1% (wt/vol) deoxycholate, and the polysomes were isolated by discontinuous sucrose gradients as previously described (8). The fraction containing polyadenylic acid [poly(A)+] was isolated by oligodeoxythymidylic acid-cellulose chromatography (20). Determination of hybridization kinetics of viral cDNA to cellular RNA.…”

“…Determination of hybridization kinetics of viral cDNA to cellular RNA. Varying amounts of RNA from 3-deazaadenosine-treated and control cells were hybridized to 3P-labeled complementary DNA (cDNA) prepared by the endogenous reverse transcriptase reaction of detergent-disrupted B77 ASV (20). Hybridization was carried out in a solution containing 0.3 M NaCl, 0.01 M Tris-hydrochloride (pH 7.5), and 0.001 M EDTA at 680C for 18 h. The resistance of the 32P-labeled cDNA to S1 nuclease was used as a measure of the extent of hybridization.…”