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• W2022742007 abstract "The 144-kDa λ2 protein of mammalian reovirus catalyzes a number of enzymatic activities in the capping of reovirus mRNA, including the transfer of GMP from GTP to the 5′ end of the 5′-diphosphorylated nascent transcript. This reaction proceeds through a covalently autoguanylylated λ2-GMP intermediate. The smaller size of RNA capping guanylyltransferases from other organisms suggested that the λ2-associated guanylyltransferase would be only a part of this protein. Limited proteinase K digestion of baculovirus-expressed λ2 was used to generate an amino-terminal M r42,000 fragment that appears to be both necessary and sufficient for guanylyltransferase activity. Although lysine 226 was identified by previous biochemical studies as the active-site residue that forms a phosphoamide bond with GMP in autoguanylylated λ2, mutation of lysine 226 to alanine caused only a partial reduction in guanylyltransferase activity at the autoguanylylation step. Alanine substitution for other lysines within the amino-terminal region of λ2 identified lysine 190 as necessary for autoguanylylation and lysine 171 as an important contributor to autoguanylylation. A novel active-site motif is proposed for the RNA guanylyltransferases of mammalian reoviruses and otherReoviridae members. The 144-kDa λ2 protein of mammalian reovirus catalyzes a number of enzymatic activities in the capping of reovirus mRNA, including the transfer of GMP from GTP to the 5′ end of the 5′-diphosphorylated nascent transcript. This reaction proceeds through a covalently autoguanylylated λ2-GMP intermediate. The smaller size of RNA capping guanylyltransferases from other organisms suggested that the λ2-associated guanylyltransferase would be only a part of this protein. Limited proteinase K digestion of baculovirus-expressed λ2 was used to generate an amino-terminal M r42,000 fragment that appears to be both necessary and sufficient for guanylyltransferase activity. Although lysine 226 was identified by previous biochemical studies as the active-site residue that forms a phosphoamide bond with GMP in autoguanylylated λ2, mutation of lysine 226 to alanine caused only a partial reduction in guanylyltransferase activity at the autoguanylylation step. Alanine substitution for other lysines within the amino-terminal region of λ2 identified lysine 190 as necessary for autoguanylylation and lysine 171 as an important contributor to autoguanylylation. A novel active-site motif is proposed for the RNA guanylyltransferases of mammalian reoviruses and otherReoviridae members. type 3 Dearing polyacrylamide gel electrophoresis dithiothreitol recombinant λ2 Mammalian reovirus, a multisegmented double-stranded RNA virus in the family Reoviridae, replicates in the cytoplasm of the eukaryotic host cell. The reovirus core particle can producem7NGpppGm2′OpC(pN)n-OH (cap 1) plus-strand RNA from each genomic double-stranded RNA segment in vitro (1.Furuichi Y. Muthukrishnan S. Tomasz J. Shatkin A.J. J. Biol. Chem. 1976; 251: 5043-5053Abstract Full Text PDF PubMed Google Scholar), indicating that it contains all of the enzymes necessary for de novo synthesis of capped mRNA. The RNA polymerase itself is likely to be the λ3 core protein (2.Bruenn J.A. Nucleic Acids Res. 1991; 18: 217-226Crossref Scopus (169) Google Scholar, 3.Starnes M.C. Joklik W.K. Virology. 1993; 193: 356-366Crossref PubMed Scopus (75) Google Scholar). Genetic and/or biochemical analyses indicate that the λ1 and μ2 core proteins have nucleoside triphosphate phosphohydrolase activity, possibly associated with an RNA helicase (4.Noble S. Nibert M.L. J. Virol. 1997; 71: 2182-2191Crossref PubMed Google Scholar, 5.Noble S. Nibert M.L. J. Virol. 1997; 71: 7728-7735Crossref PubMed Google Scholar, 6.Bisaillon M. Bergeron J. Lemay G. J. Biol. Chem. 1997; 272: 18298-18303Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The γ phosphate of the newly transcribed mRNA is thought to be removed by the RNA triphosphate phosphohydrolase activity of λ1 (7.Bisaillon M. Lemay G. J. Biol. Chem. 1997; 272: 29954-29957Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The λ2 core protein is the reovirus RNA guanylyltransferase, which adds a GMP moiety via a 5′–5′ linkage to the 5′-diphosphorylated mRNA (8.Cleveland D.R. Zarbl H. Millward S. J. Virol. 1986; 60: 307-311Crossref PubMed Google Scholar). This transfer reaction occurs through a covalent intermediate, a phosphoamide bond between the GMP of the donor GTP and a lysine of λ2 (9.Shatkin A.J. Furuichi Y. LaFiandra A.J. Yamakawa M. Compans R.W. Bishop D.J.L. Double-stranded RNA Viruses. Elsevier, New York1983: 43-54Google Scholar, 10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). Generation of this covalent bond (called “autoguanylylation” in this paper) is followed by GMP transfer from the enzyme to an acceptor, usually the 5′-diphosphorylated mRNA, although the GMP can be alternatively transferred to a 5′-triphosphorylated RNA or a di- or triphosphorylated nucleoside (11.Mao Z. Joklik W.K. Virology. 1991; 185: 377-386Crossref PubMed Scopus (67) Google Scholar). The resulting product is then sequentially methylated by RNA nucleoside-7-N- and 2′-O-methyltransferases, yielding the cap 1 mRNA (1.Furuichi Y. Muthukrishnan S. Tomasz J. Shatkin A.J. J. Biol. Chem. 1976; 251: 5043-5053Abstract Full Text PDF PubMed Google Scholar) that is released through the channel formed by the λ2 pentameric spike (12.Barlett N.M. Gillies S.C. Bullivant S. Bellamy A.R. J. Virol. 