Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Signal-dependent termination is restricted to early poxvirus genes whose transcription is catalyzed by the virion form of RNA polymerase. Two termination factors have been identified. Vaccinia termination factor/capping enzyme is a multifunctional heterodimer that also catalyzes the first three steps of mRNA cap formation and is an essential intermediate gene transcription initiation factor. Nucleoside triphosphate phosphohydrolase I (NPH I) is a single-stranded DNA-dependent ATPase. COOH-terminal deletion mutations of NPH I retain both ATPase and DNA binding activities but are unable either to terminate transcription or to act as dominant negative mutants in vitro. One appealing model posits that the COOH-terminal region of NPH I binds to one or more components in the termination complex. In an attempt to identify NPH I-related protein/protein interactions involved in transcription termination, a series of pull-down experiments were done. Among several vaccinia virus proteins tested, the H4L subunit, unique to the virion form of RNA polymerase, was shown to bind glutathione S-transferase (GST)-NPH I. To further confirm this interaction in virus-infected cells, we constructed recombinant vaccinia virus, vNPHINGST, that expresses GST-tagged NPH I. The H4L subunit of virion RNA polymerase specifically co-purified with GST-NPH I, consistent with a physical interaction. Through the analysis of a series of NH(2)- and COOH-terminal truncation mutations of H4L, the NPH I interaction site was localized to the NH(2)-terminal 195 amino acids of the H4L protein. The H4L binding site on NPH I was mapped to the COOH-terminal region between 457 and 631. Furthermore, COOH-terminal deletion mutations of NPH I failed to bind the NH(2)-terminal region of H4L, explaining their inability to support transcription termination. The COOH-terminal end of NPH I was also shown to be required for transcript release activity and for dominant negative inhibition of release. The requirement for an essential interaction between NPH I and H4L provides an explanation for the observed restriction of transcription termination to early viral genes.
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PMID:Interaction between nucleoside triphosphate phosphohydrolase I and the H4L subunit of the viral RNA polymerase is required for vaccinia virus early gene transcript release. 1083 18

Vaccinia virus early gene transcription is catalyzed by a multisubunit virion form of RNA polymerase that possesses a unique subunit, H4L. Prior studies from this laboratory showed that the NH(2)-terminal domain of H4L, containing amino acids 1-195, interacts with the COOH-terminal end of nucleoside triphosphate phosphohydrolase I (NPH I), an ATPase that is employed in early gene transcription termination. Carboxyl-terminal deletion mutations of NPH I lose both the ability to mediate transcription termination and binding to H4L, providing evidence that the interaction between NPH I and H4L is required for termination. In order to test this model further, antibodies raised against segments of H4L were tested for their ability to inhibit transcription termination in vitro. A bead-bound template was employed in these studies, which permitted us to separate transcription initiation from elongation and termination. Antibodies raised against H4L amino acids 1-256 inhibited termination in an in vitro assay using virus-infected cell extracts lacking NPH I, but antibodies raised against H4L amino acids 568-795 did not. Preincubation of anti-H4L(1-256) antibodies with H4L fragments 1-256 or 1-195 prevented antibody inhibition of termination, demonstrating that inhibition was mediated by antibody binding to one or more epitopes in the NH(2)-terminal end of H4L. Antibody inhibition of termination is reduced in wild type virus-infected cell extracts containing NPH I. Furthermore, preincubation of a NPH I minus cell extract with NPH I prior to antibody addition, or readdition of NPH I to isolated ternary complexes prepared in the absence of NPH I, prevented antibody inhibition of transcription termination. These data show that NPH I and the inhibitory antibodies compete for a binding site(s) on H4L, providing further evidence that the H4L subunit of the vaccinia virus RNA polymerase plays a direct role in transcription termination.
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PMID:The viral RNA polymerase H4L subunit is required for Vaccinia virus early gene transcription termination. 1127 16

