Poliovirus Protein Research Articles

Intracellular topology and epitope shielding of poliovirus 3A protein.

The poliovirus RNA replication complex comprises multiple viral and possibly cellular proteins assembled on the cytoplasmic surface of rearranged intracellular membranes. Viral proteins 3A and 3AB perform several functions during the poliovirus replicative cycle, including significant roles in rearranging membranes, anchoring the viral polymerase to these membranes, inhibiting host protein secretion, and possibly providing the 3B protein primer for RNA synthesis. During poliovirus infection, the immunofluorescence signal of an amino-terminal epitope of 3A-containing proteins is markedly shielded compared to 3A protein expressed in the absence of other poliovirus proteins. This is not due to luminal orientation of all or a subset of the 3A-containing polypeptides, as shown by immunofluorescence following differential permeabilization and proteolysis experiments. Shielding of the 3A epitope is more pronounced in cells infected with wild-type poliovirus than in cells with temperature-sensitive mutant virus that contains a mutation in the 3D polymerase coding region adjacent to the 3AB binding site. Therefore, it is likely that direct binding of the poliovirus RNA-dependent RNA polymerase occludes the amino terminus of 3A-containing polypeptides in the RNA replication complex.

Effects of vaccine strain mutations in domain V of the internal ribosome entry segment compared in the wild type poliovirus type 1 context.

Initiation of poliovirus (PV) protein synthesis is governed by an internal ribosome entry segment structured into several domains including domain V, which is accepted to be important in PV neurovirulence because it harbors an attenuating mutation in each of the vaccine strains developed by A. Sabin. To better understand how these single point mutations exert their effects, we placed each of them into the same genomic context, that of PV type 1. Only the mutation equivalent to the Sabin type 3 strain mutation resulted in significantly reduced viral growth both in HeLa and neuroblastoma cells. This correlated with poor translation efficiency in vitro and could be explained by a structural perturbation of the domain V of the internal ribosome entry segment, as evidenced by RNA melting experiments. We demonstrated that reduced cell death observed during infection by this mutant is due to the absence of inhibition of host cell translation. We confirmed that this shut-off is correlated principally with cleavage of eIF4GII and not eIF4GI and that this cleavage is significantly impaired in the case of the defective mutant. These data support the previously reported conclusion that the 2A protease has markedly different affinities for the two eIF4G isoforms.

Strand-specific RNA synthesis defects in a poliovirus with a mutation in protein 3A.

Substitution of a methionine residue at position 79 in poliovirus protein 3A with valine or threonine caused defective viral RNA synthesis, manifested as delayed onset and reduced yield of viral RNA, in HeLa cells transfected with a luciferase-containing replicon. Viruses containing these same mutations produced small or minute plaques that generated revertants upon further passage, with either wild-type 3A sequences or additional nearby compensating mutations. Translation and polyprotein processing were not affected by the mutations, and 3AB proteins containing the altered amino acids at position 79 showed no detectable loss of membrane-binding activity. Analysis of individual steps of viral RNA synthesis in HeLa cell extracts that support translation and replication of viral RNA showed that VPg uridylylation and negative-strand RNA synthesis occurred normally from mutant viral RNA; however, positive-strand RNA synthesis was specifically reduced. The data suggest that a function of viral protein 3A is required for positive-strand RNA synthesis but not for production of negative strands.

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required.

Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis.

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU(OH), the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU(OH), however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU(OH) synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU(OH) were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU(OH) in the asymmetric replication of poliovirus RNA.

Translation of polioviral mRNA is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3C(pro).

The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3C(pro) (and/or 3CD(pro)), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3C(pro) target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA.

Synchrotron Radiation-Induced X-Ray Emission to Identify Metal Ions in Preparations of Purified Protein

The suitability of synchrotron radiation-induced X-ray emission (SRIXE) for the detection and identification of metal ions in preparations of purified, soluble proteins was tested. Glutathione S-transferase fused to the proximal zinc finger motif of human transcription factor IIIA or to the cysteine-rich motif of poliovirus protein 2C was expressed in bacteria and purified by affinity chromatography. Aqueous samples containing the purified proteins were analyzed with SRIXE, and trace amounts of zinc and iron were detected. Mutation of the zinc-coordinating residues in the cysteine-rich motif of poliovirus protein 2C resulted in the loss of the zinc-binding ability. Relative quantities of metal in the protein preparations as determined by SRIXE corresponded well with the metal:protein ratios calculated by using a 4-(2-pyridylazo)resorcinol-based assay. We conclude that SRIXE is an accurate, sensitive, and simple method for the detection and identification of protein-bound metal ions in small amounts of sample. Thus, SRIXE may have wide use as a particularly effective method for rapidly determining trace metals in microarray samples.

Poliovirus protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface.

Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-kappaB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.

