Poliovirus Polyuridylic Acid Polymerase and RNA Replicase Have the Same Viral Polypeptide

A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [(35)S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [(35)S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex.     

Proteolysis of noncapsid protein 2 of type 3 poliovirus at the restrictive temperature: breakdown of noncapsid protein 2 correlates with loss of RNA synthesis.

Inhibition of RNA synthesis of the LH strain of type 3 poliovirus at the restrictive temperature occurs concurrently with the breakdown of noncapsid virus-specific peptide 2. This finding, along with other reported data (B.D. Korant, In Proteases and Biological Control, p. 621-644, 1975), suggests that noncapsid virus-specific peptide 2 is a component of the viral replicase.     

Antibodies against the chemically synthesized genome-linked protein of poliovirus react with native virus-specific proteins

The genome-linked protein (VPg) of poliovirus has been chemically synthesized, coupled to bovine serum albumin carrier and injected into rabbits. An antibody response was elicited not only by the full-length synthetic VPg peptide, but also by a synthetic 14-amino acid carboxy-terminal peptide. All antisera reacted with virus-specific proteins from HeLa cells infected with poliovirus. Three of these proteins have previously been implicated by others as precursors of VPg. No free cytoplasmic VPg could be detected, and the antibodies did not react with radiolabeled proteins from uninfected cells.     

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.     

The genome-linked protein of picornaviruses. VII. Genetic mapping of poliovirus VPg by protein and RNA sequence studies

The poliovirus genome-linked protein (VPg) has been subjected to radiochemical microsequence analysis. Sequence studies of virion RNA by a modification of Sanger's dideoxy method have revealed a base sequence corresponding to the amino acid analysis. This result proves that VPg is virus-encoded. The RNA sequence has allowed us to predict the total amino acid sequence of VPg and part of its precursor. VPg is, at most, 27 amino acids long. It maps within the 3' terminal segment of the viral genome that encodes the precursor polypeptide NCVP1b for the virus-specific RNA polymerase NCVP4.     

Effect of Pactamycin on Synthesis of Poliovirus Proteins: a Method for Genetic Mapping

We have studied the effect of the drug pactamycin on protein synthesis in poliovirus-infected HeLa cells. At a concentration which primarily inhibits initiation of protein synthesis, the spectrum of poliovirus proteins synthesized is markedly changed. The amount of NCVP 1, the capsid precursor, is greatly reduced relative to NCVP 2 and the amount of NCVP X is slightly reduced. Since it is believed that there is only one major site for the initiation of protein synthesis on the poliovirus genome, we interpret this differential effect on the poliovirus proteins to be an indication of their relative distance from the initiation site. On this basis, we propose a gene order for the poliovirus genome (5' --> 3') of NCVP 1, NCVP X, NCVP 2.     

Identification of multiple forms of the noncapsid parvovirus protein NCVP1 in H-1 parvovirus-infected cells.

Analysis of extracts of H-1 parvovirus-infected cells with virus-specific antiserum led to the identification of two forms of the noncapsid virus protein NCVP1. These two proteins had apparent molecular weights of 84,000 (NCVP1) and 92,000 (NCVP1') and were structurally related, based on their immunological reactivity and on peptide map analysis. Both of these proteins appeared early in the virus infection, about the same time that capsid proteins appeared. NCVP1' was a highly phosphorylated protein which was apparently derived from NCVP1 via a post-translational event. Phosphoserine was the predominant phosphorylated amino acid in NCVP1' and appeared to be localized in one site or a few sites on the protein. The possible involvement of these noncapsid proteins in parvovirus DNA replication is discussed.     

Purification of a soluble template-dependent rhinovirus RNA polymerase and its dependence on a host cell protein for viral RNA synthesis.

The soluble phase of the cytoplasm of human rhinovirus type 2-infected cells contains an enzymatic activity able to copy rhinovirion RNA without an added primer. This RNA-dependent RNA polymerase (replicase) makes a specific copy of the added rhinovirion RNA, as shown by hybridization of the product to its template RNA but not to other RNAs. The same replicase preparation also contains a virus-specific polyuridylic acid [poly(U)] polymerase activity which is dependent on added polyadenylic acid-oligouridylic acid template-primer. Both activities purify together until a step at which poly(U) polymerase but no replicase activity is recovered. Addition of a purified HeLa cell protein (host factor) to this poly(U) polymerase completely reconstitutes rhinovirus replicase activity. Host factor activity can be supplied by adding oligouridylic acid, suggesting that the host cell protein acts at the initiation step of rhinovirus RNA replication. A virus-specific 64,000-dalton protein purifies with both poly(U) polymerase and replicase activities.     

