Decapitation: poxvirus makes RNA lose its head

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TRENDS in Biochemical Sciences

Vol.32 No.7

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Decapitation: poxvirus makes RNA lose its head Alexander G. McLennan School of Biological Sciences, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK

Cellular infection by vaccinia virus involves the controlled degradation of early, intermediate and late viral mRNAs, and increased turnover of host mRNAs. A new study has identified a key mediator of both these processes. A Nudix hydrolase encoded by the viral D10 gene decaps these mRNAs, thus targeting them for destruction independently of cellular systems. This finding has several implications for virus evolution and the regulation of RNA decapping.

Regulation of gene expression by vaccinia virus Vaccinia virus (VACV) is a large, enveloped virus of the Poxviridae family and has been extensively studied as a model for understanding poxvirus biology and disease, particularly smallpox. Viral replication takes place in factories in the cytoplasm of infected cells. VACV has a double-stranded DNA genome containing 200 genes, the expression of which is tightly programmed. The sequential expression of early, intermediate and late genes not only requires positive regulatory factors to switch these genes on at the appropriate times, but the rate at which these stage-specific mRNAs decline again has indicated an equally important negative regulation via mRNA decay [1,2]. Furthermore, the rate of cellular mRNA breakdown also increases after infection to help shut down competing host-cell protein synthesis to provide precursors for viral transcription and translation, and to prevent production of cellular proteins involved in promoting an immune response to the infection. Until recently, it was unclear how VACV achieved this down-regulation of gene expression. The VACV D10 gene was thought to be a key player because both viral and cellular mRNAs persist longer in cells infected with D10 deletion mutants compared with wild type, whereas overexpression of D10 causes greatly increased mRNA turnover and decreased protein synthesis [3,4]. Moreover, this regulation seems to involve the mRNA cap, the N7-methyl, 50 –50 -linked nucleotide structure found at the 50 ends of most eukaryotic mRNAs that is required for transcript stability and conventional initiation of translation; capindependent translation of a reporter construct from an internal ribosome entry site is unaffected [4]. Now, a probable molecular explanation for these findings has been provided by Parrish et al. [5] with the demonstration that D10 encodes a Nudix hydrolase that removes the 50 cap from both viral and cellular mRNAs in vitro. Cap hydrolysis is a crucial early step in some of the mRNA-decay pathways that regulate gene expression in eukaryotes Corresponding author: McLennan, A.G. ([email protected]). Available online 10 May 2007. www.sciencedirect.com

[6] and, therefore, D10 could stimulate premature entry into these decay pathways for the host cell mRNAs. Nudix hydrolases and decapping The Nudix hydrolase superfamily comprises mainly pyrophosphohydrolases that act upon compounds with a structure consisting of a nucleoside diphosphate linked to another moiety X (NDP-X) [7–9] (Box 1). Several mRNAdecapping Nudix hydrolases have recently been characterized in yeast and metazoa [10–14]. These enzymes generate 7-methyl-GDP (m7GDP) and 50 -phosphorylated RNA from capped mRNAs (Figure 1a); they have low, or no, activity on simple cap analogues and require the RNA moiety for full activity. The Nudix catalytic motif is contained within the Nudix domain, which is itself flanked by two other regions termed Box A and Box B (Figure 1b). Box B, and possibly other C-terminal regions of the human Dcp2 decapping enzyme, which functions in the normal host-cell mRNA-decay pathway (Figure 1a), seem to be required for RNA binding, although detail is lacking [10,12]. The role of Box A is also unclear, although it might ensure the specificity of pyrophosphate-bond cleavage within the cap [13]. Like these cellular enzymes, VACV D10 also generates m7GDP and requires at least a 12nucleotide RNA for cap cleavage. The catalytic motif is essential for this decapping activity because mutation of essential glutamates to glutamine abolishes activity. D10 has a high affinity for capped RNA (Km = 3.4 nM) and uncapped RNA inhibits decapping by 50% when in 100fold molar excess. Therefore, like its cellular counterparts, it seems to interact with RNA in addition to the cap [5]. If D10 does indeed promote the differential decay of early, intermediate and late viral mRNAs and/or host mRNAs, is this achieved? Cellular and early VACV mRNAs are known to have m7GpppAm and m7GpppGm caps, whereas intermediate and late viral transcripts have only m7GpppAm caps owing to the specificity of the initiation signal at the transcriptional start sites of intermediate and late genes [1]. As D10 is itself a late gene, preferential hydrolysis of mRNAs with m7GpppGm caps by D10 would be desirable. Consistent with this, m7GpppG was found to be 2–3 times more effective than m7GpppA at inhibiting decapping in vitro, indicating that D10 might have a preference for m7GpppGm-capped cellular and early viral transcripts [5]. This study has, therefore, introduced a potentially important quantitative differential to the previous observation that D10 affects the stability of both cellular and viral mRNAs. Two other possible contributors to transcript discrimination are also highlighted by Parrish et al. [5]. First are the unusual 50 poly(A) leader sequences found specifically

