Chlorella Virus RNA Triphosphatase

Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu24, Glu26, Asp64, Arg76, Lys90, Glu112, Arg127, Lys129, Arg131, Asp142, Glu163, and Glu165) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site ofcvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors ofcvRtp1 (Ki = 0.6 μmtripolyphosphate and 2.4 μm pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate. Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu24, Glu26, Asp64, Arg76, Lys90, Glu112, Arg127, Lys129, Arg131, Asp142, Glu163, and Glu165) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site ofcvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors ofcvRtp1 (Ki = 0.6 μmtripolyphosphate and 2.4 μm pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate. African swine fever virus Chlorella virus RNA triphosphatase pyrophosphate tripolyphosphate dithiothreitol The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by three enzymatic reactions: (i) the 5′ triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA triphosphatase, (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by AdoMet:RNA (guanine-7-)methyltransferase (1Shuman S. Prog. Nucleic Acids Res. Mol. Biol. 2000; 66: 1-40Crossref Google Scholar). The 5′ ends of the mRNAs of DNA viruses such as papovaviruses, parvoviruses, adenoviruses, and herpesviruses are modified by the host cell’s capping and methylating enzymes. However, other DNA viruses, including poxviruses, African swine fever virus (ASFV),1 baculoviruses, andChlorella virus PBCV-1, encode their own cap-forming enzymes. Chlorella virus PBCV-1 is a large icosahedral DNA virus that replicates in unicellular Chlorella-like green algae (2Van Etten J.L. Meints R.H. Annu. Rev. Microbiol. 1999; 53: 447-494Crossref PubMed Scopus (179) Google Scholar). The 330-kb linear PBCV-1 genome encodes 375 polypeptides, which makes it one of the most complex viruses known. PBCV-1 encodes its own RNA triphosphatase and guanylyltransferase enzymes (3Ho C.K. Van Etten J.L. Shuman S. J. Virol. 1996; 70: 6658-6664Crossref PubMed Google Scholar, 4Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar).Chlorella virus guanylyltransferase is a 330-amino acid monomeric polypeptide that catalyzes the transfer of GMP from GTP to the 5′ diphosphate end of RNA. Its structure and mechanism have been elucidated by x-ray crystallography (5Håkansson K. Doherty A.J. Shuman S. Wigley D.B. Cell. 1997; 89: 545-553Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Chlorella virus guanylyltransferase is monofunctional and has no intrinsic triphosphatase or methyltransferase activities. It is most closely related to the monofunctional yeast RNA guanylyltransferases, more so than to the multifunctional vaccinia virus and ASFV capping enzymes or the bifunctional triphosphatase-guanylyltransferases of baculoviruses. Chlorella virus RNA triphosphatase (cvRtp1) is a 193-amino acid polypeptide that catalyzes metal-dependent hydrolysis of the γ-phosphate of triphosphate-terminated RNA (4Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar).cvRtp1 is a member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatase components of the capping enzymes of fungi (Saccharomyces cerevisiae, Candida albicans, andSchizosaccharomyces pombe), protozoan and microsporidian parasites (Plasmodium falciparum, Trypanosoma brucei, and Encephalotozoon cuniculi), and metazoan DNA viruses (poxviruses, ASFV, and baculoviruses) (6–18) (Fig. 1). The signature biochemical property of this enzyme family is the ability to hydrolyze nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate in the presence of either manganese or cobalt. The defining structural features of the metal-dependent RNA triphosphatases are two glutamate-containing motifs (β1 and β11 in Fig. 1) that are required for catalysis by the fungal, microsporidian, poxvirus, and baculovirus RNA triphosphatases. During our initial characterization of cvRtp1, we showed that alanine substitution of Glu26 in the β1 motif (VELEFRLG) abrogated the phosphohydrolase activity (4Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar). The crystal structure of the S. cerevisiae RNA triphosphatase Cet1 revealed that the active site is located within a topologically closed hydrophilic tunnel composed of eight antiparallel β strands (Fig. 2) (19Lima C.D. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The “triphosphate tunnel” has a distinctive architecture supported by an intricate network of hydrogen bonds and electrostatic interactions within the cavity, a high proportion of which are required for the triphosphatase activity of Cet1 (6Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Ho Y. Pei C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A single sulfate ion in the tunnel is coordinated by multiple basic side chains projecting into the cavity. It was proposed that the side chain interactions of the sulfate reflect contacts made by the enzyme with the γ-phosphate of the substrate (19Lima C.D. