Structural basis of suppression of host translation termination by Moloney Murine Leukemia Virus

Abstract

Retroviral reverse transcriptase (RT) of Moloney murine leukemia virus (MoMLV) is expressed in the form of a large Gag–Pol precursor protein by suppression of translational termination in which the maximal efficiency of stop codon read-through depends on the interaction between MoMLV RT and peptidyl release factor 1 (eRF1). Here, we report the crystal structure of MoMLV RT in complex with eRF1. The MoMLV RT interacts with the C-terminal domain of eRF1 via its RNase H domain to sterically occlude the binding of peptidyl release factor 3 (eRF3) to eRF1. Promotion of read-through by MoMLV RNase H prevents nonsense-mediated mRNA decay (NMD) of mRNAs. Comparison of our structure with that of HIV RT explains why HIV RT cannot interact with eRF1. Our results provide a mechanistic view of how MoMLV manipulates the host translation termination machinery for the synthesis of its own proteins.

The Complex Relationship between HTLV-1 and Nonsense-Mediated mRNA Decay (NMD)

“…The strength of these countermeasures shows the size of the threat that NMD constitutes for viral replication and the place of NMD among the cellular antiviral function. Those conclusions are also supported by comparison with several other viruses, especially retroviruses and (+) RNA viruses, which RNAs are also targeted by NMD and that evolved protective means [33,[35][36][37][39][40][41][42][43]87,[125][126][127]. It is important to note that the viruses that develop a trans-inhibition process against NMD, such as HTLV-1 and potentially HIV, might only protect themselves progressively; before the viral inhibitor is expressed at an adequate level and while viral mRNA are at low level during the early phase of infection, the NMD would inhibit optimal viral replication as demonstrated with the mouse hepatitis virus (MHV, a prototypic member of the betacoronaviridae, as SARS-CoV and MERSCoV), the plant virus potato virus X (alfaflexiviridae) and turnip crinkle virus (Tombusviridae) [37,39].…”

Tissue-specific dynamic codon redefinition in Drosophila

“…A consideration with stop codons that are read through, especially when the stop codon is followed by a second long ORF, is nonsense-mediated decay (NMD). Indeed, avoidance of NMD has been documented in a few instances of readthrough in RNA viruses (18,57). Proximity of the stop codon to a 3' splice junction, and so to exon junction complex (EJC) proteins on the mRNA is relevant in mammals where any stop greater than 50 nt upstream of an EJC is marked for NMD (58).…”

“…Nearby 3' intra-mRNA structures have long been known to be important for readthrough of the Drosophila gene, headcase (15), as has a pseudoknot for the readthrough of the Murine Leukemia Virus UAG gag terminator to yield the GagPol precursor that is the source of reverse transcriptase (16,17). The effectiveness of the pseudoknot is aided by part of the readthrough product binding eRF1 and indirectly diminishing termination (18)).…”

Using Tet-Off Cells and RNAi Knockdown to Assay mRNA Decay

“… 4. The pcTET2 constructs used in this protocol were based on constructs originally introduced by Lykke-Andersen and Steitz [6] and were previously described [5]. We find these reporters to be useful as they provide a platform to study mRNA decay, translational readthrough, and mRNP composition in parallel.…”

“…The mRNAs expressing a full-length GFP-mCherry fusion protein are highly stable, while mRNAs containing a termination codon following the GFP ORF are degraded by NMD as a result of the extended mCherry-containing 3’-UTR [4,5]. The procedures described here are equally applicable to other commonly used NMD reporters, such as those derived from the β-globin gene [6].…”