ABSTRACT
This review describes available data on the structure of viral RNA-dependent RNA polymerases (RdRP) obtained from X-ray analysis and discusses the functional significance of the structural elements of these enzymes. Because most of the studies done to date relate to RdRP structures of picorna-, flavi-, and caliciviruses, here we consider mostly the structures of RdRP of these groups of viruses, and also include information about polymerases of other virus families.
Subject(s)
RNA-Dependent RNA Polymerase/chemistry , Viral Proteins/chemistry , Viruses/enzymology , Crystallography, X-Ray , Humans , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Viruses/chemistry , Viruses/geneticsABSTRACT
The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryptophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3'-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.
Subject(s)
Mengovirus/enzymology , Mutant Proteins/metabolism , Mutation , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Mutant Proteins/genetics , Mutant Proteins/isolation & purification , RNA-Dependent RNA Polymerase/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
The ability of highly purified preparations of poliovirus RNA-dependent RNA polymerase, 3Dpol, to unwind RNA duplex structures was examined during a chain elongation reaction in vitro. Using an antisense RNA prehybridized to an RNA template, we show that poliovirus polymerase can elongate through a highly stable RNA duplex of over 1,000 bp. Radiolabeled antisense RNA was displaced from the template during the reaction, and product RNAs which were equal in length to the template strand were synthesized. Unwinding did not occur in the absence of chain elongation and did not require hydrolysis of the gamma-phosphate of ATP. The rate of elongation through the duplex region was comparable to the rate of elongation on the single-stranded region of the template. Parallel experiments conducted with avian myeloblastosis virus reverse transcriptase showed that this enzyme was not able to unwind the RNA duplex, suggesting that strand displacement by poliovirus 3Dpol is not a property shared by all polymerases.
Subject(s)
Poliovirus/enzymology , RNA, Double-Stranded/metabolism , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/metabolism , Transcription, Genetic , Adenosine Triphosphate/metabolism , Nucleic Acid Conformation , RNA, Antisense/metabolismABSTRACT
Three components of the male yellowfin Baikal sculpin pheromonal signal have been isolated from urine by diethyl ether extraction, thinayer chromatography (TLC), and high-performance liquid chromatography (HPLC). Using mass spectrometry, we have identified two of them as testosterone (T) and 11ß-hydroxytestosterone (11HT). These steroids are synthesized in testes during full spermatogenesis, and they are excreted in male urine with milt. The third component is not a steroid. It is more likely to be a polyene alcohol (farnesol). 2Z,6E-Farnesol possesses behavioral activity.
ABSTRACT
RNA template- and primer-dependent preparations of RNA polymerase were purified from encephalomyocarditis virus-infected Krebs-2 cells, using a three-step chromatographic procedure. The RNA duplex-unwinding activity of these preparations was investigated by two assays, using a partially double-stranded RNA template (encephalomyocarditis virus RNA annealed with a long segment of antisense transcript). Less purified preparations of the polymerase appeared to be able to efficiently displace, in an ATP-dependent and RNA elongation-dependent reaction, the antisense segment from the template. However, upon a more extensive purification, the unwinding activity of the RNA polymerase preparations was lost.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Encephalomyocarditis virus/enzymology , RNA Nucleotidyltransferases/metabolism , Chromatography , DNA-Directed RNA Polymerases/isolation & purification , RNA Helicases , RNA Nucleotidyltransferases/isolation & purification , RNA, Viral/metabolism , Templates, GeneticSubject(s)
Fishes/physiology , Neuronal Plasticity/physiology , Taste Buds/physiology , Afferent Pathways/drug effects , Afferent Pathways/physiology , Amino Acids/pharmacology , Animals , Feeding Behavior/physiology , Female , Male , Neuronal Plasticity/drug effects , Reproduction/physiology , Species Specificity , Taste Buds/drug effectsABSTRACT
An analysis of published nucleotide sequences of the 5'-untranslated region (5'-UTR) of 7 cardioviruses and 3 aphthoviruses has allowed us to derive a consensus secondary structure model that differs from that previously proposed for the 5'-UTR of entero- and rhinoviruses, though all these viruses belong to the same family, Picornaviridae. The theoretical model derived here was experimentally supported by investigating the accessibility of encephalomyocarditis virus RNA to modifications with dimethyl sulfate and its susceptibility to S1 and cobra venom nucleases. The possible involvement of the 5"-UTR secondary structure domains in the translational control is briefly discussed.
Subject(s)
Encephalomyocarditis virus/genetics , RNA, Viral/genetics , Animals , Base Sequence , Carcinoma, Krebs 2 , Mice , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotide Probes , RNA, Viral/isolation & purification , Species SpecificityABSTRACT
PTU-23, an effective in vivo wide-range enterovirus inhibitor, suppresses the reproduction of encephalomyocarditis (EMC) virus in Krebs-II ascites carcinoma cells at concentrations of 20-50 micrograms/ml, not affecting the cellular synthetic processes. The virus-specific RNA synthesis is distinctly inhibited. The studies on the kinetics of this effect point to its leading role in the inhibitory action the compound exerts on the production of infectious virions. The sedimentation profile in sucrose density gradient of the viral RNA isolated from PTU-23-treated cells by phenol extraction shows a clear inhibition of the synthesis of the single-stranded (ss) 37S RNA and to a lesser extent of the double-stranded (ds, RF) 20S RNA. The effect of the inhibitor on the RNA-dependent RNA polymerase in a cell-free RNA-synthesizing system has been studied, using an an enzyme preparation the 40000 g fraction of a nuclear-free extract from infected Krebs-II cells, containing the enzyme bound to the endogenous RNA template. It is established that the compound does, not affect the synthesis of the enzyme which could be probably explained by the incomplete (45-70%) inhibition of the viral RNA synthesis, its translatory function remaining unperturbed. The observed insignificant inhibition (20-26%) of the enzyme activity during the application of the inhibitor to the RNA-synthesizing reaction mixture cannot explain its effect on the viral RNA synthesis. An interaction of PTU-23 with another virus-specific protein component of the replication complex is suggested.