1974; 167: 315-326Crossref Google Scholar, 13.Yeager M. Weiner S. Coombs K.M. Biophys. J. 1996; 70 (abstr.): 116Google Scholar). Both of the methyltransferase activities appear to reside in λ2 as indicated by the finding that only the λ2 protein in cores is covalently labeled with the methyl donor S-adenosyl-l-methionine after incubation and UV cross-linking (14.Luongo C.L. Contreras C.M. Farsetta D.L. Nibert M.L. J. Biol. Chem. 1998; 273: 23773-23780Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Thus, λ2 is thought to catalyze the last three of the four reactions required for cap 1 formation on reovirus mRNA.The 144-kDa λ2 protein (15.Seliger L.S. Zheng K. Shatkin A.J. J. Biol. Chem. 1987; 262: 16289-16293Abstract Full Text PDF PubMed Google Scholar), encoded by the reovirus L2 gene, appears to contain multiple domains. The proposed guanylyltransferase active site (lysine 226) is near the amino terminus (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). There is anS-adenosyl-l-methionine-binding site that appears to span residues 827 and 829 (14.Luongo C.L. Contreras C.M. Farsetta D.L. Nibert M.L. J. Biol. Chem. 1998; 273: 23773-23780Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 16.Koonin E.V. J. Gen. Virol. 1993; 74: 733-740Crossref PubMed Scopus (200) Google Scholar). A carboxyl-terminalM r 25,000 region is expendable for capping functions but is implicated in anchoring the reovirus cell attachment protein ς1 in virions (17.Dryden K.A. Wang G. Yeager M. Nibert M.L. Coombs K.M. Furlong D.B. Fields B.N. Baker T.S. J. Cell Biol. 1993; 122: 1023-1041Crossref PubMed Scopus (289) Google Scholar, 18.Luongo C.L. Dryden K.A. Farsetta D.L. Margraf R.M. Severson T.F. Olson N.H. Fields B.N. Baker T.S. Nibert M.L. J. Virol. 1997; 71: 8035-8040Crossref PubMed Google Scholar). A multidomain structure for the λ2 protein is also consistent with what is known for other capping enzymes. The vaccinia virus capping enzyme that catalyzes the first three reactions required for cap 1 formation is a heterodimer composed of two subunits encoded by separate genes (19.Martin S.A. Paoletti E. Moss B. J. Biol. Chem. 1975; 250: 9322-9329Abstract Full Text PDF PubMed Google Scholar, 20.Morgan J.R. Cohen L.K. Roberts B.E. J. Virol. 1984; 52: 206-214Crossref PubMed Google Scholar, 21.Niles E.G. Lee-Chen G.J. Shuman S. Moss B. Broyles S.S. Virology. 1989; 172: 513-522Crossref PubMed Scopus (62) Google Scholar). By biochemical analysis of proteolytic products, the capping enzyme is separable into a region with RNA triphosphate phosphohydrolase and guanylyltransferase activity and a region with RNA nucleoside-7-N-methyltransferase activity (22.Higman M.A. Bourgeois N. Niles E.G. J. Biol. Chem. 1992; 267: 16430-16437Abstract Full Text PDF PubMed Google Scholar, 23.Shuman S. J. Biol. Chem. 1989; 264: 9690-9695Abstract Full Text PDF PubMed Google Scholar). TheSaccharomyces cerevisiae capping enzyme is a complex of two separate gene products (24.Shibagaki Y. Itoh N. Yamada H. Shibekazu N. Mizumoto K. J. Biol. Chem. 1992; 267: 9521-9528Abstract Full Text PDF PubMed Google Scholar), one having RNA triphosphate phosphohydrolase and the other having RNA guanylyltransferase activity (25.Itoh N. Yamada H. Kaziro Y. Mizumoto K. J. Biol. Chem. 1987; 262: 1989-1995Abstract Full Text PDF PubMed Google Scholar). These two examples suggest that capping enzymes can be multifunctional and that the guanylyltransferase region may be separated biochemically (vaccinia virus) or genetically (yeast) from the rest of the protein. In the case of the Chlorella virus PBCV-1, the RNA guanylyltransferase is a 330-amino acid monofunctional enzyme (26.Ho C.K. van Etten J.L. Shuman S. J. Virol. 1996; 70: 6658-6664Crossref PubMed Google Scholar). The small size of this guanylyltransferase and the guanylyltransferase regions of the other capping enzymes suggests that only a portion of the 144-kDa λ2 protein is likely to be required for its RNA guanylyltransferase activity.For most RNA guanylyltransferases, a KXDG active-site motif has been proposed based on sequence comparisons (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar). For the RNA guanylyltransferases of vaccinia virus, S. cerevisiae, and baculovirus, the identity of the active site has been confirmed by site-directed mutagenesis (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 30.Schwer B. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4328-4332Crossref PubMed Scopus (61) Google Scholar, 31.Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar). For the PBCV-1 RNA guanylyltransferase, crystallographic analysis indicates that the active-site lysine interacts with the substrate GTP and that the other residues of the consensus motif interact with the RNA or nucleotide acceptor (32.Håkansson K. Doherty A.J. Shuman S. Wigley D.B. Cell. 1997; 89: 545-553Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 33.Håkansson K. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1505-1510Crossref PubMed Scopus (56) Google Scholar). The RNA guanylyltransferases, as well as RNA and DNA ligases, are members of the RNA/DNA nucleotidyltransferase superfamily (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar), enzymes that mediate nucleotidyl transfer to RNA or DNA via a covalent intermediate. The active-site motif for this entire superfamily is KX(D/N)G (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar). The RNA guanylyltransferases of members of the family Reoviridaelack sequences that precisely match this consensus motif (15.Seliger L.S. Zheng K. Shatkin A.J. J. Biol. Chem. 1987; 262: 16289-16293Abstract Full Text PDF PubMed Google Scholar, 34.Bisaillon M. Lemay G. Virology. 