Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection

We have analyzed the divalent cation specificity of poliovirus RNA-dependent RNA polymerase, 3D(pol). The following preference was observed: Mn(2+) > Co(2+) > Ni(2+) > Fe(2+) > Mg(2+) > Ca(2+) > Cu(2+), and Zn(2+) was incapable of supporting 3D(pol)-catalyzed nucleotide incorporation. In the presence of Mn(2+), 3D(pol) activity was increased by greater than 10-fold relative to that in the presence of Mg(2+). Steady-state kinetic analysis revealed that the increased activity observed in the presence of Mn(2+) was due, primarily, to a reduction in the K(M) value for 3D(pol) binding to primer/template, without any significant effect on the K(M) value for nucleotide. The ability of 3D(pol) to catalyze RNA synthesis de novo was also stimulated approximately 10-fold by using Mn(2+), and the enzyme was now capable of also utilizing a DNA template for primer-independent RNA synthesis. Interestingly, the use of Mn(2+) as divalent cation permitted 3D(pol) activity to be monitored by following extension of 5'-(32)P-end-labeled, heteropolymeric RNA primer/templates. The kinetics of primer extension were biphasic because of the enzyme binding to primer/template in both possible orientations. When bound in the incorrect orientation, 3D(pol) was capable of efficient addition of nucleotides to the blunt-ended duplex; this activity was also apparent in the presence of Mg(2+). In the presence of Mn(2+), 3D(pol) efficiently utilized dNTPs, ddNTPs, and incorrect NTPs. On average, three incorrect nucleotides could be incorporated by 3D(pol). The ability of 3D(pol) to incorporate the correct dNTP, but not the correct ddNTP, was also observed in the presence of Mg(2+). Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3D(pol)-catalyzed nucleotide incorporation.     

Inhibitory effects of nucleoside triphosphates on nucleolar RNA synthesis.

Studies on the effects of substrates on RNA polymerase I [EC 2.7.7.6] in vitro showed that nucleolar RNA synthesis was inhibited by an excess of substrate nucleoside triphosphates in the presence of Mg2+. GTP and UTP were more inhibitory than CTP and ATP. These compounds specfically inhibited nucleolar RNA synthesis and a concentration of GTP that strongly inhibited nucleolar RNA synthesis did not inhibit RNA synthesis by partially purified RNA polymerase I. The inhibition of nucleolar RNA synthesis disappeared at pH 9.0 without any change in the apparent Km for GTP or the Vmax of RNA synthesis.     

Glycerol metabolism in higher plants: glycerol kinase

Glycerol kinase activity was identified in extracts of higher plant seeds and seedlings, and was partially purified and characterized from cucumber radicle tissue. The enzyme was localized in the post-mitochondrial supernatant of the cell, and catalyzed the formation of glycerol-3-phosphate. The pH optiumum was 9.0. ATP, CTP, GTP or UTP could be used as the phosphoryl group donor. The Km for glycerol was 55 microM and Km values for the nucleoside triphosphates were 145-620 microM. The Vmax for the reaction was 40-78 pmol product per min. Kinetic data indicate that the enzyme has a sequential mechanism.     

Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China

Enterovirus 71 (EV71), one of the major causative agents for hand-foot-and-mouth disease (HFMD), has caused more than 100 deaths among Chinese children since March 2008. The EV71 genome encodes an RNA-dependent RNA polymerase (RdRp), denoted 3D^pol, which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics. Here we report the crystal structures of EV71 RdRp (3D^pol) and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4 ? resolution. The structure of EV71 RdRp (3D^pol) has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp. And the EV71 RdRp (3D^pol) complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding. The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus (FMDV) 3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase. These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.     

De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation

The replicase activity of rotavirus open cores has been used to study the synthesis of (-) strand RNA from viral (+) strand RNA in a cell-free replication system. The last 7 nt of the (+) strand RNA, 5'-UGUGACC-3', are highly conserved and are necessary for efficient (-) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (-) strand synthesis. By preincubating open cores with (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (-) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (-) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (-) strand synthesis initiated at the 3'-terminal C residue of the (+) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 3' end of the (+) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (-) strand synthesis and that the synthesis of pGpG may occur by a template-independent process. From these data, we conclude that initiation of rotavirus (-) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (+) strand RNA, and possibly a 5'-phosphorylated dinucleotide, that is, pGpG or ppGpG.     

Interactions of calf thymus DNA polymerase alpha with primer/templates.