Molecular chaperones assist protein folding, and some chaperones are induced by heat, nutrient depletion, or pathogen invasion. This study investigates the role played by Hsp90 in the life cycle of vaccinia virus. The titer of vaccinia intracellular mature virions (IMV) was reduced by 2 orders of magnitude in RK13 cells treated with geldanamycin (GA), which blocks the ATPase activity of Hsp90. GA does not affect expression from the viral early promoter, but treatment with GA delays DNA replication and intermediate gene transcription and reduces expression from the viral late promoter. Vaccinia virus infection does not induce Hsp90 expression; however, intracellular distribution of Hsp90 is altered in virus-infected cells. Hsp90 is restricted to the cytoplasm of mock-infected cells; in contrast, Hsp90 is transiently associated with virosomes in virus-infected cells although it is not incorporated into IMV. In addition, Hsp90 interacts with viral core protein 4a, the mature form of the A10L gene product, in virus-infected cells. In conclusion, these results suggest that a cellular chaperone protein, Hsp90, is important for vaccinia virus growth in cultured cells and that viral core protein 4a associates with Hsp90-containing complexes in the infected cells.
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PMID:Molecular chaperone Hsp90 is important for vaccinia virus growth in cells. 1177 12

Vaccinia virus early gene transcription termination requires the vaccinia termination factor (VTF), NPH I, a single stranded DNA-dependent ATPase, the virion form of RNA polymerase containing the Rap 94 subunit, and the signal UUUUUNU, which resides in the nascent mRNA, located 30 to 50 bases upstream from the poly(A) addition site. Evidence indicates that a required termination factor acts through binding to the UUUUUNU signal. To further investigate the function of UUUUUNU, the ability of UUUUUNU containing oligonucleotides to inhibit transcription termination was tested. A 22-mer RNA oligonucleotide containing a central U9 sequence exhibited sequence and concentration-dependent stimulation of premature transcription termination and transcript release, in trans. Activation of premature termination required VTF, NPH I, Rap 94, and ATP, demonstrating that the normal termination machinery was employed. Premature termination was not stimulated by RNA harboring a mutant UUUUUNU, demonstrating specificity. These data are consistent with a model in which a required termination factor is converted from an inactive to an active form by binding to a UUUUUNU containing oligonucleotide. The active termination factor then interacts with the ternary complex stimulating transcription termination through the normal mechanism, independent of the nascent mRNA sequence.
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PMID:UUUUUNU oligonucleotide stimulation of vaccinia virus early gene transcription termination, in trans. 1255 20

The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous triphosphatase catalytic domain. The LEF4 triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight stranded beta barrel (the so-called 'triphosphate tunnel'). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA triphosphatase. We infer that the baculovirus and poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Synergistic activation of the LEF4 triphosphatase by manganese and magnesium suggests a two-metal mechanism of gamma phosphate hydrolysis.
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PMID:Mapping the triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two-metal mechanism. 1259 53

The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded beta barrel (the so-called "triphosphate tunnel"). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery.
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PMID:Mapping the active site of vaccinia virus RNA triphosphatase. 1272 33

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) is an essential early gene transcription termination factor. The C-terminal end of NPH I binds to the N-terminal end of the H4L subunit (RAP94) of the virion RNA polymerase. This interaction is required for transcription termination and transcript release. To refine our understanding of the specific amino acids in the C-terminal end of NPH I involved in binding to H4L, and to develop a collection of mutations exhibiting various degrees of activity to be employed in in vivo studies, we prepared a set of short deletions, and clustered substitutions of charged amino acids to alanine, or bulky hydrophobic amino acids to alanine mutations. These NPH I mutant proteins were expressed, purified, and tested for ATPase activity, binding to H4L, and transcription termination activity. Most mutations in amino acids 609 to 631 exhibited reduced activity. Deletion of the terminal five amino acids (627-631), or substitution of Y(629) with alanine or glutamic acid, dramatically reduced NPH I mediated transcription termination. Deletion of the terminal F(631), or substitution of F(631) with alanine, reduced binding to H4L and eliminated termination activity. These observations demonstrate that the terminal five amino acids directly participate in binding to RNA polymerase and in early gene transcription termination.
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PMID:Effect of selected mutations in the C-terminal region of the vaccinia virus nucleoside triphosphate phosphohydrolase I on binding to the H4L subunit of the viral RNA polymerase and early gene transcription termination in vitro. 1278 35