1997; 236: 1-7Crossref PubMed Scopus (55) Google Scholar). For example, the sequence of the proposed active site in the reovirus λ2 protein is 226KPTNG. This sequence is similar to the nucleotidyl transferase superfamily motif, but the alignment is disrupted by insertion of a proline residue after the lysine in λ2. The proposed guanylyltransferase active site for the other familyReoviridae members rotavirus (KPTGN) and bluetongue virus (KLTGN) is based on the proposed reovirus active site (34.Bisaillon M. Lemay G. Virology. 1997; 236: 1-7Crossref PubMed Scopus (55) Google Scholar).A previous effort to identify the λ2 residue that forms the phosphoamide bond with GMP used a combination of proteolysis and chemical cleavage of reovirus virions autoguanylylated with [32P]GMP to show that a lysine residue in λ2 is the site of covalent linkage to GMP (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). Immunoblot analysis of proteolyzed, [32P]GMP-labeled reovirus particles with λ2 peptide-specific antibodies indirectly identified lysine 226 as the residue to which GMP is likely attached. A similar approach involving proteolysis and chemical cleavage identified the active-site lysine of the vaccinia virus guanylyltransferase (35.Niles E.G. Christen L. J. Biol. Chem. 1993; 268: 24986-24989Abstract Full Text PDF PubMed Google Scholar), and the identity of the vaccinia virus active-site lysine was subsequently confirmed by the analysis of site-directed mutants for autoguanylylation activity (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar). To extend characterization of the reovirus RNA guanylyltransferase, we expressed recombinant λ2 and analyzed its properties. Utilizing a biochemical approach, we localized a region necessary and sufficient for guanylyltransferase activity to an amino-terminal M r 42,000 fragment of λ2. Since this region contains lysine 226, we generated mutant K226A by alanine substitution. Analysis of the mutant indicated that lysine 226 is not necessary for λ2 guanylyltransferase activity. Alanine substitution for other lysines in the M r 42,000 region identified lysine 190 as the probable site of covalent GMP linkage and lysine 171 as important for autoguanylylation activity. Based on these findings, we propose a novel active-site motif for the RNA guanylyltransferases of mammalian reoviruses and otherReoviridae family members.DISCUSSIONOur rλ2 protein expressed in insect cells from a baculovirus vector has guanylyltransferase activity using either GDP or GTP as GMP acceptor. In contrast to published observations with rλ2 expressed in mammalian cells from a vaccinia virus vector (11.Mao Z. Joklik W.K. Virology. 1991; 185: 377-386Crossref PubMed Scopus (67) Google Scholar), however, we were unsuccessful at demonstrating guanylyltransferase activity with baculovirus-expressed rλ2 using either 5′-diphosphorylated reovirus RNA or poly(A) RNA as acceptor (data not shown). Given this limitation in the activity of the baculovirus-expressed protein, which might be explained by a defect in allowing RNA molecules into the acceptor region of the enzyme, the conclusions we reach about amino acids required for GMP transfer must be considered as tentative with regard to RNA acceptors.Unlike core-associated λ2, rλ2 is hypersensitive to cleavage into complementary M r 42,000 and 100,000 fragments that dissociate in solution. Retention of both autoguanylylation and GMP transfer activities by the M r 42,000 fragment suggested that it is both necessary and sufficient to act as a guanylyltransferase. The M r 42,000 region contains the active site originally localized by direct biochemical analysis to the region between amino acids 131 and 266 (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). Further indirect biochemical analysis in the previous study identified lysine 226 as the active-site residue that forms a phosphoamide bond with GMP (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar).The previously identified active-site motif 226KPTNG in reovirus λ2 is anomalous for two reasons. First it is not conserved outside the family Reoviridae. Of the known nucleotidyl transferases, at least 13 RNA guanylyltransferases, 16 DNA ligases, and one RNA ligase have the motif KXDG (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar, 31.Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar). The two DNA ligases of African swine fever virus and the S. cerevisiaetRNA ligase have the motif KXNG (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar). The second reason is that insertion of additional residues may interfere with noncovalent bonding of one of the substrates. Based on the structure of the PBCV-1 guanylyltransferase (32.Håkansson K. Doherty A.J. Shuman S. Wigley D.B. Cell. 1997; 89: 545-553Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 33.Håkansson K. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1505-1510Crossref PubMed Scopus (56) Google Scholar) and the ATP-dependent DNA ligase of bacteriophage T7 (44.Subramanya H.S. Doherty A.J. Ashford S.R. Wigley D.B. Cell. 1996; 85: 607-615Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), the lysine in the active-site motif covalently binds GMP or AMP, respectively, and the remaining residues interact with the nucleotide acceptor in RNA for PBCV-1 or the ATP for T7 DNA ligase.As was done for the vaccinia virus RNA guanylyltransferase (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar), we chose to confirm the identity of the active-site lysine in reovirus λ2 by site-directed mutagenesis. Since alanine substitution has been used to identify the active-site lysine of the RNA guanylyltransferase of S. cerevisiae (30.Schwer B. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4328-4332Crossref PubMed Scopus (61) Google Scholar) and baculovirus (31.Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar), we generated the λ2 mutant K226A. Based on the demonstrable guanylyltransferase activity of this mutant, we concluded that lysine 226 is not the active-site residue. This conclusion has ramifications for the guanylyltransferases of the other members of the double-stranded RNA virus family Reoviridae and brings into question the functional significance of the similar motifs recently identified in the rotavirus and bluetongue virus RNA guanylyltransferases (34.Bisaillon M. Lemay G. Virology. 1997; 236: 1-7Crossref PubMed Scopus (55) Google Scholar).In addition to lysine 226, the M r 42,000 region contains seven lysines at positions 44, 89, 94, 171, 190, 197, and 372 (15.Seliger L.S. Zheng K. Shatkin A.J. J. Biol. Chem. 1987; 262: 16289-16293Abstract Full Text PDF PubMed Google Scholar). A series of six alanine substitution mutants were generated to identify the active-site residue. Lysines 44, 89, and 94 are not necessary for activity, based on mutants K44A, K89A, and K94A having approximately wild type levels of autoguanylylation activity. Lysine 197, like lysine 226, is not necessary for activity but affects the level of autoguanylylation. These two residues may function to stabilize the structure of the active site or the bound GTP, consistent with K226A having decreased autoguanylylation activity while maintaining wild type levels of GMP transfer. Lysine 171 is likely to be critical for substrate binding, since K171A showed less than 1% of wild type autoguanylylation activity. Based on the severe defect in autoguanylylation of the K190A mutant, lysine 190 is proposed to be necessary for activity and to be the active-site residue for formation of the phosphoamide bond.Lysine 190 in the reovirus λ2 protein is in a sequence context, KDLS, that lacks similarity with the consensus active-site motif (KXDG) of the well characterized class of eukaryotic and viral RNA guanylyltransferases (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar). This lack of similarity suggests that the λ2 active site may be formed via a novel protein fold. Sequences similar to the λ2 KDLS sequence were found to be widely conserved among the RNA guanylyltransferases of otherReoviridae family members within the genera Rotavirus, Orbivirus, and Phytoreovirus (data not shown) and may suggest that the enzymes from these other viruses share a novel active-site motif with reovirus λ2. Additionally, the consensus sequence KXDG is not strictly conserved among the RNA guanylyltransferases of any of these viruses, although KXXG motifs are conserved at two positions in the aligned orbivirus sequences and at one position in the aligned rotavirus sequences (data not shown). Our current hypothesis is that the reovirus RNA guanylyltransferase and perhaps also the RNA guanylyltransferases of other viruses in this family represent a distinct class of these enzymes. Clearly, biochemical and mutational analyses, as performed for mammalian reovirus λ2 in this study, are required to identify the active-site residues in the RNA guanylyltransferases of the other Reoviridae family members. Mammalian reovirus, a multisegmented double-stranded RNA virus in the family Reoviridae, replicates in the cytoplasm of the eukaryotic host cell. The reovirus core particle can producem7NGpppGm2′OpC(pN)n-OH (cap 1) plus-strand RNA from each genomic double-stranded RNA segment in vitro (1.Furuichi Y. Muthukrishnan S. Tomasz J. Shatkin A.J. J. Biol. Chem. 1976; 251: 5043-5053Abstract Full Text PDF PubMed Google Scholar), indicating that it contains all of the enzymes necessary for de novo synthesis of capped mRNA. The RNA polymerase itself is likely to be the λ3 core protein (2.Bruenn J.A. Nucleic Acids Res. 1991; 18: 217-226Crossref Scopus (169) Google Scholar, 3.Starnes M.C. Joklik W.K. Virology. 1993; 193: 356-366Crossref PubMed Scopus (75) Google Scholar). Genetic and/or biochemical analyses indicate that the λ1 and μ2 core proteins have nucleoside triphosphate phosphohydrolase activity, possibly associated with an RNA helicase (4.Noble S. Nibert M.L. J. Virol. 1997; 71: 2182-2191Crossref PubMed Google Scholar, 5.Noble S. Nibert M.L. J. Virol. 1997; 71: 7728-7735Crossref PubMed Google Scholar, 6.Bisaillon M. Bergeron J. Lemay G. J. Biol. Chem. 1997; 272: 18298-18303Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The γ phosphate of the newly transcribed mRNA is thought to be removed by the RNA triphosphate phosphohydrolase activity of λ1 (7.Bisaillon M. Lemay G. J. Biol. Chem. 1997; 272: 29954-29957Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The λ2 core protein is the reovirus RNA guanylyltransferase, which adds a GMP moiety via a 5′–5′ linkage to the 5′-diphosphorylated mRNA (8.Cleveland D.R. Zarbl H. Millward S. J. Virol. 1986; 60: 307-311Crossref PubMed Google Scholar). This transfer reaction occurs through a covalent intermediate, a phosphoamide bond between the GMP of the donor GTP and a lysine of λ2 (9.Shatkin A.J. Furuichi Y. LaFiandra A.J. Yamakawa M. Compans R.W. Bishop D.J.L. Double-stranded RNA Viruses. Elsevier, New York1983: 43-54Google Scholar, 10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). Generation of this covalent bond (called “autoguanylylation” in this paper) is followed by GMP transfer from the enzyme to an acceptor, usually the 5′-diphosphorylated mRNA, although the GMP can be alternatively transferred to a 5′-triphosphorylated RNA or a di- or triphosphorylated nucleoside (11.Mao Z. Joklik W.K. Virology. 