Processing of a cellular polypeptide by 3CD proteinase is required for poliovirus ribonucleoprotein complex formation.

Poliovirus interactions with host cells were investigated by studying the formation of ribonucleoprotein complexes at the 3' end of poliovirus negative-strand RNA which are presumed to be involved in viral RNA synthesis. It was previously shown that two host cell proteins with molecular masses of 36 and 38 kDa bind to the 3' end of viral negative-strand RNA at approximately 3 to 4 h after infection. We tested the hypothesis that preexisting cellular proteins are modified during the course of infection and are subsequently recruited to play a role in viral replication. It was demonstrated that the 38-kDa protein, either directly or indirectly, is the product of processing by poliovirus 3CD/3C proteinase. Only the modified 38-kDa protein, not its precursor protein, has a high affinity for binding to the 3' end of viral negative-strand RNA. This modification depends on proteolytically active proteinase, and a direct correlation between the levels of 3CD proteinase and the 38-kDa protein was demonstrated in infected tissue culture cells. The nucleotide (nt) 5-10 region (positive-strand numbers) of poliovirus negative-strand RNA is important for binding of the 38-kDa protein. Deletion of the nt 5-10 region in full-length, positive-strand RNA renders the RNA noninfectious in transfection experiments. These results suggest that poliovirus 3CD/3C proteinase processes a cellular protein which then plays an essential role during the viral life cycle.     

Modulation of the RNA binding and protein processing activities of poliovirus polypeptide 3CD by the viral RNA polymerase domain

To study the role of the RNA polymerase domain (3D) in the proteinase substrate recognition and RNA binding properties of poliovirus polypeptide 3CD, we generated recombinant 3C and 3CD polypeptides and purified them to near homogeneity. By using these purified proteins in in vitro cleavage assays with structural and non-structural viral polyprotein substrates, we found that 3CD processes the poliovirus structural polyprotein precursor (P1) 100 to 1000 times more efficiently than 3C processes P1. We also found that trans-cleavage of other 3CD molecules and sites within the non-structural P3 precursor is more efficiently mediated by 3CD than 3C. However, 3C and 3CD appear to be equally efficient in the processing of a non-structural polyprotein precursor, 2C3AB. Four mutated 3CD polyproteins with site-directed lesions in the 3D domain of the proteinase were analyzed for their ability to process viral polyprotein precursors and to form a ternary complex with RNA sequences encoded in the 5' terminus of the viral genome. Analysis of mutated 3CD polypeptides revealed that specific mutations within the 3D amino acid sequences of 3CD confer differential effects on 3CD activity. All four mutated 3CD proteins tested were able to process the P1 structural precursor with wild type or near wild type efficiency. However, three of the mutated enzymes demonstrated an impaired ability to process some sites within the P3 non-structural precursor, relative to wild type 3CD. One of the mutant 3CD polypeptides, 3CD-3DK127A, also displayed a defect in its ability to form a ternary ribonucleoprotein complex with poliovirus 5' RNA sequences.     

Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase.

A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.     

Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C(pro) and the viral polymerase 3D(pol) and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-A resolution and the G64S fidelity mutant of 3D(pol) at a 3.0-A resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D(pol) G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D(pol) makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.     

Site-directed mutagenesis of proteinase 3C results in a poliovirus deficient in synthesis of viral RNA polymerase.

We used a synthetic double-stranded oligonucleotide to introduce amino acid substitutions into the proteinase 3C region of a poliovirus type 1 cDNA clone. The six different mutant viruses recovered exhibited a small-plaque phenotype when assayed on HeLa cells. Further investigation revealed that all the mutations (with the exception of one) yielded P3 region proteins that displayed altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A conservative Val----Ala change at amino acid 54 of the proteinase resulted in a virus that was deficient in the production of the mature viral RNA polymerase 3D. Although this mutant achieved less than one-half of the wild-type levels of RNA synthesis during the course of infection, it still grew to nearly wild-type titers.     

Purification and characterization of RNA polymerase I from a higher plant.

RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).     

Purification and characterization of proteinase In, a trypsin-like proteinase, in Escherichia coli.

