T. Peery et al., MUTATIONAL ANALYSIS OF THE CENTRAL DOMAIN OF ADENOVIRUS VIRUS-ASSOCIATED RNA MANDATES A REVISION OF THE PROPOSED SECONDARY STRUCTURE, Journal of virology, 67(6), 1993, pp. 3534-3543
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.