The interactions of calf thymus DNA polymerase alpha (pol alpha) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol alpha (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the abilit of pol alpha to discriminate against nucleotide analogs, it did not compromise the ability of pol alpha to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol alpha strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol alpha.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: purification and enzymatic analysis of the RNA-dependent RNA polymerase 3D(pol)

The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3D(pol). Using in vitro assay systems previously described for poliovirus RNA polymerase 3D(pol) (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3D(pol) is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn(2+), and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)(15) primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5' end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.     

Calcium transport and calcium-ATPase activity in human lymphocyte plasma membrane vesicles

We have studied Ca transport and the Ca-activated Mg-ATPase in plasma membrane vesicles prepared from normal human lymphocytes. Membrane vesicles that were exposed to oxalate as a Ca-trapping agent accumulated Ca in the presence of Mg2+ and ATP. ADP, AMP, GTP, UTP, ITP, TTP, or CTP did not substitute for ATP in energizing uptake. The Vmax for Ca uptake was 2.4 pmol of Ca/micrograms of protein/min, and the Km values for Ca and ATP were 1.0 and 80 microM, respectively. One microM A23187, added initially, completely inhibited net Ca uptake and, if added later, caused the release of Ca accumulated previously. Cyanide, oligomycin, ouabain, or varying Na+ or K+ concentrations had no effect on Ca uptake. A Ca-activated ATPase was present in the same membrane vesicles, which had a Vmax of 25 pmol of Pi/micrograms of protein/min at a free Ca concentration of 4-5 microM. This Ca-ATPase had Km values for Ca and ATP of 0.6 and 90 microM, respectively. These kinetic parameters were similar to those observed for uptake of Ca by the vesicles. The Ca-ATPase activity was insensitive to azide, oligomycin, ouabain, or varying Na+ or K+ concentrations. No Ca-activated hydrolysis of GTP or UTP was observed. Both Ca transport and the Ca-ATPase activity of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-treated lymphocyte plasma membranes were stimulated 2-fold by a cytoplasmic component (calmodulin) that was purified 500-fold from lymphocyte cytoplasm. Thus, human lymphocyte plasma membranes have both a Ca transport activity and a Ca-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivities to calmodulin.     

DNA primase-DNA polymerase alpha from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates

DNA primase-DNA polymerase alpha, purified 53,000-fold from CV-1 cells, synthesized predominantly (p)ppA(pA)6-primed DNA on a poly(dT) template. About 80% of the RNA primers synthesized on an M13 DNA template were (p)ppA/G(pN)5-7, and 20% were (p)ppA/G(pN)0-4. RNA primer size was determined by gel electrophoresis after removing nascent DNA with phage T4 DNA polymerase 3'-5' exonuclease, leaving a single dNMP at the 3'-end of the RNA primer, and the terminal 5'-(p)ppN residue was determined by "capping" with [alpha-32P]GTP using vaccinia guanylyl-transferase. The processivity of DNA synthesis initiated by de novo synthesis of RNA primers was the same as that initiated on pre-existing RNA primers (10-15 dNMPs), although initiation on pre-existing primers was strongly preferred. Primers always began with A or G, even at high levels of CTP or UTP, although the ratio of A to G varied from 4:1 to 1:1 depending on the relative concentrations of ATP and GTP in the assay. ATP and GTP had no effect on primer length, but the fraction of shorter RNA primers increased 2-fold with higher concentrations of CTP or UTP. Nearest-neighbor analysis revealed a preference for purine ribonucleotides at RNA covalently linked to the 5'-end of DNA (RNA-p-DNA) junctions, and increasing the concentration of a single rNTP increased slightly its presence at RNA-p-DNA junctions. Thus, the base composition and size of RNA primers synthesized by DNA primase-DNA polymerase alpha is modulated by the relative concentrations of ribonucleoside triphosphates.     

Distinct interactions of GTP,UTP, and CTP with G(s) proteins

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G(s) protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G(s) activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of beta(2)AR-G(s) coupling than native systems. Nucleoside 5'-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP > or = UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(s) in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP > or = UTP > or = CTP > or = GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Galpha(s) than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G(s) proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach approximately 10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.     