1991; 185: 377-386Crossref PubMed Scopus (67) Google Scholar). The resulting product is then sequentially methylated by RNA nucleoside-7-N- and 2′-O-methyltransferases, yielding the cap 1 mRNA (1.Furuichi Y. Muthukrishnan S. Tomasz J. Shatkin A.J. J. Biol. Chem. 1976; 251: 5043-5053Abstract Full Text PDF PubMed Google Scholar) that is released through the channel formed by the λ2 pentameric spike (12.Barlett N.M. Gillies S.C. Bullivant S. Bellamy A.R. J. Virol. 1974; 167: 315-326Crossref Google Scholar, 13.Yeager M. Weiner S. Coombs K.M. Biophys. J. 1996; 70 (abstr.): 116Google Scholar). Both of the methyltransferase activities appear to reside in λ2 as indicated by the finding that only the λ2 protein in cores is covalently labeled with the methyl donor S-adenosyl-l-methionine after incubation and UV cross-linking (14.Luongo C.L. Contreras C.M. Farsetta D.L. Nibert M.L. J. Biol. Chem. 1998; 273: 23773-23780Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Thus, λ2 is thought to catalyze the last three of the four reactions required for cap 1 formation on reovirus mRNA. The 144-kDa λ2 protein (15.Seliger L.S. Zheng K. Shatkin A.J. J. Biol. Chem. 1987; 262: 16289-16293Abstract Full Text PDF PubMed Google Scholar), encoded by the reovirus L2 gene, appears to contain multiple domains. The proposed guanylyltransferase active site (lysine 226) is near the amino terminus (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). There is anS-adenosyl-l-methionine-binding site that appears to span residues 827 and 829 (14.Luongo C.L. Contreras C.M. Farsetta D.L. Nibert M.L. J. Biol. Chem. 1998; 273: 23773-23780Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 16.Koonin E.V. J. Gen. Virol. 1993; 74: 733-740Crossref PubMed Scopus (200) Google Scholar). A carboxyl-terminalM r 25,000 region is expendable for capping functions but is implicated in anchoring the reovirus cell attachment protein ς1 in virions (17.Dryden K.A. Wang G. Yeager M. Nibert M.L. Coombs K.M. Furlong D.B. Fields B.N. Baker T.S. J. Cell Biol. 1993; 122: 1023-1041Crossref PubMed Scopus (289) Google Scholar, 18.Luongo C.L. Dryden K.A. Farsetta D.L. Margraf R.M. Severson T.F. Olson N.H. Fields B.N. Baker T.S. Nibert M.L. J. Virol. 1997; 71: 8035-8040Crossref PubMed Google Scholar). A multidomain structure for the λ2 protein is also consistent with what is known for other capping enzymes. The vaccinia virus capping enzyme that catalyzes the first three reactions required for cap 1 formation is a heterodimer composed of two subunits encoded by separate genes (19.Martin S.A. Paoletti E. Moss B. J. Biol. Chem. 1975; 250: 9322-9329Abstract Full Text PDF PubMed Google Scholar, 20.Morgan J.R. Cohen L.K. Roberts B.E. J. Virol. 1984; 52: 206-214Crossref PubMed Google Scholar, 21.Niles E.G. Lee-Chen G.J. Shuman S. Moss B. Broyles S.S. Virology. 1989; 172: 513-522Crossref PubMed Scopus (62) Google Scholar). By biochemical analysis of proteolytic products, the capping enzyme is separable into a region with RNA triphosphate phosphohydrolase and guanylyltransferase activity and a region with RNA nucleoside-7-N-methyltransferase activity (22.Higman M.A. Bourgeois N. Niles E.G. J. Biol. Chem. 1992; 267: 16430-16437Abstract Full Text PDF PubMed Google Scholar, 23.Shuman S. J. Biol. Chem. 1989; 264: 9690-9695Abstract Full Text PDF PubMed Google Scholar). TheSaccharomyces cerevisiae capping enzyme is a complex of two separate gene products (24.Shibagaki Y. Itoh N. Yamada H. Shibekazu N. Mizumoto K. J. Biol. Chem. 1992; 267: 9521-9528Abstract Full Text PDF PubMed Google Scholar), one having RNA triphosphate phosphohydrolase and the other having RNA guanylyltransferase activity (25.Itoh N. Yamada H. Kaziro Y. Mizumoto K. J. Biol. Chem. 1987; 262: 1989-1995Abstract Full Text PDF PubMed Google Scholar). These two examples suggest that capping enzymes can be multifunctional and that the guanylyltransferase region may be separated biochemically (vaccinia virus) or genetically (yeast) from the rest of the protein. In the case of the Chlorella virus PBCV-1, the RNA guanylyltransferase is a 330-amino acid monofunctional enzyme (26.Ho C.K. van Etten J.L. Shuman S. J. Virol. 1996; 70: 6658-6664Crossref PubMed Google Scholar). The small size of this guanylyltransferase and the guanylyltransferase regions of the other capping enzymes suggests that only a portion of the 144-kDa λ2 protein is likely to be required for its RNA guanylyltransferase activity. For most RNA guanylyltransferases, a KXDG active-site motif has been proposed based on sequence comparisons (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar). For the RNA guanylyltransferases of vaccinia virus, S. cerevisiae, and baculovirus, the identity of the active site has been confirmed by site-directed mutagenesis (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 30.Schwer B. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4328-4332Crossref PubMed Scopus (61) Google Scholar, 31.Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar). For the PBCV-1 RNA guanylyltransferase, crystallographic analysis indicates that the active-site lysine interacts with the substrate GTP and that the other residues of the consensus motif interact with the RNA or nucleotide acceptor (32.Håkansson K. Doherty A.J. Shuman S. Wigley D.B. Cell. 1997; 89: 545-553Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 33.Håkansson K. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1505-1510Crossref PubMed Scopus (56) Google Scholar). The RNA guanylyltransferases, as well as RNA and DNA ligases, are members of the RNA/DNA nucleotidyltransferase superfamily (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar), enzymes that mediate nucleotidyl transfer to RNA or DNA via a covalent intermediate. The active-site motif for this entire superfamily is KX(D/N)G (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar). The RNA guanylyltransferases of members of the family Reoviridaelack sequences that precisely match this consensus motif (15.Seliger L.S. Zheng K. Shatkin A.J. J. Biol. Chem. 1987; 262: 16289-16293Abstract Full Text PDF PubMed Google Scholar, 34.Bisaillon M. Lemay G. Virology. 