Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure

Pe'ery, T., Mellits, K. H., Mathews, M. B. (June 1993) Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure. J Virol, 67 (6). pp. 3534-43. ISSN 0022-538X (Print)

URL: http://www.ncbi.nlm.nih.gov/pubmed/8098780

Abstract

Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection. The rate of initiation is maintained by a small viral RNA, virus-associated (VA) RNAI, which prevents the phosphorylation of eukaryotic initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA. The model predicts a complex stem-loop structure in the central part of the molecule, the central domain, joining two duplexed stems. The central domain is required for the inhibition of DAI activation and participates in the binding of VA RNA to DAI. To assess the significance of the postulated stem-loop structure in the central domain, we generated compensating, deletion, and substitution mutations. A substitution mutation which disrupts the structure in the central domain abolishes VA RNA function in vitro and in vivo. Base-compensating mutations failed to restore the function or structure of the mutant, implying that the stem-loop structure may not exist. To confirm this observation, we tested mutants with alterations in the hypothetical loop and short stem that constitute the main features of the wild-type model structure. The upper part of the hypothetical loop could be deleted without abolishing the ability of the RNA to block DAI activation in vitro, whereas other loop mutations were deleterious for function and caused major rearrangements in the molecule. Base-compensating mutations in the stem did not restore the expected base pairing, even though the mutant RNAs were still functional in vitro. Surprisingly, a mutant with a noncompensating substitution mutation in the stem was more effective than wild-type VA RNAI in DAI inhibition assays but was ineffective in vivo. The structural and functional consequences of these mutations do not support the proposed model structure for the central domain, and we therefore suggest an alternative structure in which tertiary interactions may play a significant role in shaping the specificity of VA RNA function in the infected cell. Discrepancies between the functionality of mutant forms of VA RNA in vivo and in vitro are consistent with the existence of additional roles for VA RNA in the cell.

Item Type: Paper
Uncontrolled Keywords: Adenoviruses, Human/ genetics Base Composition Base Sequence Chloramphenicol O-Acetyltransferase/biosynthesis Comparative Study DNA Mutational Analysis Escherichia coli/genetics Molecular Sequence Data Mutagenesis, Site-Directed Nucleic Acid Conformation Protein Biosynthesis Protein Kinases/analysis RNA, Viral/ genetics Recombinant Fusion Proteins/biosynthesis Research Support, U.S. Gov't, P.H.S. Structure-Activity Relationship Transformation, Genetic eIF-2 Kinase
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA expression
organism description > virus > adeno associated virus
organism description > bacteria > escherichia coli
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date: June 1993
Date Deposited: 18 Apr 2016 16:39
Last Modified: 18 Apr 2016 16:39
PMCID: PMC237700
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32527

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