Kinetics of a nucleotide pyrophosphatase in the plasma membrane of the fat cell

Fat cells from rat and rabbit hydrolyzed externally applied adenosine triphosphate at a rate of about 1.8 nmol times mg(-1) cells times min(-1) corresponding to about 0.3 mumol times mg(-1) protein tinus min(-1). Similar activities were found in cell homogenates. In purified adipocyte plasma membranes the rate of hydrolysis was about 1.8 mumol times mg(-1) protein times min(-1). The hydrolytic activity was dependent on divalent metal ions. Mg(2+), Mn(2+) and Ca(2+) gave highest activities. The activity was maximal at about equimolar concentrations of M(2+) and ATP. Km for MgATP was about 0.23 mM and for CaATP about 0.36 mM. Combinations of Mg(2+) and Ca(2+), or of Mg(2+), Na(+) and K(+) gave similar activities as did Mg(2+) only. At concentrations of 1 mM the following nucleotides were hydrolyzed with a decreasing rate: ATP > ITP > GTP > UTP = CTP. In isolated fat cells the beta-adrenergic drug isoproterenol and insulin slightly increased the rate of hydrolysis of external ATP, while the alpha-effector clonidine was inhibitory. The results suggest that a major portion of the ATP hydrolytic activity of the fat cell plasma membrane represents a nucleotide pyrophosphatase activity with access to externally applied ATP.     

Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.     

Potentiation by guanine nucleotides of the VIP-induced adenylate cyclase stimulation in intestinal epithelial cell membranes

The GTP analog 5′-quanylyl-imidodiphosphate Gpp(NH) p potentiated the action of VIP on adenylate cyclase from intestinal epithelial cell membranes. The other nucleotides tested were also active on adenylate cyclase with the following order of potency GTP>GDP>GMP>ITP>UTP=CTP. Guanine nucleotides act by increasing the Vmax of the enzyme activity and by decreasing the Km of enzyme activation by VIP. Activation of the peptide-induced adenylate cyclase activity by Gpp (NH) p was inhibited by GTP and the other nucleotides with the same order and range of potency than those observed for their intrinsic stimulatory effect on adenylate cyclase. These data demonstrate the potent and specific action of quanine nucleotides on the VIP-sensitive adenylate cyclase.     

Synthesis of ribonucleic acid by isolated rat liver mitochondria

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.     

Mitochondrial adenosine triphosphatase from human placenta--purification and catalytic properties

1. The purification of ATPase (EC 3.6.1.3) from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATP hydrolysis was 85 mumole per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP greater than ITP greater than ATP greater than epsilon-ATP greater than UTP greater than CTP in Tris-HCl buffer (pH 8.0), and in the order: ATP greater than GTP greater than or equal to ITP greater than epsilon-ATP greater than UTP greater than CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (Km = 0.2 mM, 0.09 mM) and monophastic in Tris-becarbonate medium (Km = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.     

GeXP多重基因表达遗传分析系统在手足口病病原分型检测中的应用

利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应(RT-PCR)方法,同时检测引起手足口病的9种常见的人肠道病毒—人肠道病毒71型(HEV71)、柯萨奇病毒A组(CVA)16、4、5、9、10型和柯萨奇病毒B组(CVB)1、3、5型。优化多重反应体系中针对5’UTR区的肠道病毒通用引物和11对针对9种血清型人肠道病毒VP1区的特异性引物的浓度比例,分别以病毒细胞培养物和阳性粪便标本来验证多重反应体系的特异性,以TCID50定量的细胞培养物和克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度。结果表明,优化后的多重检测体系,可扩增出人肠道病毒共有的保守片段的和型特异性片段,HEV71和CVA16细胞培养物的检测下限为100.5TCID50/μL,并可在103copies/μL水平同时、特异地检测出9种病毒RNA。该方法灵敏度高、特异性强,可快速对大量临床样本进行高通量检测,用于手足口病的分子流行病学调查。     

Kinetic analysis of C-terminally truncated RNA-dependent RNA polymerase of hepatitis C virus.

The biochemical properties of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) truncated with C-terminal 21 amino acids and expressed in insect cells were analyzed. The enzyme carried copy-back and de novo RNA synthesis activity but not terminal nucleotidyl transferase activity. k(pol) and K(m) for de novo RNA synthesis were calculated as 10.0 pmol/microg/h and 2.5 microM under 0.5 mM GTP and 2.0 pmol/microg/h and 3.5 microM under 50 microM GTP, respectively. Those for copy-back RNA synthesis were similar under both conditions (k(pol), 1.8 pmol/microg/h; K(m), 3.0 microM). De novo RNA synthesis was activated by 0.5 mM GTP. However, the ratio of GTP to three other NTPs was important for activation. Our HCV RdRp showed high activity for the complementary sequence of the HCV internal ribosomal entry site and a synergistic effect of Mg(2+) to Mn(2+).