Guanidine-resistant poliovirus mutants produce modified 37-kilodalton proteins

Eighteen spontaneous, guanidine-resistant mutants of poliovirus were obtained by plaque selection. Isoelectric focusing demonstrated charge changes in a 37-kilodalton protein, pX, among three of the mutants. The precursor of pX, NCVP5b , also exhibited charge changes among the three mutants. pX of 12 mutants was also examined by peptide mapping with Staphylococcus aureus V8 protease. Nine of the mutants presented modified maps, and seven of these maps were identical. The demonstration of mutational changes in pX in 12 of 18 mutants suggests a role for this protein in determining the guanidine trait of poliovirus and corroborates studies with foot-and-mouth disease virus.     

Defective interfering particles of poliovirus. II. Nature of the defect

Poliovirus defective, interfering particles in which about 15% of the standard viral RNA is deleted have been described (Cole et al., 1971). Stocks of DI³ particles more than 99% free of standard poliovirus were prepared by centrifugation of mixed preparations in CsCl gradients. Using purified DI particles, it was found that DI particles can carry out most of the standard poliovirus functions including inhibition of cellular macromolecular synthesis, production of viral RNA and production of virus-specific protein. Neither the kinetics nor extent of viral RNA or protein synthesis differed between DI particle-infected cells and standard virus-infected cells.Newly made virions, capsid proteins, and the capsid protein precursor (NCVP 1) were totally absent in DI particle-infected cells. All of the other viral proteins were present. DI-infected cells briefly labeled with amino acids also contained a new polypeptide, DI-P, which was apparently the residual fragment of NCVP 1 encoded by the DI genome. It was very unstable, being rapidly degraded to acid-soluble fragments. When the cleavage of viral proteins was inhibited with amino acid analogs, precursors of the viral proteins were generated. Those precursors which should have contained NCVP 1 had molecular weights 30,000 to 40,000 daltons lower in DI-infected cells than in standard virus-infected cells. This is the amount of protein encoded by 15% of the standard poliovirus genome which is the per cent of the standard RNA sequence not represented in DI RNA.Poliovirus DI particles therefore appear to be deletion mutants lacking RNA encoding about one-third of the capsid protein precursor. Whether the deletion is internal or terminal remains to be determined.     

Peptide analysis of the transformation-specific antigen from avian sarcoma virus-transformed cells.

Sera from rabbits bearing tumors induced by avian sarcoma virus (ASV) were ussed to immunopecipitate virus-specific proteins from extracts of chicken, hamster, and field vole cells transformed by ASV. Two virus-specific proteins having molecular weights of 76,000 and 60,000 were found in all cell lines examined. The 76,000-molecular-weight protein, Pr76, is the precursor to the internal core proteins of ASV. The 60,000-molecular-weight (60K) transformation-specific antigen from each cell line was subjected to peptide analysis, using chymotrypsin and Staphylococcus aureus V8 protease. The resulting peptide maps of the 60K protein from the different ASV-infected cell types were similar for each enzyme, strongly suggesting that the 60K protein is virus coded. Two-dimensional analysis of chymotryptic peptides from Pr76 and 60K reveals that 60K is not related to the gs antigen precursor. Radiolabeling of ASV-transformed cells with inorganic phosphate revealed that 60K is phosphorylated in vivo. The 60K proteins isolated from both ASV-transformed chicken and field vole cells were found to contain one tryptic phosphopeptide. The tryptic phosphopeptides of 60K from both cell lines migrated identically upon two-dimensional peptide analyses, and their migration differed from that of the principal phosphopeptide of Pr76.     

Cleavage of Poliovirus-Specific Polypeptide Aggregates

Zonal electrophoresis resolves two aggregates of poliovirus type 2 cytoplasmic polypeptides. The more negatively charged aggregate contains mainly noncapsid viral-specific polypeptides (NCVP) 2 and x, whereas the other consists of the capsid polypeptides (VP) 0, 1, 2, and 3 (VP0, VP1, VP2, VP3). After treatment with sodium deoxycholate (DOC), the aggregates sediment at 5 to 6S. Their electrophoretic mobilities are unaffected by DOC or RNase. The capsid polypeptide aggregate is similar in mobility to virions but can be converted to a faster electrophoretic form, resembling empty capsids, by heating. If infected HeLa cells are allowed to synthesize poliovirus polypeptides in the presence of iodoacetamide, no capsid polypeptides are produced, but rather NCVP1a (the precursor to capsid polypeptides) is accumulated, along with NCVP2 and NCVPx. When analyzed by electrophoresis and centrifugation, uncleaved NCVP1a migrates with the NCVP2-x aggregate. NCVP1a can be cleaved to capsid-like polypeptides in vitro by using extracts of infected cells, but not uninfected cells, indicating either a virus-specified protease or a cellular enzyme activated during infection. After cleavage of NCVP1a by infected cell extracts, the capsid polypeptides which are produced dissociate from the NCVP2-x complex.     

Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase.

Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.     

The host protein required for in vitro replication of poliovirus is a protein kinase that phosphorylates eukaryotic initiation factor-2

The HeLa cell protein (host factor) required for in vitro replication of poliovirus has been identified as a 67,000 dalton phosphoprotein. The purified protein displays three activities in vitro: stimulation of poliovirus RNA synthesis in the presence of poliovirus replicase, apparent self-phosphorylation, and phosphorylation of the alpha-subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). All three activities can be removed or inhibited by an antibody to host factor. Partially purified preparations of reticulocyte eIF-2 contain a similar phosphoprotein and display host factor activity in the viral RNA synthesis assay in vitro. In vitro phosphorylation of the 67 kd protein can be stimulated by low concentrations of double-stranded RNA. Addition of phosphorylated host factor in an in vitro RNA synthesis assay significantly changes the kinetics of viral RNA synthesis, indicating that protein phosphorylation may play an important role in viral RNA replication.     

Antibody to a synthetic nonapeptide corresponding to the NH2 terminus of poliovirus genome-linked protein VPg reacts with native VPg and inhibits in vitro replication of poliovirus RNA.

A synthetic nonapeptide corresponding to the N-terminal sequence of poliovirus genome-linked protein (VPg) was linked to bovine serum albumin and used to raise antibodies in rabbits. The antipeptide antibodies specifically precipitated the nonapeptide, native VPg, and VPg-linked poliovirion RNA. The antipeptide antibodies inhibited host factor-stimulated, poliovirus replicase-catalyzed in vitro synthesis of full-length (35S) RNA in response to virion RNA. Oligouridylic acid-stimulated RNA synthesis was not affected by the antipeptide antibodies. Preincubation of the antibodies with excess nonapeptide reversed the antipeptide antibody-mediated inhibition of host factor-stimulated RNA synthesis by the poliovirus replicase. A role for VPg in the in vitro replication of poliovirus RNA genome is discussed.     

Identification of mouse hepatitis virus papain-like proteinase 2 activity

Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.     

A membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNa polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNA sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNa replication complex.     

Site-specific mutagenesis of cDNA clones expressing a poliovirus proteinase

The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio-specific cleavages occur at glutamine-glycine (Q-G) pairs that are recognized by the viral-encoded proteinase 3C (formerly called P3-7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN-III-C3-7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino- and carboxy-terminal Q-G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN-III-C3-7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site-specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy-terminal Q-G cleavage site. The mutant recombinant plasmid (designated pIN-III-C3-mu 10) directs the synthesis of a bacterial-polio precursor polypeptide that is like the wild-type construct (pIN-III-C3-7c). However, unlike the wild-type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino-terminal Q-G site but not at the carboxy-terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self-cleavage at its carboxy-terminal Q-G pair.     

An antigen chimera of poliovirus induces antibodies against human papillomavirus type 16.

It has been established that the surface of poliovirus type 1 can be extensively modified to incorporate antigenic domains from other poliovirus serotypes and from unrelated viruses. The fact that the modified (chimeric) viruses exhibit dual antigenicity and immunogenicity led us to explore the possibility of using the Sabin vaccine strain of poliovirus type 1 as a vector for the presentation of antigenic domains from human papillomavirus type 16 (HPV-16), a virus associated with the development of cervical carcinoma. We report here the construction and characterization of a chimeric poliovirus containing a 16-residue sequence derived from the major capsid protein (L1) of HPV-16. This virus chimera stimulated the production in rabbits of antibodies which recognized the HPV-16-derived peptide and an L1 fusion protein synthesized in Escherichia coli and detected HPV-16 in human biopsy material by immunoperoxidase staining. The possibility that poliovirus-HPV chimeras could be used as vaccines against HPV-16 is discussed.     

Purification and properties of a host cell protein required for poliovirus replication in vitro

A host cell protein required for poliovirus RNA-dependent RNA replicase activity in vitro has been purified several thousand-fold from an uninfected HeLa cell postmitochondrial supernatant. A single protein of apparent Mr = approximately 67,000 daltons and pI 6.3 is associated with this "host factor" activity. Poly(U)-Sepharose chromatography of the template-dependent replicase isolated from poliovirus-infected cells results in the complete loss of replicase activity if a salt gradient is used to develop the column. Host factor elutes early in the salt gradient and restores replicase activity to protein fractions eluted later in the gradient. The host factor, estimated to be present at 50,000-100,000 copies/cell, interacts physically with replicase.