We previously found a trypsin-like proteinase which momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication (Kato, M., Irisawa, T., Morimoto, Y. and Muramatu, M., unpublished results). The proteinase was named proteinase In. It was purified approximately 2880-fold with a recovery of 15%. The isolated enzyme appeared homogeneous by gel filtration and electrophoresis. Its molecular mass was estimated by analytical gel filtration and SDS/PAGE as approximately 66 kDa. The isoelectric point of proteinase In is 4.9 and its optimal pH is approximately 9. Although protein In hydrolyzes fluorogenic substrate for trypsin, its hydrolytic activity seems markedly affected by amino-acid sequence lying towards the N-terminal from the P1 (lysine, arginine) residue. The proteinase does not hydrolyze N2-benzoyl-D,L-arginine-4-nitronanilide and fluorogenic substrates for chymotrypsin and elastase. The proteinase activity is inhibited by leupeptin, antipain and 4-nitrophenyl 4-guanidinobenzoate, but the effects of tosyl-L-lysine chloromethane, diisopropylfluorophosphate, benzamidine and pentamidine isethionate on the proteinase activity are weak or not inhibitory. Its activity is strongly affected in the presence of NaCl and KCl, and at a concentration of 1.5 M, these increase the activity 14-fold and 13-fold, respectively, above that without salt. Proteinase In was strongly inhibited by various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, and their inhibitory effects were roughly correlated with those on growth of E. coli. Proteinase activity was found in the cytoplasmic fraction.     

Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag

A self‐cleaving elastin‐like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP α subunit was tagged with a self‐cleaving ELP‐intein tag and coexpressed with the β, β′, and ω subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP‐intein purification. Thus, this simple ELP‐based method should be applicable for the nonchromatographic purification of other recombinant, in vivo‐assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large‐scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.     

Purification and partial characterization of a stimulatory factor for lamb thymus RNA polymerase II.

A heat-stable protein (HSF) that stimulates the activity of lamb thymus RNA polymerase II has been purified 2500-fold and partially characterized. This factor stimulates the activity of RNA polymerase II up to 13 times and retains complete activity when heated at 90 degrees C for 5 min. Stimulation is observed only in the presence of RNA polymerase II and requires native DNA as template. The stimulatory factor has a sedimentation coefficient of 2.7 S, a diffusion coefficient of 9.55 x 10(-7) cm2/s, and an isoelectric point of 8.0. Calculated from the sedimentation and diffusion data, the factor has a molecular weight of about 24,000. Electrophoresis of the purified factor on polyacrylamide gels in the presence of sodium dodecyl sulfate results in a single band corresponding to a molecular weight of 25,000. The number-average length of the RNA synthesized by RNA polymerase II is increased in the presence of the factor. Sedimentation velocity and exclusion chromatography experiments suggest that the stimulatory factor interacts with RNA polymerase II. These results suggest that the factor stimulates RNA synthesis through a direct interaction with RNA polymerase II. The stoichiometry of the HSF-RNA polymerase binding appears to be about 1:1. HSF is located in the nucleus, as determined by cell fractionation studies.     

trans rescue of a mutant poliovirus RNA polymerase function.

A series of three-nucleotide insertions was engineered into the P2 and P3 coding regions of the T7 expression plasmid pT7(tau)-PV1, which encodes a full-length copy of poliovirus type 1 (Mahoney) cDNA. When RNA derived in vitro from these mutated templates was used to transfect HeLa cells, viable virus mutants were recovered. One mutant, Sel-3D-18, which contained a single amino acid insertion in the 3Dpol coding region, was temperature sensitive for growth at 39 degrees C and showed defects in both RNA synthesis and P1 protein processing at the nonpermissive temperature. The RNA replication defect in Se1-3D-18 was identified at the level of RNA chain elongation. A highly specific and sensitive method was developed for analyzing the ability of mutant RNA templates to replicate in the presence or absence of helper functions provided in trans. This approach was used to demonstrate that RNA synthesis in Se1-3D-18 can be rescued by helper functions provided in trans.     

Purification and characterization of RNA polymerase I from a higher plant

RNA polymerase I was purified from chromatin isolated from auxintreated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200–300 nmol/mg/30 min at 28°C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).     

Functional oligomerization of poliovirus RNA-dependent RNA polymerase.

Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis.     

Purification and characterization of proteinase yscJ, a new yeast peptidase.

A newly recognized peptidase, designated proteinase yscJ, was purified from the yeast Saccharomyces cerevisiae. The enzyme is of non-vacuolar origin and cleaves the Tyr-Lys bond of the synthetic peptide substrate Cbz-Tyr-Lys-Arg-NH-Ph (Cbz, benzyloxycarbonyl; NH-Ph, 4-nitroanilide) and the Glu-Lys bond of the substrate Boc-Glu-Lys-Lys-NH-Mec (Boc, butoxycarbonyl; Mec, 4-methylcoumarinyl) with high efficiency. Optimum pH for cleavage of Cbz-Tyr-Lys-Arg-NH-Ph is in the range 7.0-7.5. The purified enzyme has a molecular mass of approximately 58 kDa, as judged by gel filtration on a Superose 12 FPLC column. Mercury compounds and EDTA were found to be potent inhibitors of proteinase yscJ activity.