Myristate-protein interactions in poliovirus: interactions of VP4 threonine 28 contribute to the structural conformation of assembly intermediates and the stability of assembled virions.

The VP4 capsid protein of poliovirus is N-terminally modified with myristic acid. Within the poliovirus structure, a hydrogen bond is observed between the myristate carbonyl and the hydroxyl side chain of threonine 28 of VP4. This interaction is between two fivefold symmetry-related copies of VP4 and is one of several myristoyl-mediated interactions that appears to structurally link the promoters within the pentamer subunit of the virus particle. Site-specific substitutions of the threonine residue were constructed to investigate the biological relevance of these myristate-protein interactions. Replacement of the threonine with glycine or lysine is lethal, generating nonviable viruses. Substitution with serine or valine led to viable viruses, but these mutants displayed anomalies during virus assembly. In addition, both assembled serine- and valine-substituted virion particles showed reduced infectivity and were more sensitive to thermal inactivation and antibody neutralization. Thus the threonine residue provides interactions necessary for efficient assembly of the virus and for virion stability.     

Mechanism of poliovirus inactivation by bromine chloride

The mechanism of poliovirus inactivation by BrCl was determined by exposing poliovirus to various concentrations of BrCl and correlating the loss of virus infectivity with structural changes of the virus. Concentrations of 0.3 to 5 mg of BrCl per liter resulted in 95% to total inactivation of poliovirus. However, the inactivated virus retained structural integrity, as determined by buoyant density measurements of poliovirus labeled with radioactivity. However, at concentrations of 10 to 20 mg of BrCl per liter, total inactivation of poliovirus was associated with the degradation of the structural integrity of the virus. Since infectious ribonucleic acid at similar concentrations could be recovered from untreated poliovirus and poliovirus treated with 0.3 mg of BrCl per liter, it was concluded that BrCl as HOBr or bromamines inactivates poliovirus by reacting with the protein coat of the virus. Moreover, this inactivating reaction does not result in the degradation of the structure of the virion, nor does it affect the biological activity of the internal ribonucleic acid of the virus.     

Mechanism of poliovirus inactivation by bromine chloride.

The mechanism of poliovirus inactivation by BrCl was determined by exposing poliovirus to various concentrations of BrCl and correlating the loss of virus infectivity with structural changes of the virus. Concentrations of 0.3 to 5 mg of BrCl per liter resulted in 95% to total inactivation of poliovirus. However, the inactivated virus retained structural integrity, as determined by buoyant density measurements of poliovirus labeled with radioactivity. However, at concentrations of 10 to 20 mg of BrCl per liter, total inactivation of poliovirus was associated with the degradation of the structural integrity of the virus. Since infectious ribonucleic acid at similar concentrations could be recovered from untreated poliovirus and poliovirus treated with 0.3 mg of BrCl per liter, it was concluded that BrCl as HOBr or bromamines inactivates poliovirus by reacting with the protein coat of the virus. Moreover, this inactivating reaction does not result in the degradation of the structure of the virion, nor does it affect the biological activity of the internal ribonucleic acid of the virus.     

Evidence for secondary structure within the virion RNA of echovirus 22.

Phenol-extracted echovirus 22 virion RNA is infectious, but unlike poliovirus virion RNA, it resists digestion with pancreatic RNase and nuclease P-1, a 3' exonuclease selective for single-stranded RNA. These data indicate the presence of an enzyme-resistant portion somewhere in the RNA molecule and suggest that it is a double-stranded or base-paired region distant from the unblocked 3' terminus. Equilibrium density gradient centrifugation of native echovirus 22 virion RNA results in a single peak with a density of 1.63 g/cm3. When sheared before centrifugation, the molecule is resolved into two RNA species: one with an approximate density of 1.70 to 1.71 g/cm3, as is observed also for single-stranded poliovirus virion RNA, and the other with a density of 1.58 to 1.59 g/cm3. Data obtained from rate zonal centrifugation may be used to calculate an approximate sedimentation coefficient corrected to water at 20 degrees C of 34 and a molecular weight of 2.4 X 10(6) for the virion RNA. We propose a model for echovirus 22 RNA composed of a linear RNA molecule with a 5' hairpin.     

Poliovirus variants selected on mutant receptor-expressing cells identify capsid residues that expand receptor recognition.

Mutations in the predicted C'-C"-D edge of the first immunoglobulin-like domain of the poliovirus receptor were previously shown to eliminate poliovirus binding. To identify capsid residues that expand receptor recognition, 16 poliovirus suppressor mutants were selected that replicate in three different mutant receptor-expressing cell lines as well as in cells expressing the wild-type receptor. Sequence analysis of the mutant viruses revealed three capsid residues that enable poliovirus to utilize defective receptors. Two residues are in regions of the capsid that are known to regulate receptor binding and receptor-mediated conformational transitions. A third residue is located in a highly exposed loop on the virion surface that controls poliovirus host range in mice by influencing receptor recognition. One of the suppressor mutations enables the primate-restricted P1/Mahoney strain to paralyze mice by enabling the virus to recognize a receptor in the mouse central nervous system. Capsid mutations that suppress receptor defects may exert their effect at the binding site or may improve receptor binding by regulating structural transitions of the capsid.     

