Analysis of Moloney murine leukemia virus revertants mutated at the gag-pol junction

Odawara, Takashi; Yasuda, Hiroshi; Ohshima, Masamichi; Танака, Тадатсугу; Jones, David S.; Nemoto, Fumiko; Kuchino, Yoshiyuki; Iwamoto, Aikichi

doi:10.1128/jvi.65.11.6376-6379.1991

Cited by 9 publications

(14 citation statements)

References 25 publications

(23 reference statements)

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“…Two (UAA and SP1) led to a decrease of 1.7-to 3.1-fold, three (SP2, L3.1, and L3.2) led to decreases between 4-and 10-fold, and the stem destabilizations (S1d and S2d) were greatly inhibitory (27-to 65-fold down in both cell types). Changing the stop codon to UGA had essentially no effect on readthrough (as seen before) (7,37,38), and the stem 1 stabilizing mutation (S1GC) indeed stimulated readthrough (an up mutant with about a 2-fold increase; 13 to 16% depending on cell type), consistent with a role for pseudoknot stem 1 stability in recoding (39,40).…”

Section: Modulation Of Mulv Readthrough Efficiency Through Site-specisupporting

confidence: 62%

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

Csibra

Brierley

Irigoyen

2014

J Virol

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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.

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Section: Modulation Of Mulv Readthrough Efficiency Through Site-specisupporting

confidence: 62%

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

Csibra

Brierley

Irigoyen

2014

J Virol

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“…We tested luciferase constructs in which the WT UAG terminator was replaced by UGA and UAA stop codons. Viruses carrying these alternative stop codons are known to express Gag-Pol, with the insertion of distinct amino acids in the readthrough protein (Feng et al, 1989b(Feng et al, , 1990, and are at least partially replication competent (Jones et al, 1989;Odawara et al, 1991). G418 and paromomycin both increased readthrough with both alternative stop codons (Fig.…”

Section: Aminoglycosides Increase Momlv Readthrough In Vitromentioning

confidence: 99%

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

Green

Goff²

2015

Journal of General Virology

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Translational readthrough-promoting drugs enhance the incorporation of amino acids at stop codons and can thus bypass premature termination during protein synthesis. The polymerase (Pol) proteins of Moloney murine leukemia virus (MoMLV) are synthesized as a large Gag-Pol fusion protein, formed by the readthrough of a stop codon at the end of the gag ORF. The downstream pol ORF lacks its own start codon, and Pol protein synthesis is wholly dependent on translation of the upstream gag gene and the readthrough event for expression. Here, we explored the effects of readthrough-promoting drugs -aminoglycoside antibiotics and the small molecule ataluren -on the efficiency of readthrough of the stop codon in the context of the MoMLV genome. We showed that these compounds increased readthrough of the stop codon at the MoMLV gag-pol junction in vivo above the already high basal level and that the resulting elevated gag-pol readthrough had deleterious effects on virus replication. We also showed that readthrough efficiency could be driven to even higher levels in vitro, and that the combination of the small molecules and the RNA structure at the MoMLV stop codon could achieve extremely high readthrough efficiencies.

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“…Constructs pGE 6.4 -hmB and p⌬wt-hmB have been described previously (24). pMLV-B(CAG)-neo was constructed by ligating the Neo r gene to the previously described pArMLV-B(CAG) (21). pGEBstE-hmB was constructed by digestion at the BstEII site (nucleotide [nt] 5923 according to the numbering scheme of Shinnick et al [28]) of pGE 6.4 -hmB, and the gap was filled with 5 bases (TA-ACC) to stop the premature translation of env.…”

Section: Methodsmentioning

confidence: 99%

“…When the UAG codon at the gag-pol junction was changed to a glutamine codon, CAG, the mutant virus, MLV-B(CAG), produced only the Gag-Pol fusion protein from the unspliced viral RNA and became replication incompetent. The mutant could replicate if it was supplied in trans with the Gag precursor, Pr65 gag , which served as the substrate for the protease present in the Gag-Pol fusion protein (8,21).…”

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confidence: 99%

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Threshold Number of Provirus Copies Required per Cell for Efficient Virus Production and Interference in Moloney Murine Leukemia Virus-Infected NIH 3T3 Cells

Odawara

Oshima

Doi

et al. 1998

J Virol

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The gag-pol readthrough mutant of Moloney murine leukemia virus, MLV-B(CAG) (T. Odawara, H. Yoshikura, M. Oshima, T. Tanaka, D. S. Jones, F. Nemoto, Y. Kuchino, and A. Iwamoto, J. Virol. 65:6376–6379, 1991), was poorly complemented by a mutant encoding only Gag. This is because with all the genetic elements necessary for env expression present in MLV-B(CAG), insufficient Env protein was produced by the cells expressing MLV-B(CAG) for efficient virus production. Since the envmRNA expression per provirus in the MLV-B(CAG)- and wild-type-MLV-producing cells were the same and since the cells expressing the former contained eightfold fewer proviral copies, the insufficient Env expression by the former was found to be due to insufficient proviral copies in the cells. Examination of the cell clones having various proviral copies of Δwt MLV (M. Oshima, T. Odawara, T. Matano, H. Sakahira, Y. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286–2295, 1996) showed that mRNA level was proportional to the number of proviral copies while interference and virus production followed a sigmoid curve with a sharp rise at the threshold number of proviral copies of around four per cell. Multicycle infection probably continues until the threshold level of proviral copies is attained in natural infection too.

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Analysis of Moloney murine leukemia virus revertants mutated at the gag-pol junction

Cited by 9 publications

References 25 publications

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

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

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

Threshold Number of Provirus Copies Required per Cell for Efficient Virus Production and Interference in Moloney Murine Leukemia Virus-Infected NIH 3T3 Cells

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