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Box 1. Nudix hydrolases – a brief overview The first Nudix hydrolase to be described was the Escherichia coli MutT protein, which, in vitro, hydrolyses mutagenic 8-oxo-dGTP to 8-oxo-dGMP and PPi with a low, submicromolar Km. Numerous other Nudix hydrolases have now been isolated, some of high substrate specificity and others with a broad substrate range [7–9]. Most degrade compounds with the structure NDP–X, usually yielding NMP and P–X, although the precise site of bond cleavage can vary. Substrates include (d)NTPs (canonical and modified), dinucleoside polyphosphates (e.g. Ap4A and Ap6A), nucleotide sugars (e.g. ADPribose and GDP-mannose), nucleotide coenzymes (e.g. NADH and CoA) and capped RNAs, in addition to (d)NDPs and some nonnucleotide metabolites such as inositol pyrophosphates, thiamine pyrophosphate and phosphoribosyl pyrophosphate. Catalysis depends on a conserved 23-residue sequence motif GX5EX7REUXEEXGU (where U is a bulky hydrophobic residue; PROSITE PS00893) within a larger Nudix domain or structural fold, comprising an a–b–a sandwich (Pfam entry PF00293). The metal-binding glutamate residues are central to the mechanism [9]. Among prokaryotes, the number of Nudix hydrolase genes varies more or less in line with genome size, from none in most mycoplasmas to 30 in streptomycetes [8]. Saccharomyces cerevisiae has six Nudix hydrolases, whereas the human genome encodes 24 [8]; these are found in various subcellular compartments and include the Dcp2 mRNAdecapping enzyme and NUDT16, which binds small nucleolar RNAs with high affinity and removes their hypermethylated m2,2,7GDP cap [21]. Nudix hydrolases function mainly to degrade potentially toxic nucleotide metabolites and to regulate nucleotide supplies; however, RNA decapping is a good example of an essential regulatory role for some family members. Nudix genes are found in all the virus families within the monophyletic assemblage known as the nucleo-cytoplasmic large DNA viruses (NCLDV), which includes poxviruses, asfarviruses, iridoviruses, phycodnaviruses and mimivirus [22], indicating a possible common origin and, conceivably, common function. The unrelated lepidopteran baculoviruses also have a single, Nudix hydrolase associated with budded virions [23]; this enzyme has ADP-ribose pyrophosphatase activity [24].

in intermediate and late VACV transcripts and that are believed to arise through RNA-polymerase slippage during initiation. These could bind poly(A)-binding protein, and

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possibly other proteins, to the exclusion of D10. A precedent for this might be the equally unusual trans-spliced leader sequences found at the 50 ends of 70% of Caenorhabditis elegans mRNAs that reduce the decapping activity of the nematode Dcp2 by tenfold [10]. Second is the apparent diffuse localization of D10 in the cytoplasm, rather than in the discrete viral factories where intermediate and late genes are transcribed and translated [3,5]. These are obviously avenues worthy of further investigation. Viral strategies VACV is only the second virus shown to use direct decapping as part of its strategy to promote viral transcript use in preference to host mRNAs. The Gag coat protein of the yeast double-stranded RNA L-A virus decaps cellular RNA so that its own uncapped mRNA is protected by simple competition from degradation by cellular exoribonucleases that target uncapped transcripts [15]. However, its mechanism is different from D10, with m7GMP being removed and covalently attached to a histidine residue on Gag itself, rather like the (albeit unrelated) cellular DcpS protein (Figure 1a). Other, distinct cap-related strategies have also been reported [6]. By contrast, a- and g-herpesviruses promote a global shut-off of cellular gene expression through combinations of different viral nucleases and modifications to host-cell systems [16]. This raises an interesting evolutionary question – why did VACV and related large DNA viruses adopt this particular means of regulating mRNA turnover? Does the proper stage-dependent control of mRNA stability during infection require some limited degree of interaction with host components that normally stimulate or inhibit Dcp2? Concluding remarks and future directions Regulation of decapping by the 50 !30 pathway in mammalian cells is an intricate process in which the