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). A manganese ion within the tunnel cavity is coordinated with octahedral geometry to a sulfate, to the side chain carboxylates of the two glutamates in β1, and to a glutamate in β11. The sequence similarity between the Chlorella virus RNA triphosphatase and the catalytic domains of the S. cerevisiae, C. albicans, and S. pombe RNA triphosphatases, especially the β strands that comprise the triphosphate tunnel (Fig. 1), prompted a suggestion that the active site fold of the Chlorella virus and fungal RNA triphosphatases might be conserved as β barrels. In contrast, the RNA triphosphatases of poxviruses, ASFV, and baculoviruses have only limited primary structure similarity to the fungal andChlorella virus enzymes. The poxvirus, ASFV, and baculovirus triphosphatases contain the diagnostic metal-binding motifs composed of alternating glutamate/hydrophobic side chains (the presumptive equivalents of strands β1 and β11 in the Cet1 structure), and the available mutational studies of the poxvirus and baculovirus proteins are consistent with an essential catalytic role for the conserved glutamates in metal binding (7Yu L. Martins A. Deng L. Shuman S. J. Virol. 1997; 71: 9837-9843Crossref PubMed Google Scholar, 8Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar, 9Gross C.H. Shuman S. J. Virol. 1998; 72: 10020-10028Crossref PubMed Google Scholar, 10Martins A. Shuman S. J. Biol. Chem. 2001; 276: 45522-45529Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). However, the poxvirus and baculovirus enzymes do not have obvious equivalents of the other six β strands that comprise the tunnel found in the fungal enzyme. The structural and mechanistic relatedness among the various DNA virus-encoded mRNA capping systems is of interest in light of the proposal that these diverse families of large DNA viruses (poxviruses, ASFV, and Chlorella virus) have a common evolutionary origin (24Iyer L.M. Aravind L. Koonin E.V. J. Virol. 2001; 75: 11720-11734Crossref PubMed Scopus (430) Google Scholar). Our hypothesis is that the Chlorella virus RNA triphosphatase is more closely related to the cellular triphosphatases of fungi than to the other DNA virus-encoded RNA triphosphatases. To test the structural and mechanistic relatedness of theChlorella virus and yeast RNA triphosphatases, we have conducted a mutational analysis of cvRtp1 using the tunnel of Cet1 as a guide. We used the alanine scanning approach to identify 12 new amino acids (in addition to Glu26) that are essential or important for triphosphatase activity. The relevant structural features of these functional groups were then clarified by conservative amino acid substitutions. Our results underscore the fundamental similarities between the active sites of the fungal andChlorella virus triphosphatases, but they also indicate that the functional contributions of some conserved residues are context-dependent. We show that tripolyphosphate is a potent competitive inhibitor of cvRtp1 that binds the enzyme with higher affinity than ATP. cvRtp1 has a weak tripolyphosphatase activity, indicating that a nucleoside is not strictly required for phosphohydrolase chemistry. Amino acid substitution mutations and diagnostic restriction sites were introduced into the cvRTP1gene (viral open reading frame A449R) by the two-stage overlap extension method (21Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). pET-A449R (5Håkansson K. Doherty A.J. Shuman S. Wigley D.B. Cell. 1997; 89: 545-553Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) was used as the template for the first-stage amplification. The mutated full-length cvRTP1genes generated in the second-stage amplification were digested withNdeI and and then into The of the was to the mutations and the of amplification or The were introduced into of were at in the The were on for and then to and for at with the were by and the were at were performed at were in of A and was to a of and the was on for and then to and to the of the was by The were to of that had been with A The were with the and then with and and The polypeptide of the were by The wild-type and proteins were on the and in the of The enzyme were at were using the with as a and of either wild-type or cvRtp1 proteins and were for at The were by of of the were to which were with acid and was by scanning the with a were performed for and the of were as a of The were from the of the The activity for the enzymes were then to the activity of wild-type tripolyphosphate pyrophosphate or and cvRtp1 as were for at The were by of green from was by and the to a phosphate of inorganic phosphate of the of or from the of a reaction were from the for the The crystal structure of S. cerevisiae Cet1 that the active site is located within a topologically closed β to as the “triphosphate tunnel” (19Lima C.D. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The β strands of the tunnel and are displayed the Cet1 amino acid sequence in of the hydrophilic amino acids that comprise the active site of yeast RNA triphosphatase and are important for its catalytic activity (6Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Ho Y. Pei C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, M. Shuman S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) are also in To relationships for we the effects of single alanine mutations at the conserved side chains by in Fig. Asp64, Arg76, Lys90, Glu112, Arg127, Lys129, Arg131, Asp142, Glu163, and one amino acid was mutated in of the of the β strands that comprise the Cet1 triphosphate tunnel. The mutations were introduced into and the were as in with wild-type The proteins were from by analysis of the polypeptide of the revealed similar of (Fig. cvRtp1 was the polypeptide in The triphosphatase of the wild-type and cvRtp1 proteins were by the from in the presence of The of the to the wild-type activity, are in substitutions at of the at a in catalytic activity. residues Asp64, Arg76, Lys90, Glu112, Arg127, Lys129, Arg131, and mutational effects were also for of wild-type activity) and of We these residues as essential for the phosphohydrolase activity of cvRtp1 by a for of a activity by side chain at which alanine substitution reduced activity to of wild-type in were deemed to be important but not whereas residues at which alanine substitutions than a and were not to be important for triphosphatase activity. Fig. a of the effects of alanine mutations at the amino acids of cvRtp1, S. cerevisiae Cet1, The the of the and proteins in hydrolysis of (6Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Ho Y. Pei C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Y. K. L. Shuman S. Nucleic Acids Res. 2000; PubMed Scopus Google Scholar, 20Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). of the generated in cvRtp1 have in Cet1, and of the have been in The of the is that most of the mutational effects are in three metal-dependent Nonetheless, are at of cvRtp1 Cet1 are Asp64, Lys90, Arg127, and in The mutational be in light of the crystal structure of We have the essential and important active site residues of Cet1 into three functional M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). residues of Cet1 in catalysis of the γ-phosphate in and and in or the essential metal in β1 and in six amino acids in this are essential for the triphosphatase of cvRtp1 and We that the equivalents of the residues Lys129, Arg131, Glu26, and Glu165) are in of the γ-phosphate and the residues of Cet1 contacts with the γ-phosphate in and in or the metal in and The two residues that the metal are essential for cvRtp1 and The conserved glutamate in that to the phosphate is also essential and However, the conserved in is more for the of cvRtp1 has of wild-type triphosphatase activity) than it is for either Cet1 has activity) or has residues of Cet1 in catalysis their interactions with other essential side chains their of the tunnel residues in in in and in β11. It is in this functional that the Chlorella virus and fungal enzymes displayed the in alanine substitution the reduced the activity of Cet1 to whereas the mutations in cvRtp1 and their triphosphatase activity (Fig. The of Cet1 activity) was also than the in cvRtp1 activity) and the in (3% the other whereas was essential for Cet1 has of wild-type the of the basic side chain at the of cvRtp1 had only a on activity of that was not to be In the crystal structure of Cet1, is located on the side of the tunnel and is not in to the it was proposed that is not a and that its for Cet1 its role in which is on the for cvRtp1, we that a basic in is not required to the in for its role in metal the role for the basic is context-dependent. The in has not been to mutational is by or in other members of the metal-dependent phosphohydrolase family (Fig. 1). It is that introducing a or in of cerevisiae Cet1 reduced triphosphatase activity to of the wild-type M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The essential role of at this to be to the S. in β1 of cvRtp1 is conserved as a or in of the triphosphatases in Fig. In the Cet1 crystal the the essential in we found that no role in catalysis which with for in Cet1 and in (Fig. in of cvRtp1 is conserved as a or in of the six triphosphatases in Fig. alanine mutations at this had no or Cet1 triphosphatase activity (Fig. in β11 of cvRtp1 to of Cet1, and side chain is this is in with the of at this in other members of the triphosphatase substitutions were introduced at of the 12 residues by the alanine scan as essential or important for cvRtp1 We also introduced conservative substitutions for Glu26, which had been to be essential for activity (4Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar). A of proteins with conservative were and from by (Fig. The ATPase of the conservative were by and the activity to that of are in of conservative substitutions activity. The ATPase of conservative cvRtp1 were from the of the and then to the activity of wild-type cvRtp1 as The effects of conservative substitutions at the of Cet1 on ATPase activity as the of the wild-type ATPase activity) are to the of of the three essential ofcvRtp1 Arg127, and by to the triphosphatase to of the wild-type and in only the activity of the We that cvRtp1 a side chain at and not The of the and in cvRtp1 is in with the of to for the and in Cet1 and whereas the effects of conservative mutations Cet1 are discordant (Fig. The two essential and were by and The and proteins were with and of wild-type activity, The a of activity that of the alanine but the had no effect. cvRtp1 