Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation terminates within the coding region. In mammalian cells, this is presumably due to a downstream signal deposited during pre-mRNA splicing. In contrast, unspliced retroviral RNA undergoes NMD in chicken cells when premature termination codons (PTCs) are present in the gag gene. Surprisingly, deletion of a 401-nt 3¢ UTR sequence immediately downstream of the normal gag termination codon caused this termination event to be recognized as premature. We termed this 3¢ UTR region the Rous sarcoma virus (RSV) stability element (RSE). The RSE also stabilized the viral RNA when placed immediately downstream of a PTC in the gag gene. Deletion analysis of the RSE indicated a smaller functional element. We conclude that this 3¢ UTR sequence stabilizes termination codons in the RSV RNA, and termination codons not associated with such an RSE sequence undergo NMD.

After reverse transcription of the retroviral RNA genome and integration of the DNA provirus into the host genome, host machinery is used for viral gene expression along with viral proteins and RNA regulatory elements. Here, we discuss co-transcriptional and posttranscriptional regulation of retroviral gene expression, comparing simple and complex retroviruses. Cellular RNA polymerase II synthesizes full-length viral primary RNA transcripts that are capped and polyadenylated. All retroviruses generate a singly spliced env mRNA from this primary transcript, which encodes the viral glycoproteins. In addition, complex viral RNAs are alternatively spliced to generate accessory proteins, such as Rev, which is involved in posttranscriptional regulation of HIV-1 RNA. Importantly, the splicing of all retroviruses is incomplete; they must maintain and export a fraction of their primary RNA transcripts. This unspliced RNA functions both as the major mRNA for Gag and Pol proteins and as the packaged genomic RNA. Different retroviruses export their unspliced viral RNA from the nucleus to the cytoplasm by either Tap-dependent or Rev/CRM1-dependent routes. Translation of the unspliced mRNA involves frame-shifting or termination codon suppression so that the Gag proteins, which make up the capsid, are expressed more abundantly than the Pol proteins, which are the viral enzymes. After the viral polyproteins assemble into viral particles and bud from the cell membrane, a viral encoded protease cleaves them. Some retroviruses have evolved mechanisms to protect their unspliced RNA from decay by nonsense-mediated RNA decay and to prevent genome editing by the cellular APOBEC deaminases.

In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3 untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5-to 7-kb 3 UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5 end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.Organisms have developed multiple ways to regulate the abundance and quality of their gene products. The RNA stability and level of protein production of many mRNAs are dictated by elements located in their untranslated regions (UTRs). Binding sites for translation-inhibiting microRNAs and AU-rich element binding proteins that determine RNA stability are often located in 3Ј UTRs (4, 12). Additionally, when the 3Ј UTR is abnormally long, the mRNA is often rapidly turned over by the cellular nonsense-mediated mRNA decay (NMD) pathway (7,26,30). Retroviruses have developed mechanisms to splice only a fraction of their primary RNA transcripts and to export and translate both spliced and unspliced mRNAs (5). The resulting unspliced gag mRNA has a very long 3Ј UTR (Ͼ5 kb) (32), yet it is stable, with a half-life of greater than 7 h (31), suggesting that it somehow evades cellular mRNA surveillance mechanisms.We are studying the mechanism by which the Rous sarcoma virus (RSV) unspliced mRNA is immune to NMD. Previous work has elucidated a cis-acting RNA sequence, called the RSV stability element (RSE), downstream of the gag termination codon (36). When the RSE is deleted, the ensuing RNA degradation requires RNA translation and the critical NMD factor Upf1, implicating the NMD pathway (36). While premature termination codons (PTCs) in the RSV gag gene also trigger NMD (2,3,19), insertion of the RSE downstream of the PTC stabilizes the RNA (36). Thus, when the RSE is downstream of a termination codon, it defines the termination event as correct. The RSE is a novel element, and its characterization may teach us more about the relationship between translation termination and NMD.