Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain.     

The interaction between Z-DNA and the Zab domain of double-stranded RNA adenosine deaminase characterized using fusion nucleases.

Zab is a structurally defined protein domain that binds specifically to DNA in the Z conformation. It consists of amino acids 133-368 from the N terminus of human double-stranded RNA adenosine deaminase, which is implicated in RNA editing. Zab contains two motifs with related sequence, Zalpha and Zbeta. Zalpha alone is capable of binding Z-DNA with high affinity, whereas Zbeta alone has little DNA binding activity. Instead, Zbeta modulates Zalpha binding, resulting in increased sequence specificity for alternating (dCdG)n as compared with (dCdA/dTdG)n. This relative specificity has previously been demonstrated with short oligonucleotides. Here we demonstrate that Zab can also bind tightly to (dCdG)n stabilized in the Z form in supercoiled plasmids. Binding was assayed by monitoring cleavage of the plasmids using fusion nucleases, in which Z-DNA-binding peptides from the N terminus of double-stranded RNA adenosine deaminase are linked to the nuclease domain of FokI. A fusion nuclease containing Zalpha shows less sequence specificity, as well as less conformation specificity, than one containing Zab. Further, a construct in which Zbeta has been replaced in Zab with Zalpha, cleaves Z-DNA regions in supercoiled plasmids more efficiently than the wild type but with little sequence specificity. We conclude that in the Zab domain, both Zalpha and Zbeta contact DNA. Zalpha contributes contacts that produce conformation specificity but not sequence specificity. In contrast, Zbeta contributes weakly to binding affinity but discriminates between sequences of Z-DNAs.     

Interaction of adenosine deaminase with inhibitors. Chemical modification by diethyl pyrocarbonate

The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors— erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine—was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme–inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors—derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.     

Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4⁺ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1.     

3'-Beta-ethynyl and 2'-deoxy-3'-beta-ethynyl adenosines: first 3'-beta-branched-adenosines substrates of adenosine deaminase

The 3'-C-branched-adenosine and 2'-deoxyadenosine analogues 1-7 were tested as substrate of adenosine deaminase. The 9-(3'-C-ethynyl-beta-D-ribo-pentofuranosyl)adenine 1 and its 2'-deoxy analogue 7 were deaminated by the enzyme while the vinyl and ethyl derivatives 2 and 3 were not. The 9-(3'-C-branched-beta-D-xylo-pentofuranosyl)adenines 4-6 were deaminated by the deaminase.     

Double-stranded RNA adenosine deaminase as a potential mammalian RNA editing factor

Double-stranded RNA (dsRNA) adenosine deaminase, or DRADA, is a cellular enzyme that modifies adenosine residues to inosines in dsRNA by hydrolytic deamination, replacing A-U with mismatched I-U base pairs. Since it alters the base composition in its substrate RNA, one possible role played by DRADA is to participate in RNA editing. In this article, a brief review is given of characteristics of DRADA. Its possible involvement in RNA editing is also discussed in detail, including specific cases in which DRADA has been implicated as an RNA editing factor.     

Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.

Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.     

In vitro selection of packaging sites in a double-stranded RNA virus.

The Saccharomyces cerevisiae double-stranded RNA virus ScVL1 recognizes a small sequence in the viral plus strand for both packaging and replication. Viral particles will bind to this viral binding sequence (VBS) with high affinity in vitro. An in vitro selection procedure has been used to optimize binding, and the sequences isolated have been analyzed for packaging and replication in vivo. The selected sequence consists of a stem with a bulged A residue topped by a loop of several bases. Four residues of the 18 bases are absolutely conserved for tight binding. These all fall in regions that appear to be single stranded. Eight more residues have preferred identities, and six of these are in the stem. The VBS is similar to the R17 bacteriophage coat protein binding site. Packaging and replication require tight binding to viral particles.     

Tryptophan environment in adenosine deaminase. I. Enzyme modification with N-bromosuccinimide in the presence of adenosine and EHNA analogues

Adenosine deaminase from bovine cerebral hemisphere (white and gray matter) and spleen was treated with N-bromosuccinimide, a reagent known to oxidize selectively tryptophan residues in proteins. Spectrally observable tryptophan modification was accompanied by enzyme inactivation. Tsow graphics revealed that two Trps are essential for the activity of enzyme from both tissues. Enzyme inhibitors and substrate analogues, derivatives of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine, were able to protect Trp against modification, and this effect correlated in general with the enzyme activity protection. In the presence of adenosine deaza analogues (the noninhibitor tubercidin among them) only two Trps were modified in the fully inactivated enzyme. In the presence of EHNA and its deaza analogues, full inactivation of the enzyme was accompanied by the modification of four Trps. The obtained data confirm the previous hypothesis about the presence on the enzyme of different binding sites for adenosine and EHNA derivatives that are responsible for the different effects on the enzyme conformation elicited by the corresponding derivatives. Moreover, these data allow us to suggest that Trp residues, still unidentified by X-ray analysis, are essential for the functioning of the enzyme.     

Erythrocyte adenosine deaminase in Malaysians.

944 adenosine deaminase phenotypings of Malay, Chinese, and Indian blood donors and newborns at Kuala Lumpur, Malaysia, yielded ADA1 gene frequency estimates of 0.885 for the Malays, 0.939 for the Chinese, and 0.853 for the Indians.