JOURNAL OF VIROLO(G, Nov. 1993, p. 6605-6617 Vol. 67, No. 11 0022-538X/93/1 16605- 13$02.00/0 Copyright © 1993, American Society for Microbiology Comparative Analysis of the Structure and Function of Adenovirus Virus-Associated RNAs YULIANG MA' 2 AND MICHAEL B. MATHEWSI* Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, New York 11724,' and Molecular Microbiology Program, State University of New York, Stony Brook, New York 117942 Received 10 June 1993/Accepted 7 August 1993 The protein kinase DAI is an important component of the interferon-induced cellular defense mechanism. In cells infected by adenovirus type 2 (Ad2), activation of the kinase is prevented by the synthesis of a small, highly ordered virus-associated (VA) RNA, VA RNA,. The inhibitory function of this RNA depends on its structure, which has been partially elucidated by a combination of mutagenesis and RNase sensitivity analysis. To gain further insight into the structure and function of this regulatory RNA, we have compared the primary sequences, secondary structures, and functions of seven VA RNA species from five human and animal adenoviruses. The sequences exhibit variable degrees of homology, with a particularly close relationship between the VA RNA,, species of Ad2 and Ad7 and notably divergent sequence for the avian (CELO) virus VA RNA. Apart from two pairs of mutually complementary tetranucleotides which are highly conserved, homologies are limited to transcription signals located within the RNA sequence and at its termini. Secondary structure analysis indicated that all seven RNAs conform to the model in which VA RNA possesses three main structural regions, a terminal stem, an apical stem-loop, and a central domain, although these elements vary in size and other details. The apical stem is implicated in binding to DAI, and the central domain is essential for inhibition of DAI activation. One of the pairs of conserved tetranucleotides (CCGG:C/UCGG) provides further evidence for the existence of the apical stem, but the other conserved pair (GGGU:ACCC) strongly suggests a revised structure for the central domain. In two functional assays conducted in vivo, the VA RNA, species of Ad2 and Ad7 were the most active, their corresponding VA RNA,, species displayed little activity, and the single VA RNAs of Adl2 and simian adenovirus type 7 exhibited intermediate activity. Correlation of the structural and functional data suggests that the VA RNA,, species adopt a structure different from those of the other VA RNA species and may play a different role in the life cycle of the virus. The virus-associated (VA) RNA, of adenovirus type 2 (Ad2) Ad2 VA RNA, is a short ('-160 nucleotides), unmodified, is a positive regulator of virus multiplication which acts at the nonpolyadenylated transcript which is synthesized by RNA translational level to neutralize an interferon-induced cellular polymerase III (Pol ITT) and accumulates to high levels in the defense mechanism (reviewed by Mathews and Shenk [51]). cytoplasm at late times of infection. Several lines of evidence The defense mechanism involves a protein kinase known as indicate that it is a highly ordered molecule and that its DAI, the double-stranded RNA (dsRNA)-activated inhibitor function is closely associated with its secondary and tertiary of protein synthesis, also referred to as PKR, P1, and p68. structure. Nuclease sensitivity analysis revealed three main Infection with a mutant virus, AdS d1331, defective in produc- structural features (Fig. IA): a terminal stem, an apical ing VA RNA,, leads to the phosphorylation by DAI of the stem-loop, and a central domain (19, 53). Mutagenic studies translational initiation factor eIF-2 on its oX subunit. This confirmed the existence of the apical stem-loop and suggested results in the entrapment of a second initiation factor (GEF or that it is required for efficient binding to DAI, while a correctly eIF-2B) and the consequent blockade of protein synthesis in structured central domain is necessary for inhibition of DAI the infected cell (60, 79). DAI appears to be activated by activation (52, 53, 55, 61). As a means to identify structural dsRNA produced by symmetrical transcription of the viral features and functional elements, these approaches suffer from genome at late times in virus infection (46). Synthesis of the some limitations, however; for example, some regions of the enzyme is induced by interferon, and many viruses have molecule are not susceptible to probing by nucleases, and of its developed the means to circumvent the consequences mutations may disrupt the overall structure of a compact RNA VA activation (reviewed by Sonenberg [74] and Mathews [49]). molecule rather than simply