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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni(2+)-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris.
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PMID:Expression of functional influenza virus RNA polymerase in the methylotrophic yeast Pichia pastoris. 1075 19

The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system. These studies revealed that the C-terminal 20-amino-acid region of P is absolutely required for transcription in vitro and luciferase expression in vivo, suggesting its critical role in viral RNA synthesis. The N-terminal 40-amino-acid region, on the other hand, is essential for luciferase expression but dispensable for transcription in vitro. Consistent with these findings, the C-terminal domain is required for binding of P protein to the N-RNA template involved in both transcription and replication, whereas the N-terminal domain is required for the formation of soluble N-P complex involved in encapsidation of nascent RNA chains during replication. Coimmunoprecipitation analysis showed that the P protein forms a stable homooligomer (perhaps a trimer) that is present in L-P and N-P complexes in the higher oligomeric forms (at least a pentamer). Interestingly, coexpression of a large excess of N- or C-terminally deleted P with wild-type P had no effect on minigenome replication in vivo, notwithstanding the formation of heterooligomeric complexes. These data indicate that P protein with a deleted terminal domain can function normally within the P heterooligomeric complex to carry out transcription and replication in vivo.
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PMID:Role of NH(2)- and COOH-terminal domains of the P protein of human parainfluenza virus type 3 in transcription and replication. 1084 69

The paramyxovirus P protein is an essential component of the viral RNA polymerase composed of P and L proteins. In this study, we characterized the physical and functional interactions between P and L proteins using human parainfluenza virus type 1 (hPIV1) and its counterpart Sendai virus (SV). The hPIV1 P and SV L proteins or the SV P and hPIV1 L proteins formed complexes detected by anti-P antibodies. Functional analysis using the minigenome SV RNA containing CAT gene indicated that the hPIV1 P--SV L complex, but not the SV P--hPIV1 L complex, was biologically active. Mutant SV P or hPIV1 P cDNAs, which do not express C proteins, showed the same phenotype with wild-type P cDNAs, indicating that C proteins are not responsible for the dysfunction of SV P--hPIV1 L polymerase complex. Using the chimeric hPIV1/SV P cDNAs, we identified two regions (residues 387--423 and 511--568) on P protein, which are required for the functional interaction with hPIV1 L. These regions overlap with a previously identified domain for oligomer formation and binding to nucleocapsids. Our results indicate that in addition to a P--L binding domain, hPIV1 L requires a specific region on P protein to be biologically functional as a polymerase.
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PMID:Two regions of the P protein are required to be active with the L protein for human parainfluenza virus type 1 RNA polymerase activity. 1133 55

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. For the molecular anatomy of this multifunctional enzyme, we have established a simultaneous expression of three P proteins in cultured insect cells using recombinant baculoviruses. For purification of P protein complexes, the PA protein was expressed as a fusion with a histidine tag added at its N terminus. By using affinity chromatography, a complex consisting of the three P proteins was isolated from nuclear extracts of virus-infected cells. The affinity-purified 3P complex showed the activities of capped RNA binding, capped RNA cleavage, viral model RNA binding, model RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of the viral RNA polymerase with the catalytic specificity of transcriptase is formed in recombinant baculovirus-infected insect cells. Using the viral RNA-free 3P complex, we found that the capped RNA cleavage takes place in the presence of vRNA but not of cRNA, indicating that the vRNA functions as a regulatory factor for the specificity control of viral RNA polymerase as well as a template for transcription. The structural elements of RNA directing the expression of RNA polymerase functions were analyzed using variant forms of the model RNA templates.
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PMID:Differential roles of viral RNA and cRNA in functional modulation of the influenza virus RNA polymerase. 1137 86

