Role of the apical stem in maintaining the structure and function of adenovirus virus-associated RNA

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To explore the structure and function of a small regulatory RNA, we examined the virus-associated (VA) RNA species of all 47 known human adenovirus serotypes and of one simian virus, SA7. The VA RNA gene regions of 43 human adenoviruses were amplified and sequenced, and the structures of 10 representative VA RNAs were probed by nuclease sensitivity analysis. Most human viruses have two VA RNA species, VA RNA I and VA RNA II , but nine viruses (19%) have a single VA RNA gene. Sequence alignments classified the RNAs into eight families, corresponding broadly to the known virus groups, and three superfamilies. One superfamily contains the single VA RNAs of groups A and F and the VA RNA I species of group C; the second contains the VA RNA I species of groups B1, D, and E and the unclassified viruses (adenovirus types 42 to 47), as well as the single VA RNAs of group B2; and the third contains all VA RNA II species. Fourteen regions of homology occur throughout the molecule. The longest of these correspond to transcription signals; most of the others participate in RNA secondary structure. The previously identified tetranucleotide pair, GGGU:ACCC, is nearly invariant, diverging slightly (to GGGU:ACCU) only in the two group F viruses and forming a stem in the central domain that is critical for VA RNA structure and function. Secondary structure models which accommodate the nuclease sensitivity data and sequence variations within each family were generated. The major structural features-the terminal stem, apical stem-loop, and central domain-are conserved in all VA RNAs, but differences exist in the apical stem and central domains, especially of the VA RNA II species. Sequence analysis suggests that an ancestral VA RNA gene underwent duplication during the evolution of viruses containing two VA RNA genes. Although the VA RNA II gene seems to have been lost or inactivated by secondary deletion events in some viruses, the high degree of homology among the VA RNA II species implies that this RNA may play an undiscovered role in virus survival. We speculate that the VA RNA genes originated from cellular sequences containing multiple tRNA genes.

mentioning

confidence: 97%

Structure, function, and evolution of adenovirus-associated RNA: a phylogenetic approach

Mathews

1996

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“…The VAI RNA binds to PKR and inactivates it. To study the structural requirements for the VAI RNA to bind to PKR, our group and also that of Mathews have mutagenized the RNA extensively and analyzed it in vivo (both in the context of viral chromosome and in transient assays) and also in vitro for its capacity to bind to and block the autophosphorylation of PKR (1,6,7,(19)(20)(21)24). Although there are some minor differences between the results of the two laboratories, in general these results indicate that the VAI RNA binding to PKR correlates with function and that the central domain and the proximal part of the apical stem are the critical parts of the molecule both for function and for binding.…”

Section: Discussionmentioning

confidence: 99%

“…Some of the patterns are faint in this figure, but they have been repeated to confirm the cleavages. We and others have used this approach previously to predict the folding of the mutant VAI RNAs (6,8,21,24). The results presented in Fig.…”

Section: Introduction Of Single-base Substitution Mutations In the Cementioning

confidence: 99%

“…VAI RNA binds to and irreversibly inactivates PKR and thereby protects and maintains the eIF-2 activity (4,8,12,14,22). The structural requirements of VAI RNA for its function have been investigated by introducing deletions and linker-scan and base-compensatory substitutions into the VAI RNA and analyzing such mutants in vivo in virus infections and transient assays and also in vitro for their capacities to bind to and block the autophosphorylation activity of PKR (1,4,5,7,8,(19)(20)(21)24). These studies showed that mutant molecules would retain function as long as the integrity of the central domain was maintained.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Effect of single-base substitutions in the central domain of virus-associated RNA I on its function

Rahman

Malhotra

Dhar

et al. 1995

Adenoviruses use virus-associated RNA I (VAI RNA) to counteract the cellular antiviral response mediated by the interferon-induced, double-stranded-RNA-activated protein kinase PKR. VAI RNA is a highly structured small RNA which consists of two long duplex regions connected at the center by a complex, short stem-loop. This short stem-loop and the adjacent base-paired regions, referred to as the central domain, bind to PKR and inactivate it. Currently it is not known whether binding of VAI RNA to PKR is dependent solely on the secondary (and tertiary) structure of the central domain or whether nucleotide sequences in the central domain are also critical for this interaction. To address this question, 54 VAI mutants with single-base substitution mutations in the central domain of the RNA were constructed, and their capacities to inhibit the autophosphoryation of PKR in vitro were determined. It was found that although about half of the mutants inhibited PKR activity as efficiently as the wild type, a significant number of mutants lost the inhibitory activity substantially, without a perceptible change in their secondary structures. These results indicate that, in addition to secondary structure, at least some nucleotides in the central domain may be critical for the efficient function of VAI RNA.

“…Several observations support this argument. First, several deletion mutants with large truncations of the apical stem-loop retain full biologic activity as long as the central short stem-loop is preserved in these mutants (4,12,38,39). Second, mutants with WT apical stem-loop or extended apical stem-loop but without the central short stemloop (sub743, sub745, sub746, sub747, and sub748) cannot function in vivo or in vitro.…”

Section: Vw -Fmentioning

confidence: 99%

In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain

Ghadge

Malhotra

Furtado

et al. 1994

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants