Lewis T L, Matsui S M
Program in Cancer Biology, Stanford University School of Medicine, California 94305-5487, USA.
Adv Exp Med Biol. 1997;412:323-30. doi: 10.1007/978-1-4899-1828-4_53.
Human astroviruses use a (-1) ribosomal frameshift mechanism to regulate expression of the viral RNA-dependent RNA polymerase gene. To evaluate the efficiency of the astrovirus frameshift signal in cell culture, different regions of the frameshift signal were cloned into the rhesus rotavirus VP4 gene and expressed in an infection-transfection transient expression cell-culture system. The various astrovirus-VP4 constructs were transfected into BHK-21 cells infected with a recombinant vaccinia virus that expresses T7 RNA polymerase (vTF7-3). All constructs contain a T7 promoter, a picornavirus internal ribosome entry site, and a T7 terminator. Frameshifted and non-frameshifted proteins were distinguished by immunoprecipitation with monoclonal antibodies specific for either the VP4 amino- or carboxy-terminus. Frameshifting efficiency was calculated as the ratio of radioactive counts in the frameshifted protein to the total counts in both the frameshifted and nonframeshifted proteins as determined by Phosphorimager analysis. We found the efficiency of astrovirus frameshifting in intact cells to be 25-28%, significantly greater than the 5-7% efficiency reported previously in a cell-free uncoupled translation system. Since the transfected plasmid is expressed in the cytoplasm in the infection-transfection system, the frameshifting efficiency determined by this assay may be a more accurate reflection of the level of frameshifting for this RNA virus in which transcription and translation are likely coupled in the cytoplasm of infected cells. This hypothesis is supported by the observation that the level of astrovirus frameshifting is increased three-fold when evaluated in a cell-free coupled transcription-translation system. These studies also confirm in intact cells what was previously determined in cell-free studies: the shifty heptamer is an absolute requirement for astrovirus ribosomal frameshifting, but deletion of sequences downstream of the stem-loop that are potentially involved in pseudoknot formation does not affect the efficiency of frameshifting.
人星状病毒利用（-1）核糖体移码机制来调控病毒RNA依赖性RNA聚合酶基因的表达。为了评估星状病毒移码信号在细胞培养中的效率，将移码信号的不同区域克隆到恒河猴轮状病毒VP4基因中，并在感染-转染瞬时表达细胞培养系统中进行表达。将各种星状病毒-VP4构建体转染到感染了表达T7 RNA聚合酶的重组痘苗病毒（vTF7-3）的BHK-21细胞中。所有构建体均包含一个T7启动子、一个微小RNA病毒内部核糖体进入位点和一个T7终止子。通过用针对VP4氨基末端或羧基末端的单克隆抗体进行免疫沉淀来区分移码和未移码的蛋白质。移码效率通过磷光成像分析确定的移码蛋白中的放射性计数与移码和未移码蛋白中的总计数之比来计算。我们发现完整细胞中星状病毒移码的效率为25%-28%，显著高于先前在无细胞非偶联翻译系统中报道的5%-7%的效率。由于转染的质粒在感染-转染系统的细胞质中表达，通过该测定法确定的移码效率可能更准确地反映了这种RNA病毒的移码水平，在这种病毒中，转录和翻译可能在受感染细胞的细胞质中偶联。这一假设得到了以下观察结果的支持：在无细胞偶联转录-翻译系统中评估时，星状病毒移码水平增加了三倍。这些研究还在完整细胞中证实了先前在无细胞研究中确定的结果：易变七聚体是星状病毒核糖体移码的绝对必要条件，但删除茎环下游可能参与假结形成的序列不会影响移码效率。