Structural differences between poxvirus and human mRNA capping enzymes recommend cap formation as a target for antipoxviral drug discovery. Genetic and pharmacologic analysis of the poxvirus capping enzymes requires in vivo assays in which the readout depends on the capacity of the viral enzyme to catalyze cap synthesis. Here we have used the budding yeast Saccharomyces cerevisiae as a genetic model for the study of poxvirus cap guanine-N7 methyltransferase. The S. cerevisiae capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components. All three activities are essential for cell growth. We report that the methyltransferase domain of vaccinia virus capping enzyme (composed of catalytic vD1-C and stimulatory vD12 subunits) can function in lieu of yeast Abd1. Coexpression of both vaccinia virus subunits is required for complementation of the growth of abd1Delta cells. Previously described mutations of vD1-C and vD12 that eliminate or reduce methyltransferase activity in vitro either abolish abd1Delta complementation or elicit conditional growth defects. We have used the yeast complementation assay as the primary screen in a new round of alanine scanning of the catalytic subunit. We thereby identified several new amino acids that are critical for cap methylation activity in vivo. Studies of recombinant proteins show that the lethal vD1-C mutations do not preclude heterodimerization with vD12 but either eliminate or reduce cap methyltransferase activity in vitro.
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PMID:Yeast-based genetic system for functional analysis of poxvirus mRNA cap methyltransferase. 1280 28

Here, we present the genome sequence, with analysis, of a poxvirus infecting Nile crocodiles (Crocodylus niloticus) (crocodilepox virus; CRV). The genome is 190,054 bp (62% G+C) and predicted to contain 173 genes encoding proteins of 53 to 1,941 amino acids. The central genomic region contains genes conserved and generally colinear with those of other chordopoxviruses (ChPVs). CRV is distinct, as the terminal 33-kbp (left) and 13-kbp (right) genomic regions are largely CRV specific, containing 48 unique genes which lack similarity to other poxvirus genes. Notably, CRV also contains 14 unique genes which disrupt ChPV gene colinearity within the central genomic region, including 7 genes encoding GyrB-like ATPase domains similar to those in cellular type IIA DNA topoisomerases, suggestive of novel ATP-dependent functions. The presence of 10 CRV proteins with similarity to components of cellular multisubunit E3 ubiquitin-protein ligase complexes, including 9 proteins containing F-box motifs and F-box-associated regions and a homologue of cellular anaphase-promoting complex subunit 11 (Apc11), suggests that modification of host ubiquitination pathways may be significant for CRV-host cell interaction. CRV encodes a novel complement of proteins potentially involved in DNA replication, including a NAD(+)-dependent DNA ligase and a protein with similarity to both vaccinia virus F16L and prokaryotic serine site-specific resolvase-invertases. CRV lacks genes encoding proteins for nucleotide metabolism. CRV shares notable genomic similarities with molluscum contagiosum virus, including genes found only in these two viruses. Phylogenetic analysis indicates that CRV is quite distinct from other ChPVs, representing a new genus within the subfamily Chordopoxvirinae, and it lacks recognizable homologues of most ChPV genes involved in virulence and host range, including those involving interferon response, intracellular signaling, and host immune response modulation. These data reveal the unique nature of CRV and suggest mechanisms of virus-reptile host interaction.
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PMID:Genome of crocodilepox virus. 1664 Dec 89

The vaccinia virus-encoded D5 protein is an essential ATPase involved in viral DNA replication. We have expanded the genotypic and phenotypic analysis of six temperature-sensitive (ts) D5 mutants (Cts17, Cts24, Ets69, Dts6389 [also referred to as Dts38], Dts12, and Dts56) and shown that at nonpermissive temperature all of the tsD5 viruses exhibit a dramatic reduction in DNA synthesis and virus production. For Cts17 and Cts24, this restriction reflects the thermolability of the D5 proteins. The Dts6389, Dts12, and Dts56 D5 proteins become insoluble at 39.7 degrees C, while the Ets69 D5 protein remains stable and soluble and retains the ability to oligomerize and hydrolyze ATP when synthesized at 39.7 degrees C. To investigate which structural features of D5 are important for its biological and biochemical activities, we generated targeted mutations in invariant residues positioned within conserved domains found within D5. Using a transient complementation assay that assessed the ability of D5 variants to sustain ongoing DNA synthesis during nonpermissive Cts24 infections, only a wtD5 allele supported DNA synthesis. Alleles of D5 containing targeted mutations within the Walker A or B domains, the superfamily III helicase motif C, or the AAA+ motif lacked biological competency. Furthermore, purified preparations of these variant proteins revealed that they all were defective in ATP hydrolysis. Multimerization of D5 appeared to be a prerequisite for enzymatic activity and required the Walker B domain, the AAA+ motif, and a region located upstream of the catalytic core. Finally, although multimerization and enzymatic activity are necessary for the biological competence of D5, they are not sufficient.
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PMID:Biochemical and genetic analysis of the vaccinia virus d5 protein: Multimerization-dependent ATPase activity is required to support viral DNA replication. 1709 87


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