Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus

Abstract

Translational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial.IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.

Translational Control in Virus-Infected Cells

“…A 7-nucleotide slippery site sequence, the spacer region, and structural barriers that stall the ribosome, impact PRF efficiency. Retroviruses generate high levels of Gag and low levels of a Gag-Pol fusion from the same transcript via PRF or by readthrough of a stop codon (Hung et al 1998;Csibra et al 2014). Perturbations in this ratio affect viral assembly, RNA packaging and maturation.…”

Functional and structural characterization of the chikungunya virus translational recoding signals

“…Ϫ1 PRF also presents a target for alphaviruses: in mice, ablation of Ϫ1 PRF attenuated the symptoms of Sindbis virus (20) and VEEV (21) infections. Although less well-studied than Ϫ1 PRF, five published studies report that alteration of TCR also has negative impacts on virus replication (15,(55)(56)(57)(58). Three of these studies (55)(56)(57) examined the impact of TCR ablation in a retrovirus (murine leukemia virus).…”

“…Although less well-studied than Ϫ1 PRF, five published studies report that alteration of TCR also has negative impacts on virus replication (15,(55)(56)(57)(58). Three of these studies (55)(56)(57) examined the impact of TCR ablation in a retrovirus (murine leukemia virus). Pertinent to this study, TCR ablation was shown to reduce the pathogenicity of two alphaviruses, O'nyong-nyong and Sindbis viruses (15,58).…”

Translational readthrough-promoting drugs enhance pseudoknot-mediated suppression of the stop codon at the Moloney murine leukemia virus gag–pol junction

“…An abnormally high production of Gag-Pol protein has been shown to have negative effects on assembly and maturation of infectious particles (Felsenstein & Goff, 1988;Shehu-Xhilaga et al, 2001). Manipulating the Gag : Gag-Pol ratio downward by mutation has been shown to be strongly deleterious to virus replication, while increasing the ratio by separate expression of these proteins, by silencing expression of the termination factors eRF1 or -3, or with aminoglycosides, was well tolerated in one study (Csibra et al, 2014).…”