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.
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DOI：
10.1128/jvi.00898-14
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发表时间：
2014-09
影响因子：
5.4
通讯作者：
Irigoyen N
中科院分区：
文献类型：
--
作者：
Csibra E;Brierley I;Irigoyen N
翻译通读抑制终止密码子的终止被利用在几种病毒的复制周期，并代表了抗病毒干预的潜在目标。在以莫洛尼鼠白血病病毒（MuLV）为代表的γ逆转录病毒中，gag和pol在相同的阅读框中，由UAG终止密码子分开，并且终止密码子通读是表达病毒Gag-Pol融合蛋白所必需的。在这里，我们研究了调节通读效率对MuLV复制的影响。我们首先在感染性病毒克隆的背景下操纵通读信号，以产生一系列MuLV变体，其中通读被刺激或减少。在仔细控制的感染性测定中，发现MuLV通读效率仅降低4倍会导致明显缺陷，而降低10倍基本上消除了复制。然而，高达8.5倍的通读刺激（高达60%的通读）被病毒很好地耐受。这些高水平的通读是使用双质粒系统实现的，其中Gag和Gag-Pol从单独的感染性克隆表达。我们还通过沉默真核释放因子1和3（eRF1和eRF3）的表达或通过将氨基糖苷类引入细胞来调节通读。获得的数据表明，γ逆转录病毒耐受大量过量的病毒Gag-Pol合成，但对这种多蛋白水平的降低非常敏感。因此，与核糖体移码的情况一样，靶向通读的抗病毒疗法与抑制剂可能是最有益的。重要性许多致病性RNA病毒和逆转录病毒使用核糖体移码或终止密码子通读来调节其复制酶的表达。这些翻译的"重编码"过程是抗病毒干预的潜在靶点，但我们对调节重编码效率对病毒复制的影响只有有限的了解，特别是对于那些采用通读的病毒。在本文中，我们描述了第一个系统的分析增加或减少通读效率对病毒复制的影响，使用γ逆转录病毒MuLV作为模型系统。我们意外地发现，MuLV复制仅受到通读频率大幅增加的轻微抑制，但与使用重编码策略的其他病毒一样，复制对即使是适度的减少也非常敏感。这些研究为通读过程和MuLV复制提供了见解，并对抗γ逆转录病毒的抗病毒药物的选择具有影响。
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.