changing its sequence or altering binds to RNA, expressed by wild-type adenovirus DAI (20, 27, local base pairing. The limitations are well illustrated in the 35, 40) and prevents it from being activated (37, 60), thereby case of the central domain of Ad2 VA RNA,, which remains ensuring that viral polypeptide synthesis proceeds unimpeded. poorly understood. Thus, alternative secondary structures for VA RNA, may also contribute to the selective translation of the central domain have been proposed (19, 53, 61), and there to cellular the host shutoff viral mRNAs relative mRNAs, is disagreement as to the effects of mutations in the central occurs at late times of infection and phenomenon that (48, 59), domain on the ability of VA RNA to bind to DAI (11, 22, 52). of cotransfected in transient assays, it increases the expression The phylogenetic approach offers an alternative way to study plasmids (36, 76). These effects are both mediated, at least in the relationship between RNA structure and function and has part, by the interaction of VA RNA, with DAI (2). been successfully applied to complex RNA molecules such as 16S rRNA (29, 82) and RNase P RNA (8). This method relies on comparisons among a cohort of functionally and evolution- * Corresponding author. arily related molecules from different organisms to reveal 6605 6606 MA AND MATHEWS J. VIROL. A Apical Stem-loop U-A G-C140 G- C 4o 15G A 15G A 171j-A TU-A G- C G-C C-G C-G C-G C- G OU G Terminal Stem IoUC. G U- G U G C-G C-G U-A U-A C- Gso C- Gio A C A C C- GS C G; G- C G-C Ge U Ge U 5'PG- CCUUUn 5P G-CCUUUn FIG. 1. Secondary structure models for Ad2 VA RNAs. (A) The original model structure for Ad2 VA RNA, (53), showing the three principal regions of the molecule. (B) Revised Ad2 VA RNA, structure proposed on the basis of the work described here, especially the postulated GGGU:ACCC pairing (highlighted in stippled boxes). Cleavage sites for single-strand-specific nucleases and nuclease VI are marked with filled and open arrowheads, respectively. Large arrowheads, pronounced cleavages; small arrowheads, weak cleavages. conserved features of primary sequence and higher-order upstream of the gene for VA RNA,, and is separated from it structure, through, for example, compensating base changes. by a spacer of about 100 nucleotides (1, 50). A similar Nearly 50 human adenoviruses have been isolated and classi- organization seems to exist in Ad7, a representative of group B fied into six groups, A to F, on the basis of biological, of the human adenoviruses (15, 58), but the group A virus serological, and biochemical criteria (30). In addition, several Adl2 and simian adenovirus type 7 (SA7) apparently contain adenoviruses that infect animals are known. Although most only a single VA RNA gene at this locus (16, 42, 69). Chicken work has been done on the group C human viruses, Ad2 and adenovirus type I (also known as CELO virus) carries a gene AdS, VA RNAs have also been identified in some other encoding two shorter VA RNAs which are overlapping tran- adenoviruses. Moreover, in addition to the major species, VA scripts from a single gene (41). Unlike the mammalian genes, RNA,, Ad2 encodes a second species, VA RNA,J (1, 47, 50), however, the avian VA RNA gene is located at 90 map units which is transcribed at a slower rate (73), accumulates to a and is transcribed in the leftward direction. Nevertheless, lesser extent (47, 62), and seems to be less effective than VA CELO virus VA RNA appears to function like Ad2 VA RNAJ, RNA, in suppressing DAI activation (6). Both VA RNA genes albeit considerably less efficiently (41). Thus, VA RNA genes are located in the vicinity of map unit 30 on the Ad2 genome are carried by all the adenovirus serotypes examined, although and, like the major late transcription unit, are transcribed in they differ in number, sequence, location, and possibly func- the rightward direction (47). The gene for VA RNA, is tion. VC)L. 67, 19t93 VA RNA STRUCTURE-FUNCTION COMPARISONS 6607 VA RNA X VA RNA / 52,55K Ad2 %- b-~ -L. - - Yh am__ TaA B - Msc E Apa pTP VA RNA I VA RNA 1i 52,55K Ad7 iIm 6~~~~~~~~~~-~ 1I p- Fsp <TpIP E Hind E Pst 52,55K VA RNA [F:> Adl 2 B.gill <pTP B- 0- Nsc 52,55K VA RNA SA7 l~~~~~~~= Xbav B _aApa pTP VA RNAs CELO Fok -"S Kpn 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 800 900 1000 (nts) FIG. 2. VA RNA genes. Depicted are the VA RNA genes (solid arrows) of three human adenoviruses (Ad2, Ad7, and Ad12), a simian adenovirus (SA7), and an avian adenovirus (CELO virus). The individual genes were subcloned into pUCl 18 or pUCl 19 vector at the restriction sites denoted with standard three-letter abbreviations to give plasmids that could be transcribed with RNA Pol III.