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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 plays a key role in initiating viral replication and is an important target for developing anti-HIV drugs. Our previous study showed that two mutations (Y271A and I274A) in the turn RT (Gln(269)-Arg(277)) abrogated viral replication, but the replication capacity and RT activity was discordant. In this study, we further investigated why alanine substitutions at these two sites would affect viral replication. We found that both RT activity and RT protein were almost undetectable in viral particles of these two mutants, although the Pr160(gag-pol) mutants were properly expressed, transported and incorporated. Using protease inhibition assay, we demonstrated a correlation between the degradation of the RT mutants and the activity of viral protease. Our native gel analysis indicated that the mutations at 271 and 274 amino acids might cause conformational changes, leading to the formation of higher order oligomers instead of dimers, resulting in increased protein instability and susceptibility to viral protease. Thus, residues 271 and 274 are critical to RT stability and resistance to viral protease. The conservation of the two amino acid residues among different strains of HIV-1 lent further support to this conclusion. The knowledge gained here may prove useful in drug design.
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PMID:The y271 and i274 amino acids in reverse transcriptase of human immunodeficiency virus-1 are critical to protein stability. 1957 44

Nucleoside reverse transcriptase inhibitors (NRTIs) are employed in first line therapies for the treatment of human immunodeficiency virus (HIV) infection. They generally lack a 3'-hydroxyl group, and thus when incorporated into the nascent DNA they prevent further elongation. In this report we show that 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), a nucleoside analog that retains a 3'-hydroxyl moiety, inhibited HIV-1 replication in activated peripheral blood mononuclear cells with an EC(50) of 0.05 nm, a potency several orders of magnitude better than any of the current clinically used NRTIs. This exceptional antiviral activity stems in part from a mechanism of action that is different from approved NRTIs. Reverse transcriptase (RT) can use EFdA-5'-triphosphate (EFdA-TP) as a substrate more efficiently than the natural substrate, dATP. Importantly, despite the presence of a 3'-hydroxyl, the incorporated EFdA monophosphate (EFdA-MP) acted mainly as a de facto terminator of further RT-catalyzed DNA synthesis because of the difficulty of RT translocation on the nucleic acid primer possessing 3'-terminal EFdA-MP. EFdA-TP is thus a translocation-defective RT inhibitor (TDRTI). This diminished translocation kept the primer 3'-terminal EFdA-MP ideally located to undergo phosphorolytic excision. However, net phosphorolysis was not substantially increased, because of the apparently facile reincorporation of the newly excised EFdA-TP. Our molecular modeling studies suggest that the 4'-ethynyl fits into a hydrophobic pocket defined by RT residues Ala-114, Tyr-115, Phe-160, and Met-184 and the aliphatic chain of Asp-185. These interactions, which contribute to both enhanced RT utilization of EFdA-TP and difficulty in the translocation of 3'-terminal EFdA-MP primers, underlie the mechanism of action of this potent antiviral nucleoside.
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PMID:Mechanism of inhibition of HIV-1 reverse transcriptase by 4'-Ethynyl-2-fluoro-2'-deoxyadenosine triphosphate, a translocation-defective reverse transcriptase inhibitor. 1983 73

The emergence of the extremely aggressive influenza recently has highlighted the urgent need for new effective treatments. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer including PA, PB1 and PB2 has crucial roles in viral RNA replication and transcription. The highly conserved PB1 binding site on PA can be considered as a novel potential drug target site. The interaction between PB1 binding site and PA is crucial to many functions of the virus. In this study, to understand the detailed interaction profile and to characterize the binding hot spots in the interactions of the PA-PB1 complex, an 8 ns molecular dynamics simulation of the subunit PA-PB1 combined with MM-PBSA (molecular mechanics Poisson-Boltzmann surface area), MM-GBSA (molecular mechanics generalized Born surface area) computations and virtual alanine scanning were performed. The results from the free energy decomposition indicate that the intermolecular van der Waals interaction and the nonpolar solvation term provide the driving force for binding process. Through the pair interaction analysis and virtual alanine scanning, we identified the binding hot spots of PA and the basic binding motif of PB1. This information can provide some insights for the structure-based RNA-dependent RNA polymerase inhibitors design. The identified binding motif can be used as the starting point for the rational design of small molecules or peptide mimics. This study will also lead to new opportunities toward the development of new generation therapeutic agents exhibiting specificity and low resistance to influenza virus.
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PMID:Molecular basis of the interaction for an essential subunit PA-PB1 in influenza virus RNA polymerase: insights from molecular dynamics simulation and free energy calculation. 1988 12

