Biomaterial Database

Studies of the astrovirus signal that induces (-1) ribosomal frameshifting. 1997

T L Lewis, and S M Matsui

Program in Cancer Biology, Stanford University School of Medicine, California 94305-5487, USA.

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.

UI	MeSH Term	Description	Entries
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D011233	Precipitin Tests	Serologic tests in which a positive reaction manifested by visible CHEMICAL PRECIPITATION occurs when a soluble ANTIGEN reacts with its precipitins, i.e., ANTIBODIES that can form a precipitate.	Precipitin Test,Test, Precipitin,Tests, Precipitin
D002474	Cell-Free System	A fractionated cell extract that maintains a biological function. A subcellular fraction isolated by ultracentrifugation or other separation techniques must first be isolated so that a process can be studied free from all of the complex side reactions that occur in a cell. The cell-free system is therefore widely used in cell biology. (From Alberts et al., Molecular Biology of the Cell, 2d ed, p166)	Cellfree System,Cell Free System,Cell-Free Systems,Cellfree Systems,System, Cell-Free,System, Cellfree,Systems, Cell-Free,Systems, Cellfree
D002478	Cells, Cultured	Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.	Cultured Cells,Cell, Cultured,Cultured Cell
D006224	Cricetinae	A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS.	Cricetus,Hamsters,Hamster
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001257	Mamastrovirus	A genus of small, circular RNA viruses in the family ASTROVIRIDAE. They cause GASTROENTERITIS and are found in the stools of several vertebrates including humans. Transmission is by the fecal-oral route and there are at least eight human serotypes. The type species is Human astrovirus.	Human astrovirus,Human astroviruses,Mamastroviruses,astroviruses, Human
D012270	Ribosomes	Multicomponent ribonucleoprotein structures found in the CYTOPLASM of all cells, and in MITOCHONDRIA, and PLASTIDS. They function in PROTEIN BIOSYNTHESIS via GENETIC TRANSLATION.	Ribosome
D012324	RNA-Dependent RNA Polymerase	An enzyme that catalyses RNA-template-directed extension of the 3'- end of an RNA strand by one nucleotide at a time, and can initiate a chain de novo. (Enzyme Nomenclature, 1992, p293)	Nucleoside-Triphosphate:RNA Nucleotidyltransferase (RNA-directed),RNA Replicase,RNA-Dependent RNA Replicase,RNA-Directed RNA Polymerase,RNA Dependent RNA Polymerase,RNA Dependent RNA Replicase,RNA Directed RNA Polymerase,RNA Polymerase, RNA-Dependent,RNA Polymerase, RNA-Directed,RNA Replicase, RNA-Dependent,Replicase, RNA,Replicase, RNA-Dependent RNA