Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

Abstract

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain ofgag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.High levels of retroviral RNAs are generated by efficient transcription from regulatory sequences in the viral long terminal repeat (LTR) and maintained as a result of their high intracellular stability (reviewed in references 9 and 20). We are studying the mechanism of stabilization of Rous sarcoma virus (RSV) RNA. Premature termination of translation within the 5' proximal gag gene of RSV was previously shown to lead to reduced accumulation of unspliced RSV RNA because of its increased degradation rate (2, 4). In contrast, the level of spliced RSV mRNAs was not affected by nonsense codons within the gag gene (4). The introduction of nonsense codons within the coding region of a number of eukaryotic cellular genes has also been associated with decreased mRNA levels. In the cases of dihydrofolate reductase (dhfr) (22), triosephosphate isomerase (7), and ,-globin (5) RNAs, it appears that prematurely terminated RNA may be degraded either in the nucleus or during transport from the nucleus to the cytoplasm.The primary RNA transcript of RSV (5' gag-pol-env-src 3') is transcribed and processed by cellular machinery (9,20). This RNA is polyadenylated; however, only a fraction of it is spliced to generate env and src mRNAs. Maintenance and nucleocytoplasmic transport of a portion of the unspliced RNA is critical to viral replication; the unspliced RNA is the form of the viral genome that is packaged into virions, and it is also the mRNA for Gag and Gag-Pol polyproteins. The major translation product of the unspliced RSV RNA is the 76-kDa Gag precursor polyprotein, which is subsequently processed into several virion structural proteins and a protease. Approximately 5% of ribosomes progress beyond the normal gag termination codon via a translational frameshift (12, 13), producing a 180-kDa Gag-Pol polyprotein, which is processed to generate the viral reverse transcriptase and integrase. We have proposed that a cis-acting interac...

A 3′ UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus

“…Previous results have shown that termination codons placed throughout the gag coding region destabilize the unspliced RSV transcript (Barker and Beemon 1991) by the NMD pathway (LeBlanc and Beemon 2004). Knowing that the pol gene is translated much less frequently than gag (Swanstrom and Wills 1997), we asked whether termination codons in pol would cause degradation of the unspliced RNA.…”

“…This deletion prevents packaging of the viral unspliced RNA. Barker and Beemon (1994) reported that a termination codon placed early in the pol gene (nt 2598) had no substantial effect on unspliced RNA stability. However, this could be due to the infrequent À1 frameshift, which leads to infrequent translation of the pol gene.…”

“…In contrast to the paradigm in mammalian cells that NMD is linked to splicing, Rous sarcoma virus (RSV) RNA undergoes NMD in chicken cells in the absence of splicing (Barker andBeemon 1991, 1994;LeBlanc and Beemon 2004). The 9.3-kb unspliced viral RNA is exported to the cytoplasm where it serves as both the genome of progeny virions and the mRNA template for the Gag and the Gag-Pol polyproteins (Swanstrom and Wills 1997).…”

“…Approximately 5% of the time, a À1 programmed ribosomal frameshift moves the ribosome into the reading frame of the second viral gene, pol, resulting in synthesis of a Gag-Pol fusion protein (Swanstrom and Wills 1997). Barker andBeemon (1991, 1994) reported that premature termination codons (PTCs) in the gag gene lead to a substantial decrease in unspliced cytoplasmic viral RNA. This decay diminishes gradually when the PTC is in the final 150 nt of the gag gene, approaching the natural termination codon (Barker and Beemon 1994).…”

“…Barker andBeemon (1991, 1994) reported that premature termination codons (PTCs) in the gag gene lead to a substantial decrease in unspliced cytoplasmic viral RNA. This decay diminishes gradually when the PTC is in the final 150 nt of the gag gene, approaching the natural termination codon (Barker and Beemon 1994). Additionally, LeBlanc and Beemon (2004) showed that this decay depends on translation and the critical NMD factor Upf1, implicating the NMD machinery.…”