Figure 1. Mechanisms and structures of decapping enzymes. (a) Two main pathways for hydrolysis of cellular mRNA caps. In the 50 !30 mRNA-decay pathway, the 50 cap on 30 deadenylated mRNA is removed by a complex that includes the Nudix hydrolase Dcp2, and generates m7GDP; in the 30 !50 pathway, the unrelated DcpS (a histidine triad family protein) produces m7GMP. DcpS can also hydrolyse m7GDP [6,17,18]. (b) Alignment of cellular Dcp2 decapping enzymes with VACV D10 and D9 proteins and African Swine Fever g5R protein. Sequences are aligned around the conserved glycine at the start of the catalytic Nudix motif (arrowed; see Box 1). The Nudix domain (yellow) is shown as defined by the NCBI BLAST server. Conserved Box A (blue) and Box B regions (green) [12] are shown along with regions of low similarities (mottled) in the viral sequences. First and last residues and the start of the Nudix domain are numbered. Unlike the viral proteins, most cellular Dcp2 enzymes have N- or C-terminal extensions that could interact with other proteins. www.sciencedirect.com

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Dcp2 decapping enzyme interacts with many other proteins in a multiprotein complex within cytoplasmic processing bodies (P-bodies) [17,18]. So far, no interacting proteins for D10 have been described and so it is possible that it can initiate a more unregulated and indiscriminate destruction of cellular mRNA, at least in part, independently of the normal host machinery. It will be important now to identify the cellular and/or viral proteins responsible for the remaining aspects of viral- and host-mRNA degradation in infected cells. In addition, D10 might only be half the story, at least for chordopoxviruses such as VACV. This subfamily possesses a second Nudix hydrolase, D9, that shares 25% sequence identity with D10 (Figure 1b). D9 is encoded by an early gene and overexpression of D9 also decreases the level of capped mRNAs, although to a lesser extent than D10 [4]. Although both D9- and D10-deletion mutants are viable, a double mutant has not been isolated, indicating that they might show functional compensation. It is tempting to suggest that the target specificity of D9 is different to that of D10 within the pool of host mRNAs and early, intermediate and late viral transcripts, and this now requires investigation in the same way as has been done for D10. Verification of these roles for D10 and D9, particularly with regard to m7GpppXm discrimination, could include a quantitative microarray analysis of individual VACV transcripts during the course of infection using a combination of different deletion and overexpression viral constructs. The role of the RNA moiety of capped mRNA in promoting D10 activity and the residues involved in RNA binding also need to be identified. The Box B region present in cellular decapping enzymes is not so clearly conserved in D10, although it does have a similar number of basic amino acids that could contribute to RNA binding (Figure 1b). An examination of the properties of mutated and truncated forms of D10 should resolve this issue. Finally, a potentially novel mode of regulating decapping that could stem from this work and that could be of much wider importance depends on a possible functional relationship between VACV D10 and the single Nudix hydrolase (g5R) encoded by the distantly related asfarvirus, African Swine Fever Virus (Figure 1b and Box 1). When originally assayed, g5R had only low activity with nucleotide cap analogues, particularly methylated analogues, but instead degraded the inositol pyrophosphate diphosphoinositol pentakisphosphate with a Km of 1.2 mM [19]. However, we now know that all decapping Nudix hydrolases are inefficient with cap structures unless these are attached to an oligoribonucleotide representing at least part of the mRNA. Because g5R shows the strongest sequence similarity (64% and 44% in two regions within the Nudix domain) to the Schizosaccharomyces pombe Dcp2 decapping enzyme [13,19], it is still possible that it could decap intact mRNAs. Equally, VACV D10 might also recognize alternative substrates such as inositol pyrophosphates; these have not been tested as possible substrates for any of the known decapping Nudix hydrolases. If this proved to be the case, then inositol pyrophosphates might be novel regulators of both viral and cellular decapping in addition to their numerous other roles [20] and could conceivably participate in differential decapping. www.sciencedirect.com