Novel double-stranded RNAs (approximately 8 kbp) were isolated from threecornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA polymerase (RdRp). Expression of the 3'-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3.
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PMID:Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses. 2054 86

The NS3-NS5B interaction of classical swine fever virus (CSFV) is important for viral replication. For characterisation of the interaction between the NS3 and NS5B, a series of NS5B mutants with deletion of N-, C-terminal amino acids and quadruple alanine substitution mutations were produced. GST pull-down assays and immunoprecipitation analyses showed that NS5B and some NS5B mutants have NS3 binding activity. Further experimental data indicated that CSFV NS5B might contain two NS3 binding sites, one covering amino acids 63-99 located at the N-terminal end, another covering amino acids 611-642 at the C-terminal end. Assays for RNA-dependent RNA polymerase (RdRp) activity revealed that CSFV NS3 is able to enhance the RdRp activity of NS5B and some NS5B mutants in vitro. The enhancement might be obtained by NS3 binding to the two terminal sequences of NS5B, which could be attractive targets for drug development against CSFV.
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PMID:Characterisation of interaction between NS3 and NS5B protein of classical swine fever virus by deletion of terminal sequences of NS5B. 2123 22

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.
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PMID:Functional analysis of two cavities in flavivirus NS5 polymerase. 2134 34

The viral RNA-dependent RNA polymerase (vRdRp) of paramyxovirus consists of the large (L) protein and the phosphoprotein (P). P is heavily phosphorylated, and it is thought that the phosphorylation of P plays a role in regulating viral RNA synthesis. However, no phosphorylation site within the P protein in paramyxovirus has been identified as playing a positive role in viral RNA synthesis in virus infection. Using mass spectrometry analysis, the threonine residue at position 286 of P of parainfluenza virus 5 (PIV5) was found phosphorylated. Mutation of T286 to alanine (T286A), aspartic acid (T286D), or glutamic acid (T286E) reduced minigenome activity. Recombinant virus containing a mutation at the T286 position (rPIV5-P-T286A) grew slower than wild-type virus; viral mRNA synthesis and protein expression of rPIV5-P-T286A were delayed. Biochemical studies showed that the binding of NP or L protein with the P mutants or tetramer formation by the mutant P proteins was unaltered from that for wild-type P. While we failed to rescue rPIV5-P-T286E virus, several revertant viruses were obtained. All non-wild-type revertants had mutations at T286 and showed defects in both minigenome activity and viral growth. This is the first time that a phosphorylation site within the P protein in paramyxovirus has been found to play a positive role in viral mRNA synthesis and virus growth.
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PMID:Identification of a phosphorylation site within the P protein important for mRNA transcription and growth of parainfluenza virus 5. 2168 May 23