Proteins attached to viral genomes are multifunctional

Publisher Summary This chapter discusses the nature and function of eukaryotic viral genome-linked proteins (VPgs) and outlines some of the remarkable features of these proteins. The first indications that a viral protein could form a stable complex with the genome of eukaryotic viruses were based on electron microscopy analyses and various biochemical techniques and were obtained with Adenoviridae. In RNA viruses, the term viral genome-linked protein was coined for the poliovirus protein and has been adopted for virtually all the proteins linked to RNA viruses. For the sake of simplification and to facilitate comprehension, the RNA and the DNA viruses whose genome possesses a covalently bound protein have been treated separately. In positive single-stranded RNA viruses, the VPg is always synthesized as part of a larger polyprotein from which it is excised by a viral proteinase that is also part of the polyprotein. The genomes of certain phages and viruses have long been known to covalently bind a viral protein. This feature was subsequently shown to occur also in cell systems. It is thus not an exclusive property of viruses. Indeed, extensive work on cellular topoisomerases, and the tumor suppressor p53 has demonstrated that these proteins can be covalently bound to a nucleic acid.

MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A.

The effects of poliovirus 3A protein expression and poliovirus infection on the presentation of hepatitis C virus antigens in cultured chimpanzee cells were examined. Expression of poliovirus 3A protein inhibits protein secretion when expressed in isolation and was sufficient to protect chimpanzee cells from lysis by hepatitis C virus-specific cytotoxic T cells in standard (51)Cr-release assays. Poliovirus infection also inhibited antigen presentation, as determined by decreased cytotoxic T cell activation. A mutation in 3A that abrogates the inhibition of protein secretion also abolished the effects of poliovirus on antigen presentation. These results demonstrate that the inhibition of secretion observed in poliovirus-infected cells substantially reduces the presentation of new antigens on the cell surface. These observations may reflect a general mechanism by which nonenveloped viruses such as poliovirus and other viruses that do not require a functional protein secretory apparatus can evade detection by the cellular immune response.

The antiviral compound 5-(3,4-dichlorophenyl) methylhydantoin inhibits the post-synthetic cleavages and the assembly of poliovirus in a cell-free system

The mode of action of the antiviral drug 5-(3,4-dichlorophenyl) methylhydantoin (hydantoin) was studied in a cell-free system allowing de novo synthesis of poliovirus. This cell-free system, which is programmed with viral RNA, is suitable for the study of the late stages of poliovirus replication and, thus, for a study of antiviral compounds acting on these late stages. It was shown that, apart from the known inhibition of the assembly of poliovirus, hydantoin also blocks post-synthetic cleavages of poliovirus proteins. Our data demonstrate that the cell-free system is a sensitive tool to study the mode of action of antiviral compounds.

Mutations in the 2C region of poliovirus responsible for altered sensitivity to benzimidazole derivatives.

MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (mrgr), while 1 was susceptible (mrgs). Sequence analysis of the 2C region revealed that the 14 mrgr mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. The mrgs mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL-1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine-resistant and -dependent mutants. Two guanidine-resistant mutants were cross-resistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect.

A cysteine-rich motif in poliovirus protein 2C(ATPase) is involved in RNA replication and binds zinc in vitro.

Protein 2C(ATPase) of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C(ATPase) engages in these processes are not known. To characterize functional domains of 2C(ATPase), we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2C(ATPase). This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2C(ATPase) as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.

Characterization of the Nucleoside Triphosphatase Activity of Poliovirus Protein 2C Reveals a Mechanism by Which Guanidine Inhibits Poliovirus Replication

The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a Km of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.

Isolation and biological characterization of 3(2H)-isoflavene-resistant and -dependent poliovirus type 2 Sabin mutants.

Poliovirus type 2 Sabin mutants were selected for drug resistance and dependence by plating on HeLa cell monolayers in the presence of 3(2H)-isoflavene, a compound related to dichloroflavan, which prevents the shut-off of host translation and poliovirus RNA and protein synthesis. The drug-resistant mutants grew equally well in the presence and in the absence of the drug, while the drug-dependent mutants only grew in the presence of the compound. One dependent and one resistant mutant were characterized biologically in more detail. The resistant mutant did not exhibit thermolability. The mild thermolability exhibited by the dependent mutant was not affected by the addition of 3(2H)-isoflavene, indicating that the substance does not bind the poliovirus type 2 Sabin capsid. The translation of viral proteins and the shut-off of host protein translation during cell infection were not inhibited in either mutant. In the absence of the drug, the cleavage of the precursor VPO, a step in virus protein processing, was affected in the dependent mutant. The dependence of the mutant on the drug was due to the inability of 75S empty particles to reach maturation: our results strongly suggest that this phenomenon is strictly dependent on the reduction of RNA synthesis, confirming the existence of a dynamic equilibrium between RNA production and genome encapsidation during the poliovirus replication cycle.

Genetic analysis of poliovirus protein 3A: characterization of a non-cytopathic mutant virus defective in killing Vero cells.