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Given the substrate promiscuity of many Nudix hydrolases (Box 1), other untested nucleotides might also compete with capped RNA. This new insight into viral regulation of gene expression through decapping should be a launch pad for many new and exciting discoveries in both virology and enzymology. References 1 Baldick, C.J. and Moss, B. (1993) Characterization and temporal regulation of messenger RNAs encoded by vaccinia virus intermediate-stage genes. J. Virol. 67, 3515–3527 2 Broyles, S.S. (2003) Vaccinia virus transcription. J. Gen. Virol. 84, 2293–2303 3 Parrish, S. and Moss, B. (2006) Characterization of a vaccinia virus mutant with a deletion of the D10R gene encoding a putative negative regulator of gene expression. J. Virol. 80, 553–561 4 Shors, T. et al. (1999) Down regulation of gene expression by the vaccinia virus D10 protein. J. Virol. 73, 791–796 5 Parrish, S. et al. (2007) Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression. Proc. Natl. Acad. Sci. U. S. A. 104, 2139–2144 6 Cougot, N. et al. (2004) ‘Cap-tabolism’. Trends Biochem. Sci. 29, 436–444 7 Bessman, M.J. et al. (1996) The MutT proteins or ‘nudix’ hydrolases, a family of versatile, widely distributed, ‘housecleaning’ enzymes. J. Biol. Chem. 271, 25059–25062 8 McLennan, A.G. (2006) The Nudix hydrolase superfamily. Cell. Mol. Life Sci. 63, 123–143 9 Mildvan, A.S. et al. (2005) Structures and mechanisms of Nudix hydrolases. Arch. Biochem. Biophys. 433, 129–143 10 Cohen, L.S. et al. (2005) Dcp2 decaps m2,2,7 GpppN-capped RNAs, and its activity is sequence and context dependent. Mol. Cell. Biol. 25, 8779–8791 11 Dunckley, T. and Parker, R. (1999) The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J. 18, 5411–5422 12 Piccirillo, C. et al. (2003) Functional characterization of the mammalian mRNA decapping enzyme hDcp2. RNA 9, 1138–1147 13 She, M. et al. (2006) Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe. Nat. Struct. Mol. Biol. 13, 63–70 14 Wang, Z. et al. (2002) The hDcp2 protein is a mammalian mRNA decapping enzyme. Proc. Natl. Acad. Sci. U. S. A. 99, 12663–12668 15 Blanc, A. et al. (1994) His-154 is involved in the linkage of the Saccharomyces cerevisiae L-A double-stranded RNA virus Gag protein to the cap structure of mRNAs and is essential for M1 satellite virus expression. Mol. Cell. Biol. 14, 2664–2674 16 Glaunsinger, B.A. and Ganem, D.E. (2006) Messenger RNA turnover and its regulation in herpesviral infection. Adv. Virus Res. 66, 337–394 17 Bail, S. and Kiledjian, M. (2006) More than 1+2 in mRNA decapping. Nat. Struct. Mol. Biol. 13, 7–9 18 Simon, E. et al. (2006) New insights into the control of mRNA decapping. Trends Biochem. Sci. 31, 241–243 19 Cartwright, J.L. et al. (2002) The g5R (D250) gene of African swine fever virus encodes a nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates. J. Virol. 76, 1415–1421 20 Bennett, M. et al. (2006) Inositol pyrophosphates: metabolism and signaling. Cell. Mol. Life Sci. 63, 552–564 21 Ghosh, T. et al. (2004) Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme. Mol. Cell 13, 817–828 22 Iyer, L.M. et al. (2006) Evolutionary genomics of nucleo-cytoplasmic large DNA viruses. Virus Res. 117, 156–184 23 Wang, D. et al. (2005) Characterization of Helicoverpa armigera nucleopolyhedrovirus orf33 that encodes a novel budded virion derived protein, BV-e31. Arch. Virol. 150, 1505–1515 24 Ge, J. et al. (2007) AcMNPV ORF38 protein has the activity of ADP-ribose pyrophosphatase and is important for virus replication. Virology 361, 204–211

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