The phosphoprotein (P) of human respiratory syncytial virus (RSV) is an essential component of the viral RNA polymerase, along with the large polymerase (L), nucleocapsid (N), and M2-1 proteins. By screening a randomly mutagenized P gene cDNA library, two independent mutations, one with a substitution of glycine at position 172 by serine (G172S) and the other with a substitution of glutamic acid at position 176 by glycine (E176G), were identified to result in the loss of N-P interaction at 37 degrees C in the yeast two-hybrid assay. Both P mutants exhibited greatly reduced activity in supporting the replication and transcription of an RSV minigenome replicon at 37 and 39 degrees C. The G172S and E176G mutations were introduced individually into the RSV A2 (rA2) antigenomic cDNA, and recombinant viruses, rA2-P172 and rA2-P176, were obtained. Both viruses replicate as well as wild-type A2 virus in both Vero and HEp-2 cells at 33 degrees C, but each mutant virus exhibited temperature-sensitive replication in both cell lines. rA2-P176 is more temperature sensitive than rA2-P172. Coimmunoprecipitation of the N protein with each P mutant from virus-infected cells demonstrates that N-P interaction is impaired at 37 degrees C. In addition, the levels of replication of rA2-P172 and rA2-P176 in the lungs of mice and cotton rats were reduced. As is the case with the in vitro assays, rA2-P176 is more restricted in replication in the lower respiratory tract of mice and cotton rats than rA2-P172. During in vitro passage at 37 degrees C, the E176G mutation in rA2-P176 was rapidly changed from glycine to predominantly aspartic acid; mutations to cysteine or serine were also detected. All of the revertants lost the temperature-sensitive phenotype. To analyze the importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro. A double mutant containing both G172S and E176G changes in the P gene, substitution of the three charged residues at positions 174 to 176 by alanine, and a deletion of residues from positions 161 to 180 completely abolished the P protein function in the minigenome assay. Thus, the amino acids at positions 172 and 176 and the adjacent charged residues play critical roles in the function of the P protein.
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PMID:Identification of temperature-sensitive mutations in the phosphoprotein of respiratory syncytial virus that are likely involved in its interaction with the nucleoprotein. 1186 54

The RNA polymerase complex of human parainfluenza virus type 3 (HPIV 3), a member of the family Paramyxoviridae, is composed of two virally encoded polypeptides: a multifunctional large protein (L, 255 kDa) and a phosphoprotein (P, 90 kDa). From extensive deduced amino acid sequence analyses of the cDNA clones of a number of L proteins of nonsegmented negative-strand RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. Here, we have focused on the NH(2)-terminal domain of HPIV 3 L protein that is also highly conserved. Following mutational analyses within this domain, we examined the ability of the mutant L proteins to (i) transcribe an HPIV 3 minireplicon, (ii) transcribe the viral RNA in vitro using the HPIV 3 nucleocapsid RNA template, and (iii) interact with HPIV 3 P protein. Our results demonstrate that the first 15 amino acids of the NH(2)-terminal domain spanning a highly conserved motif is directly involved in transcription of the genome RNA and in forming a functional complex with the P protein. Substitution of eight nonconserved amino acids within this domain by the corresponding Sendai virus L protein residues yielded mutants with variable transcriptional activities. However, one mutant in which all eight amino acids were replaced with the corresponding residues of Sendai virus L protein failed to both transcribe the minireplicon and interact with HPIV 3 P and the Sendai virus P protein. The possible functional significance of the NH(2)-terminal domain of paramyxovirus L protein is discussed.
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PMID:Role of a highly conserved NH(2)-terminal domain of the human parainfluenza virus type 3 RNA polymerase. 1213 15

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo. We have previously shown that the P protein of VSV, expressed in E. coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII). In the present study we present evidence that the P protein, in addition to being phosphorylated, binds covalently to GTP only when it is phosphorylated. Competition experiments show that ATP, ADP, GTP, and GDP can compete for the binding site(s) of GTP but not AMP, GMP, CTP, or UTP. Interestingly, once GTP is bound to P protein it cannot be displaced by unlabeled GTP. The GTP binding site has been mapped within the domain where the phosphorylation of P protein by CKII occurs. Finally, we show that phosphorylation negative P mutants P3A (P60A, P62A, P64A), P3E (P60E, P62E, P64E), and P3R (P60R, P62R, P64R) failed to bind to GTP, indicating that phosphorylation of P is indeed essential for binding to GTP. Although the precise role of binding of GTP to P is unclear, it appears that phosphorylation of P may initiate a structural change within the P protein allowing GTP to bind, thus manifesting biological function to the transcription factor.
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PMID:Novel binding of GTP to the phosphoprotein (P) of vesicular stomatitis virus. 1217 45