1997; 236: 1-7Crossref PubMed Scopus (55) Google Scholar). For example, the sequence of the proposed active site in the reovirus λ2 protein is 226KPTNG. This sequence is similar to the nucleotidyl transferase superfamily motif, but the alignment is disrupted by insertion of a proline residue after the lysine in λ2. The proposed guanylyltransferase active site for the other familyReoviridae members rotavirus (KPTGN) and bluetongue virus (KLTGN) is based on the proposed reovirus active site (34.Bisaillon M. Lemay G. Virology. 1997; 236: 1-7Crossref PubMed Scopus (55) Google Scholar). A previous effort to identify the λ2 residue that forms the phosphoamide bond with GMP used a combination of proteolysis and chemical cleavage of reovirus virions autoguanylylated with [32P]GMP to show that a lysine residue in λ2 is the site of covalent linkage to GMP (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). Immunoblot analysis of proteolyzed, [32P]GMP-labeled reovirus particles with λ2 peptide-specific antibodies indirectly identified lysine 226 as the residue to which GMP is likely attached. A similar approach involving proteolysis and chemical cleavage identified the active-site lysine of the vaccinia virus guanylyltransferase (35.Niles E.G. Christen L. J. Biol. Chem. 1993; 268: 24986-24989Abstract Full Text PDF PubMed Google Scholar), and the identity of the vaccinia virus active-site lysine was subsequently confirmed by the analysis of site-directed mutants for autoguanylylation activity (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar). To extend characterization of the reovirus RNA guanylyltransferase, we expressed recombinant λ2 and analyzed its properties. Utilizing a biochemical approach, we localized a region necessary and sufficient for guanylyltransferase activity to an amino-terminal M r 42,000 fragment of λ2. Since this region contains lysine 226, we generated mutant K226A by alanine substitution. Analysis of the mutant indicated that lysine 226 is not necessary for λ2 guanylyltransferase activity. Alanine substitution for other lysines in the M r 42,000 region identified lysine 190 as the probable site of covalent GMP linkage and lysine 171 as important for autoguanylylation activity. Based on these findings, we propose a novel active-site motif for the RNA guanylyltransferases of mammalian reoviruses and otherReoviridae family members. DISCUSSIONOur rλ2 protein expressed in insect cells from a baculovirus vector has guanylyltransferase activity using either GDP or GTP as GMP acceptor. In contrast to published observations with rλ2 expressed in mammalian cells from a vaccinia virus vector (11.Mao Z. Joklik W.K. Virology. 1991; 185: 377-386Crossref PubMed Scopus (67) Google Scholar), however, we were unsuccessful at demonstrating guanylyltransferase activity with baculovirus-expressed rλ2 using either 5′-diphosphorylated reovirus RNA or poly(A) RNA as acceptor (data not shown). Given this limitation in the activity of the baculovirus-expressed protein, which might be explained by a defect in allowing RNA molecules into the acceptor region of the enzyme, the conclusions we reach about amino acids required for GMP transfer must be considered as tentative with regard to RNA acceptors.Unlike core-associated λ2, rλ2 is hypersensitive to cleavage into complementary M r 42,000 and 100,000 fragments that dissociate in solution. Retention of both autoguanylylation and GMP transfer activities by the M r 42,000 fragment suggested that it is both necessary and sufficient to act as a guanylyltransferase. The M r 42,000 region contains the active site originally localized by direct biochemical analysis to the region between amino acids 131 and 266 (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). Further indirect biochemical analysis in the previous study identified lysine 226 as the active-site residue that forms a phosphoamide bond with GMP (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar).The previously identified active-site motif 226KPTNG in reovirus λ2 is anomalous for two reasons. First it is not conserved outside the family Reoviridae. Of the known nucleotidyl transferases, at least 13 RNA guanylyltransferases, 16 DNA ligases, and one RNA ligase have the motif KXDG (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar, 31.Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar). The two DNA ligases of African swine fever virus and the S. cerevisiaetRNA ligase have the motif KXNG (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar). The second reason is that insertion of additional residues may interfere with noncovalent bonding of one of the substrates. Based on the structure of the PBCV-1 guanylyltransferase (32.Håkansson K. Doherty A.J. Shuman S. Wigley D.B. Cell. 1997; 89: 545-553Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 33.Håkansson K. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1505-1510Crossref PubMed Scopus (56) Google Scholar) and the ATP-dependent DNA ligase of bacteriophage T7 (44.Subramanya H.S. Doherty A.J. Ashford S.R. Wigley D.B. Cell. 1996; 85: 607-615Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), the lysine in the active-site motif covalently binds GMP or AMP, respectively, and the remaining residues interact with the nucleotide acceptor in RNA for PBCV-1 or the ATP for T7 DNA ligase.As was done for the vaccinia virus RNA guanylyltransferase (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar), we chose to confirm the identity of the active-site lysine in reovirus λ2 by site-directed mutagenesis. Since alanine substitution has been used to identify the active-site lysine of the RNA guanylyltransferase of S. cerevisiae (30.Schwer B. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4328-4332Crossref PubMed Scopus (61) Google Scholar) and baculovirus (31.Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar), we generated the λ2 mutant K226A. Based on the demonstrable guanylyltransferase activity of this mutant, we concluded that lysine 226 is not the active-site residue. This conclusion has ramifications for the guanylyltransferases of the other members of the double-stranded RNA virus family Reoviridae and brings into question the functional significance of the similar motifs recently identified in the rotavirus and bluetongue virus RNA guanylyltransferases (34.Bisaillon M. Lemay G. Virology. 1997; 236: 1-7Crossref PubMed Scopus (55) Google Scholar).In addition to lysine 226, the M r 42,000 region contains seven lysines at positions 44, 89, 94, 171, 190, 197, and 372 (15.Seliger L.S. Zheng K. Shatkin A.J. J. Biol. Chem. 1987; 262: 16289-16293Abstract Full Text PDF PubMed Google Scholar). A series of six alanine substitution mutants were generated to identify the active-site residue. Lysines 44, 89, and 94 are not necessary for activity, based on mutants K44A, K89A, and K94A having approximately wild type levels of autoguanylylation activity. Lysine 197, like lysine 226, is not necessary for activity but affects the level of autoguanylylation. These two residues may function to stabilize the structure of the active site or the bound GTP, consistent with K226A having decreased autoguanylylation activity while maintaining wild type levels of GMP transfer. Lysine 171 is likely to be critical for substrate binding, since K171A showed less than 1% of wild type autoguanylylation activity. Based on the severe defect in autoguanylylation of the K190A mutant, lysine 190 is proposed to be necessary for activity and to be the active-site residue for formation of the phosphoamide bond.Lysine 190 in the reovirus λ2 protein is in a sequence context, KDLS, that lacks similarity with the consensus active-site motif (KXDG) of the well characterized class of eukaryotic and viral RNA guanylyltransferases (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar). This lack of similarity suggests that the λ2 active site may be formed via a novel protein fold. Sequences similar to the λ2 KDLS sequence were found to be widely conserved among the RNA guanylyltransferases of otherReoviridae family members within the genera Rotavirus, Orbivirus, and Phytoreovirus (data not shown) and may suggest that the enzymes from these other viruses share a novel active-site motif with reovirus λ2. Additionally, the consensus sequence KXDG is not strictly conserved among the RNA guanylyltransferases of any of these viruses, although KXXG motifs are conserved at two positions in the aligned orbivirus sequences and at one position in the aligned rotavirus sequences (data not shown). Our current hypothesis is that the reovirus RNA guanylyltransferase and perhaps also the RNA guanylyltransferases of other viruses in this family represent a distinct class of these enzymes. Clearly, biochemical and mutational analyses, as performed for mammalian reovirus λ2 in this study, are required to identify the active-site residues in the RNA guanylyltransferases of the other Reoviridae family members. Our rλ2 protein expressed in insect cells from a baculovirus vector has guanylyltransferase activity using either GDP or GTP as GMP acceptor. In contrast to published observations with rλ2 expressed in mammalian cells from a vaccinia virus vector (11.Mao Z. Joklik W.K. Virology. 1991; 185: 377-386Crossref PubMed Scopus (67) Google Scholar), however, we were unsuccessful at demonstrating guanylyltransferase activity with baculovirus-expressed rλ2 using either 5′-diphosphorylated reovirus RNA or poly(A) RNA as acceptor (data not shown). Given this limitation in the activity of the baculovirus-expressed protein, which might be explained by a defect in allowing RNA molecules into the acceptor region of the enzyme, the conclusions we reach about amino acids required for GMP transfer must be considered as tentative with regard to RNA acceptors. Unlike core-associated λ2, rλ2 is hypersensitive to cleavage into complementary M r 42,000 and 100,000 fragments that dissociate in solution. Retention of both autoguanylylation and GMP transfer activities by the M r 42,000 fragment suggested that it is both necessary and sufficient to act as a guanylyltransferase. The M r 42,000 region contains the active site originally localized by direct biochemical analysis to the region between amino acids 131 and 266 (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). Further indirect biochemical analysis in the previous study identified lysine 226 as the active-site residue that forms a phosphoamide bond with GMP (10.Fausnaugh J. Shatkin A.J. J. Biol. Chem. 1990; 265: 7669-7672Abstract Full Text PDF PubMed Google Scholar). The previously identified active-site motif 226KPTNG in reovirus λ2 is anomalous for two reasons. First it is not conserved outside the family Reoviridae. Of the known nucleotidyl transferases, at least 13 RNA guanylyltransferases, 16 DNA ligases, and one RNA ligase have the motif KXDG (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar, 31.Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar). The two DNA ligases of African swine fever virus and the S. cerevisiaetRNA ligase have the motif KXNG (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar). The second reason is that insertion of additional residues may interfere with noncovalent bonding of one of the substrates. Based on the structure of the PBCV-1 guanylyltransferase (32.Håkansson K. Doherty A.J. Shuman S. Wigley D.B. Cell. 1997; 89: 545-553Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 33.Håkansson K. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1505-1510Crossref PubMed Scopus (56) Google Scholar) and the ATP-dependent DNA ligase of bacteriophage T7 (44.Subramanya H.S. Doherty A.J. Ashford S.R. Wigley D.B. Cell. 1996; 85: 607-615Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), the lysine in the active-site motif covalently binds GMP or AMP, respectively, and the remaining residues interact with the nucleotide acceptor in RNA for PBCV-1 or the ATP for T7 DNA ligase. As was done for the vaccinia virus RNA guanylyltransferase (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar), we chose to confirm the identity of the active-site lysine in reovirus λ2 by site-directed mutagenesis. Since alanine substitution has been used to identify the active-site lysine of the RNA guanylyltransferase of S. cerevisiae (30.Schwer B. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4328-4332Crossref PubMed Scopus (61) Google Scholar) and baculovirus (31.Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar), we generated the λ2 mutant K226A. Based on the demonstrable guanylyltransferase activity of this mutant, we concluded that lysine 226 is not the active-site residue. This conclusion has ramifications for the guanylyltransferases of the other members of the double-stranded RNA virus family Reoviridae and brings into question the functional significance of the similar motifs recently identified in the rotavirus and bluetongue virus RNA guanylyltransferases (34.Bisaillon M. Lemay G. Virology. 1997; 236: 1-7Crossref PubMed Scopus (55) Google Scholar). In addition to lysine 226, the M r 42,000 region contains seven lysines at positions 44, 89, 94, 171, 190, 197, and 372 (15.Seliger L.S. Zheng K. Shatkin A.J. J. Biol. Chem. 1987; 262: 16289-16293Abstract Full Text PDF PubMed Google Scholar). A series of six alanine substitution mutants were generated to identify the active-site residue. Lysines 44, 89, and 94 are not necessary for activity, based on mutants K44A, K89A, and K94A having approximately wild type levels of autoguanylylation activity. Lysine 197, like lysine 226, is not necessary for activity but affects the level of autoguanylylation. These two residues may function to stabilize the structure of the active site or the bound GTP, consistent with K226A having decreased autoguanylylation activity while maintaining wild type levels of GMP transfer. Lysine 171 is likely to be critical for substrate binding, since K171A showed less than 1% of wild type autoguanylylation activity. Based on the severe defect in autoguanylylation of the K190A mutant, lysine 190 is proposed to be necessary for activity and to be the active-site residue for formation of the phosphoamide bond. Lysine 190 in the reovirus λ2 protein is in a sequence context, KDLS, that lacks similarity with the consensus active-site motif (KXDG) of the well characterized class of eukaryotic and viral RNA guanylyltransferases (27.Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar, 28.Fresco L.D. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6624-6628Crossref PubMed Scopus (64) Google Scholar, 29.Wang S.P. Deng L. Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9573-9578Crossref PubMed Scopus (99) Google Scholar). This lack of similarity suggests that the λ2 active site may be formed via a novel protein fold. Sequences similar to the λ2 KDLS sequence were found to be widely conserved among the RNA guanylyltransferases of otherReoviridae family members within the genera Rotavirus, Orbivirus, and Phytoreovirus (data not shown) and may suggest that the enzymes from these other viruses share a novel active-site motif with reovirus λ2. Additionally, the consensus sequence KXDG is not strictly conserved among the RNA guanylyltransferases of any of these viruses, although KXXG motifs are conserved at two positions in the aligned orbivirus sequences and at one position in the aligned rotavirus sequences (data not shown). Our current hypothesis is that the reovirus RNA guanylyltransferase and perhaps also the RNA guanylyltransferases of other viruses in this family represent a distinct class of these enzymes. Clearly, biochemical and mutational analyses, as performed for mammalian reovirus λ2 in this study, are required to identify the active-site residues in the RNA guanylyltransferases of the other Reoviridae family members. We are grateful to W. K. Joklik for providing the original clone of the reovirus T3D L2 gene, A. Khimani for assistance in generating plasmid subclones containing this gene, and K. L. Tyler and H. W. Virgin IV for providing a purified preparation of monoclonal antibody 7F4. We are also grateful to J. Rush and P. Rahaim at the Howard Hughes Medical Institute and Harvard Medical School Biopolymer Facility and C. Nicolet and colleagues at the University of Wisconsin Biotechnology Center DNA Sequencing Facility for automated DNA sequencing. We thank R. L. Margraf and S. J. Harrison for technical assistance and other members of our laboratories for helpful discussions." @default.
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• W2022742007 title "Identification of the Guanylyltransferase Region and Active Site in Reovirus mRNA Capping Protein λ2" @default.
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