MUTATIONAL ANALYSIS OF THE CENTRAL DOMAIN OF ADENOVIRUS VIRUS-ASSOCIATED RNA MANDATES A REVISION OF THE PROPOSED SECONDARY STRUCTURE

Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection. The rate of initiation is maintained by a sm all viral RNA, virus-associated (VA) RNA(I), which prevents the phosph orylation of eukaryotic initiation factor eIF-2 by a double-stranded R NA-activated protein kinase, DAI. On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA. The mo del 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 participat es in the binding of VA RNA to DAI. To assess the significance of the postulated stem-loop structure in the central domain, we generated com pensating, deletion, and substitution mutations. A substitution mutati on 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 ste m-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 up per 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 rearran gements in the molecule. Base-compensating mutations in the stem did n ot restore the expected base pairing, even though the mutant RNAs were still functional in vitro. Surprisingly, a mutant with a noncompensat ing substitution mutation in the stem was more effective than wild-typ e VA RNA(I) in DAI inhibition assays but was ineffective in vivo. The structural and functional consequences of these mutations do not suppo rt the proposed model structure for the central domain, and we therefo re 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 muta nt forms of VA RNA in vivo and in vitro are consistent with the existe nce of additional roles for VA RNA in the cell.