A mutational and genetic analysis of the poliovirus protein 3A has led to the identification of a single amino acid mutant virus with a restrictive phenotype to form plaques in Vero cells. This mutant (I46T 3A) can be grown and amplified in HeLa cells, where virus replication takes place at wild-type levels. However, Vero cells infected with this virus cannot complete the growth cycle. I46T 3A virus has a defect in the ability to kill Vero cells, as estimated by FACS analysis of propidium iodide uptake by dead cells. Since these defects are observed under conditions where no abnormalities in the rate of synthesis and processing of the mutant polyprotein occur, the inability to induce the cytopathic effect in infected Vero cells denotes the existence of a defect in the activity of 3A, but not the level of expression of the viral genome. As a consequence of this impaired capability to generate the cytopathic effect, I46T 3A mutant viruses cannot be titrated by plaque assay in Vero cells. Only revertant viruses with the wild-type sequence arise and form lysis plaques in Vero cells. Our results suggest a role for the 3A protein (or a precursor thereof) in the virus-induced cytopathic effect. The mutant virus characterized in this work may be a useful tool to understand how poliovirus kills infected cells and carries out the final step of its life-cycle, the release of virus progeny.

Construction and characterization of encapsidated poliovirus replicons that express biologically active murine interleukin-2.

Poliovirus genomes have been constructed in which the capsid genes have been substituted with the murine gene encoding interleukin-2 (IL-2) (referred to as replicons). One replicon contained the gene for IL-2 in place of the poliovirus capsid VP2 and VP3 genes, and a second replicon was constructed that contained the murine IL-2 substituted for the poliovirus VP3 and VP1 genes. The IL-2 genes were cloned into the replicon so as to maintain the translational reading frame with the remaining poliovirus proteins. Transfection of either replicon into cells resulted in the expression of replicon-encoded proteins and replication of replicon RNA. Using a procedure developed in this laboratory, we have encapsidated these replicons into authentic polio virions by passaging the replicons in the presence of a recombinant vaccinia virus, VVP1, which expresses the capsid precursor, P1, protein. Using a quantitative immunoassay, we determined that the majority of the IL-2 produced remained intracellular, with approximately 1%-2% released from the infected cells, and that the IL-2 was biologically active. The results of these studies demonstrate the utility of poliovirus replicons for expression of small bioactive molecules and are discussed with respect to future applications as immune adjuvants as well as potential new tumor therapies.

Amino-terminal region of poliovirus 2C protein is sufficient for membrane binding

The poliovirus-encoded, membrane associated polypeptide 2C is required for viral replication. We have previously established that, while the 2C protein lacks a defined membrane binding domain, the N-terminal region containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In order to determine whether the N-terminal region is sufficient for membrane binding, we have made fusion constructs between this region of 2C (amino acids 1–72 and 1–88) and a soluble protein, chloramphenicol acetyltransferase (CAT). The ability of CAT and the fusion polypeptides to bind to membranes was examined by in vitro translation in the presence of microsomal membrane. While CAT was found in the soluble fraction, both 2C/CAT fusion constructs (1–72/CAT and 1–88/CAT) were membrane associated, suggesting that the N-terminal region of 2C was sufficient to impart membrane binding. To confirm these results in vivo, CAT, 1–72/CAT, and 1–88/CAT were expressed in HeLa cells and their localization was examined using indirect immunofluorescence. Results presented here demonstrate that, while CAT is expressed throughout the cell, 1–72/CAT and 1–88/CAT constructs are capable of localizing to the endoplasmic reticulum (ER) area in transfected cells in the absence of other poliovirus proteins. These results suggest that the first 72 amino acids of 2C contain a membrane binding domain that is capable of targeting soluble proteins to the ER region of the cell.

A protein linkage map of the P2 nonstructural proteins of poliovirus.

The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2A(pro), 2BC, 2C, and 2B. Specifically, 2A(pro) can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. transdominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome.

Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: genetic and ultrastructural analysis.

Poliovirus protein 3A, only 87 amino acids in length, is a potent inhibitor of protein secretion in mammalian cells, blocking anterograde protein traffic from the endoplasmic reticulum (ER) to the Golgi complex. The function of viral protein 3A in blocking protein secretion is extremely sensitive to mutations near the N terminus of the protein. Deletion of the first 10 amino acids or insertion of a single amino acid between amino acids 15 and 16, a mutation that causes a cold-sensitive defect in poliovirus RNA replication, abrogates the inhibition of protein secretion although wild-type amounts of the mutant proteins are expressed. Immunofluorescence light microscopy and immunoelectron microscopy demonstrate that 3A protein, expressed in the absence of other viral proteins, colocalizes with membranes derived from the ER. The precise topology of 3A with respect to ER membranes is not known, but it is likely to be associated with the cytosolic surface of the ER. Although the glycosylation of 3A in translation extracts has been reported, we show that tunicamycin, under conditions in which glycosylation of cellular proteins is inhibited, has no effect on poliovirus growth. Therefore, glycosylation of 3A plays no functional role in the viral replicative cycle. Electron microscopy reveals that the ER dilates dramatically in the presence of 3A protein. The absence of accumulated vesicles and the swelling of the ER-derived membranes argues that ER-to-Golgi traffic is inhibited at the step of vesicle formation or budding from the ER.