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. The PB1 subunit plays a key role in both the assembly of three P protein subunits and the catalytic function of RNA polymerization. We have established a simultaneous expression system of three P proteins in various combinations using recombinant baculoviruses, and isolated the PA-PB1-PB2 ternary (3P) complex and two kinds of the binary (2P) complex, PA-PB1 and PB1-PB2. The affinity-purified 3P complex showed all of the catalytic properties characteristic of the transcriptase, including capped RNA-binding, capped RNA cleavage, model viral RNA binding, model viral RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. The PB1-PB2 binary complex showed essentially the same catalytic properties as does the 3P complex, whereas the PA-PB1 complex catalyzed de novo initiation of RNA synthesis in the absence of primers. Taken together we propose that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA.
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PMID:Minimum molecular architectures for transcription and replication of the influenza virus. 1227 Nov 17

The phosphoprotein (P) of human parainfluenza virus type 3 (HPIV 3) plays a central role in the viral genome RNA transcription and replication. It acts as an essential cofactor of the RNA polymerase (L) by forming a functional L-P complex, binds to the genomic N-RNA template to recruit the L-P complex for RNA synthesis, and interacts with the nucleocapsid protein (N) to form the encapsidation complex (N-P). We have earlier demonstrated that the P protein forms oligomers (B. P. De, M. A. Hoffman, S. Choudhary, C. C. Huntley, and A. K. Banerjee, 2000, J. Virol. 74, 5886-5895) and in this article we identified the putative oligomerization domain of the P protein and studied the role of this domain in transcription. By computer analyses, we have localized a high-score coiled-coil motif characteristic of oligomerization domain residing between the amino acid residues 423 and 457 of the P protein. Deletion of 12 amino acid residues within this coiled-coil motif (P Delta 439-450) completely abrogated oligomerization, whereas deletion in other regions outside the motif had no significant effect. The mutant P Delta 439-450 was both defective in mRNA synthesis in vitro and minigenome transcription in vivo. Interestingly, the mutant interacted with L to form L-P complex, albeit less efficiently, while its interaction with N protein to form N-P complex and with N-RNA template was similar to the wt P protein. Our results indicate that oligomerization provides a key function to the P protein in the transcription of HPIV 3 genome RNA.
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PMID:Characterization of the oligomerization domain of the phosphoprotein of human parainfluenza virus type 3. 1244 Oct 81

Our laboratory's recent observations that transcriptionally inactive phosphoprotein (P) mutants can efficiently function in replicating vesicular stomatitis virus (VSV) defective interfering particle in a three-plasmid-based (L, P, and N) reverse genetics system in vivo (A. K. Pattnaik, L. Hwang, T. Li, N. Englund, M. Mathur, T. Das, and A. K. Banerjee, J. Virol. 71:8167-8175, 1997) led us to propose that a tripartite complex consisting of L-(N-P) protein may represent the putative replicase for synthesis of the full-length genome RNA. In this communication we demonstrate that such a complex is indeed detectable in VSV-infected BHK cells. Furthermore, coexpression of L, N, and P proteins in Sf21 insect cells by recombinant baculovirus containing the respective genes also resulted in the formation of a tripartite complex, as shown by immunoprecipitation with specific antibodies. A basic amino acid mutant of P protein, P260A, previously shown to be inactive in transcription but active in replication (T. Das, A. K. Pattnaik, A. M. Takacs, T. Li, L. N. Hwang, and A. K. Banerjee, Virology 238:103-114, 1997) was also capable of forming the mutant [L-(N-Pmut)] complex in both insect cells and BHK cells. Sf21 extract containing either the wild-type P protein or the mutant P protein along with the L and N proteins was capable of synthesizing 42S genome-sense RNA in an in vitro replication reconstitution reaction. Addition of N-Pmut or wild-type N-P complex further stimulated the synthesis of the genome-length RNA. These results indicate that the transcriptase and replicase complexes of VSV are possibly two distinct entities involved in carrying out capped mRNAs and uncapped genome and antigenome RNAs, respectively.
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PMID:Identification of a novel tripartite complex involved in replication of vesicular stomatitis virus genome RNA. 1247 78


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