Positive-strand RNA viruses within the Picornaviridae family express an RNA-dependent RNA polymerase, 3D(pol), that is required for viral RNA replication. Structures of 3D(pol) from poliovirus, coxsackievirus, human rhinoviruses, and other picornaviruses reveal a putative template RNA entry channel on the surface of the enzyme fingers domain. Basic amino acids and tyrosine residues along this entry channel are predicted to form ionic and base stacking interactions with the viral RNA template as it enters the polymerase active site. We generated a series of alanine substitution mutations at these residues in the poliovirus polymerase and assayed their effects on template RNA binding, RNA synthesis initiation, rates of RNA elongation, elongation complex (EC) stability, and virus growth. The results show that basic residues K125, R128, and R188 are important for template RNA binding, while tyrosines Y118 and Y148 are required for efficient initiation of RNA synthesis and for EC stability. Alanine substitutions of tyrosines 118 and 148 at the tip of the 3D(pol) pinky finger drastically decreased the rate of initiation as well as EC stability, but without affecting template RNA binding or RNA elongation rates. Viable poliovirus was recovered from HeLa cells transfected with mutant RNAs; however, mutations that dramatically inhibited template RNA binding (K125A-K126A and R188A), RNA synthesis initiation (Y118A, Y148A), or EC stability (Y118A, Y148A) were not stably maintained in progeny virus. These data identify key residues within the template RNA entry channel and begin to define their distinct mechanistic roles within RNA ECs.
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PMID:A template RNA entry channel in the fingers domain of the poliovirus polymerase. 2232 98

Caliciviridae are RNA viruses with a single-stranded, positively oriented polyadenylated genome, responsible for a broad spectrum of diseases such as acute gastroenteritis in humans. Recently, analyses on the structures and functionalities of the RNA-dependent RNA polymerase (RdRp) from several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in synthesis and amplification of additional subgenomic RNA. Starting from the crystal structures of human Norovirus (hNV) RdRp, we performed an in silico docking search to identify synthetic compounds with predicted high affinity for the enzyme active site. The best-ranked candidates were tested in vitro on murine Norovirus (MNV) and hNV RdRps to assay their inhibition of RNA polymerization. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: Suramin and NF023, both symmetric divalent molecules hosting two naphthalene-trisulfonic acid heads. We report here the crystal structure of MNV RdRp alone and in the presence of the two identified inhibitors. Both inhibitory molecules occupy the same RdRp site, between the fingers and thumb domains, with one inhibitor head close to residue 42 and to the protein active site. To further validate the structural results, we mutated Trp42 to Ala in MNV RdRp and the corresponding residue (i.e., Tyr41 to Ala) in hNV RdRp. Both NF023 and Suramin displayed reduced inhibitory potency versus the mutated hNV RdRp, thus hinting at a conserved inhibitor binding mode in the two polymerases.
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PMID:Structure-based inhibition of Norovirus RNA-dependent RNA polymerases. 2244 84

Rubella virus (RUBV), a positive-strand RNA virus, replicates its RNA within membrane-associated replication complexes (RCs) in the cytoplasm of infected cells. RNA synthesis is mediated by the nonstructural proteins (NSPs) P200 and its cleavage products, P150 and P90 (N and C terminal within P200, respectively), which are processed by a protease residing at the C terminus of P150. In this study of NSP maturation, we found that early NSP localization into foci appeared to target the membranes of the endoplasmic reticulum. During maturation, P150 and P90 likely interact within the context of P200 and remain in a complex after cleavage. We found that P150-P90 interactions were blocked by mutational disruption of an alpha helix at the N terminus (amino acids [aa] 36 to 49) of P200 and that these mutations also had an effect on NSP targeting, processing, and membrane association. While the P150-P90 interaction also required residues 1700 to 1900 within P90, focus formation required the entire RNA-dependent RNA polymerase (aa 1700 to 2116). Surprisingly, the RUBV capsid protein (CP) rescued RNA synthesis by several alanine-scanning mutations in the N-terminal alpha helix, and packaged replicon assays showed that rescue could be mediated by CP in the virus particle. We hypothesize that CP rescues these mutations as well as internal deletions of the Q domain within P150 and mutations in the 5' and 3' cis-acting elements in the genomic RNA by chaperoning the maturation of P200. CP's ability to properly target the otherwise aggregated plasmid-expressed P200 provides support for this hypothesis.
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PMID:Determinants in the maturation of rubella virus p200 replicase polyprotein precursor. 2249 63


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