triphosphatase activity a at and for at the Cet1 residues and (Fig. The two essential acids of cvRtp1 and were by glutamate and The and proteins had and of wild-type activity, similar to the activity of the We that ATPase activity is strictly on a functional and that of by the glutamate side relationships were for the of Cet1 (Fig. of a glutamate in of to of wild-type activity, with activity for the The also activity, only to of the wild-type We that activity on a functional at of cvRtp1 and that the active site the glutamate side The activity by the of that this makes hydrogen with a coordinated to the as in the structure of Cet1 (Fig. that the relationships at this and The acid is important for Cet1 activity not essential as in and the Cet1 ATPase was was with either glutamate or (Fig. The three essential glutamates of cvRtp1 (Glu24, Glu26, and Glu165) to the three metal-binding side chains of Cet1 and were by and The and were as as the alanine mutations (Fig. are with the conservative mutational effects for Cet1, and they suggest that and Glu26 of cvRtp1 are likely to the metal and that cvRtp1 its structure to an its chain to into the metal of cvRtp1 with had no with the alanine whereas introducing a of to of wild-type activity (Fig. The activity of the the of interactions of this side relationships were for the of Cet1, the ATPase activity. The essential of of cvRtp1 not be by either of wild-type activity) or acid (3% activity) (Fig. In contrast, the of the side chain of Cet1, which with a coordinated by the metal (Fig. was by acid of wild-type not by Cet1 is more than cvRtp1 of a of the chain to at this The side chain of cvRtp1 was as important on the activity of the The in Cet1 a with to essential in which is to the tunnel architecture (Fig. The conservative of cvRtp1 activity) was more that the whereas the in a of to of the wild-type (Fig. The high activity by the that a to is not essential and that interactions to the fold of the active site and the side chain for with the of the to the chain (in or electrostatic on cvRtp1 that not the was introduced into it was that the essential of in cvRtp1 not be by whereas substitution with phosphohydrolase activity to of the wild-type (Fig. The side chain of Cet1 a that is in coordinated by the sulfate at the active site. It was proposed that this is the that the and that as a to the M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). is consistent with the that replacement of with which a from in a in Cet1 phosphohydrolase activity. The activity of the ofcvRtp1 in the catalytic mechanism of cvRtp1 Cet1, of the interactions to an activity whereas transfer to the only a of activity. We the effects of various phosphate on the ability ofcvRtp1 to hydrolyze in the presence of which is a of the phosphohydrolase activity by at and by at (Fig. not pyrophosphate was a more potent inhibitor than phosphate at μm whereas tripolyphosphate was more potent than pyrophosphate at μm (Fig. The that tripolyphosphate and pyrophosphate at and than and and than manganese the that these by as to with for the metal A for the potent is that more than to the within the active site tunnel. The mechanism of was by analysis of the effects of and on hydrolysis at three different of the substrate and The activity for of were into a of the of or revealed a of that a to the of the this is of competitive Scholar). The were 2.4 μm for pyrophosphate and 0.6 μm for tripolyphosphate (Fig. We that of cvRtp1 for is μm (4Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar). the from the in Fig. that pyrophosphate and especially tripolyphosphate more to the cvRtp1 active site than the The showed that tripolyphosphate and pyrophosphate to the active site but they not these might be for We a to of inorganic phosphate from or in the presence of of as a The of from was to the of cvRtp1 the of the we an ATPase of which with the using the of hydrolysis (4Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar). The was that cvRtp1 also the of from than it hydrolyzed as from the to the in the (Fig. Nonetheless, the showed that the of at of cvRtp1 was of the of that the reaction the of to and The for the tripolyphosphatase activity of cvRtp1 was of its ATPase activity. with the proposed tripolyphosphatase reaction we no hydrolysis of to the of enzyme The tripolyphosphatase activity of cvRtp1 was strictly on a (Fig. of tripolyphosphate was at and at not the metal (Fig. the of the tripolyphosphatase for manganese is the as that of the ATPase activity of cvRtp1 (4Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar). that the tripolyphosphatase activity was intrinsic to was by the that the which was for was also for the hydrolysis of not The results of the mutational analysis of Chlorella virus RNA triphosphatase suggest that its active site is located within a hydrophilic tunnel similar to that of S. cerevisiae Cet1 and that the constellation of functional groups responsible for phosphohydrolase reaction is the in theChlorella virus and fungal enzymes. However, there are in relationships at we to be of cvRtp1 and Cet1 that suggest mechanistic as as a for some of the side chains that are proposed to catalysis