Unspliced Rous Sarcoma Virus Genomic RNAs Are Translated and Subjected to Nonsense-Mediated mRNA Decay before Packaging

Abstract

Retroviruses package full-length, unspliced RNAs into progeny virions as dimerized RNA genomes. They also use unspliced RNAs as mRNAs to produce the gag and pol gene products. We asked whether a single Rous sarcoma virus (RSV) RNA can be translated and subsequently packaged or whether genomic packaging requires a nontranslated population of RNAs. We addressed this issue by utilizing the translation-dependent nonsense-mediated mRNA decay (NMD) pathway. NMD is the selective destruction of mRNAs bearing premature termination codons (PTCs). The pathway has been shown to be associated with splicing in higher eukaryotes. Here, we demonstrate that both translation and the cellular factor Upf1 are required for the decay of unspliced, PTC-bearing RSV RNA by the NMD pathway. To address the relationship between RNA translation and packaging, we examined virus produced in cells cotransfected with PTC-bearing retroviral clones and wild-type viral clones. We observed that PTC-bearing transcripts are packaged into viral particles at levels three-to fivefold less than those of control RNAs. Since PTC-mediated degradation requires translation, we conclude that RSV can package progeny virion particles using previously translated RNAs.

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 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. Since this regulation occurs on an unspliced RNA, it contrasts with previous reports in higher eukaryotes that splicing is necessary to distinguish a PTC from a normal termination codon (Maquat 2004).…”

“…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).…”

Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

“…Initially, the presence of EJCs downstream from TCs was thought to be an essential signal to identify the TC as premature and elicit NMD (Maquat 2004), but it is meanwhile clear that mammalian NMD can also occur in the absence of an EJC downstream from the PTC (Zhang et al 1998;Rajavel and Neufeld 2001;Delpy et al 2004;LeBlanc and Beemon 2004;Buhler et al 2006;Matsuda et al 2007;Eberle et al 2008;Singh et al 2008). Since NMD is triggered upon aberrant translation termination, which can only occur after at least one ribosome has passed the entire coding sequence and thereby removed the EJCs, NMD-targeted mRNAs without introns downstream from the TC are most likely devoid of any bound EJCs, and we therefore refer to this NMD pathway as "EJC-independent NMD."…”

Retrovirus Translation Initiation: Issues and Hypotheses Derived from Study of HIV-1

“…Instead, unspliced HIV-1 RNA functions interchangeably as an mRNA template for translation and as genomic RNA that is packaged [16]. A similar conclusion was reached for Rous sarcoma virus (RSV) in a study that evaluated the relationship between translation-dependent nonsense mediated decay and RNA packaging [48].…”