Thermal inactivation of oral polio vaccine: contribution of RNA and protein inactivation.

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.     

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.     

Engineered picornavirus VPg-RNA substrates: analysis of a tyrosyl-RNA phosphodiesterase activity

Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed "unlinkase," that recognizes and cleaves the unique 5' tyrosyl-RNA phosphodiester bond found at the 5' end of picornavirus virion RNAs. This bond connects VPg, a viral-encoded protein primer essential for RNA replication, to the viral RNA; it is cleaved from virion RNA prior to its engaging in protein synthesis as mRNA. Due to VPg retention on nascent RNA strands and replication templates, but not on viral mRNA, we hypothesize that picornaviruses utilize unlinkase activity as a means of controlling the ratio of viral RNAs that are translated versus those that either serve as RNA replication templates or are encapsidated. To test our hypothesis and further characterize this enzyme, we have developed a novel assay to detect unlinkase activity. We demonstrate that unlinkase activity can be detected using this assay, that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells, and that unlinkase activity is unaffected by the presence of exogenous VPg or anti-VPg antibodies. Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate.     

Cold-adapted poliovirus mutants bypass a postentry replication block.

In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.     

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.     

Inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles.

The entry of animal viruses into cells is associated with permeabilization of the infected cells to protein toxins such as alpha-sarcin (C. Fernández-Puentes and L. Carrasco, Cell 20:769-775, 1980). This phenomenon has been referred to as "the early permeabilization by animal viruses" (L. Carrasco, Virology 113:623-629, 1981). A number of inhibitors of poliovirus growth such as WIN 51711 6-(3,4-dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile (DEPC) and Ro 09-0410 specifically block the uncoating step of poliovirus but have no effect on attachment or entry of poliovirus particles into cells. These agents are potent inhibitors of the early permeabilization induced by poliovirus to the toxin alpha-sarcin. Thus, the uncoating of poliovirus is required for the permeabilization of cell membranes to proteins. The increased entry of labeled heparin promoted by virus entry is not blocked by these agents, indicating that poliovirus binds to its receptor and is internalized along with heparin in endosomes in the presence of WIN 51711, DEPC, or Ro 09-0410. We conclude that the delivery to the cytoplasm of some molecules that coenter with virion particles does not take place if the uncoating process is hindered.     

Animal viruses promote the entry of polysaccharides with antiviral activity into cells

Charged polysaccharides such as heparin and carrageenan show potent antiviral activity in cultured cells. Labelled [3H]heparin binds to several virion particles as evidenced by Sepharose 6B chromatography. This binding is inhibited by carrageenan. The complex [3H]heparin-HSV1 virions is able to enter cells. Thus, almost no entry of [3H]heparin is observed in control HeLa cells, whereas in the presence of HSV1 or poliovirus the amount of radioactivity internalized is enhanced. This internalization is inhibited by carrageenan, suggesting that these two sulphated polysaccharides bind to the same sites on virion particles and may share a similar antiviral mechanism of action.     

Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor.

Poliovirus initiates infection by binding to its cell receptor and undergoing a receptor-mediated conformational alteration. To identify capsid residues that control these interactions, we have isolated and characterized poliovirus mutants that are resistant to neutralization by a soluble form of the poliovirus receptor. Twenty one soluble receptor-resistant (srr) mutants were identified which still use the poliovirus receptor to infect cells. All but one srr mutant contain a single amino acid change at one of 13 different positions, either on the surface or in the interior of the virion. The results of binding and alteration assays demonstrate that both surface and internal capsid residues regulate attachment to the receptor and conformational change of the virus. Mutations that reduce alteration also affect receptor binding, suggesting a common structural basis for early events in poliovirus infection.     

Poliovirus 2A(pro) induces the nucleic translocation of poliovirus 3CD and 3C' proteins

Poliovirus genomic RNA replication, protein translation, and virion assembly are performed in the cytoplasm of host cells. However, this does not mean that there is no relationship between poliovirus infection and the cellular nucleus. In this study, recombinant fluorescence-tagged poliovirus 3CD and 3C' proteins were shown to be expressed mainly in the cytoplasm of Vero cells in the absence of other viral proteins. However, upon poliovirus infection, many of these proteins redistributed to the nucleus, as well as to the cytoplasm. A series of transfection experiments revealed that the poliovirus 2A(pro) was responsible for the same redistribution of 3CD and 3C' proteins to the nucleus. Furthermore, a mutant 2A(pro) protein lacking protease activity abrogated this effect. The poliovirus 2A(pro) protein was also found to co-localize with the Nup153 protein, a component of the nuclear pore complexes on the nuclear envelope. These data provide further evidence that there are intrinsic interactions between poliovirus proteins and the cell nucleus, despite that many processes in the poliovirus replication cycle occur in the cytoplasm.     

Intestinal trypsin can significantly modify antigenic properties of polioviruses: implications for the use of inactivated poliovirus vaccine.

It was recently reported that the intestinal protease trypsin cleaves in vitro the VP1 protein of type 3 poliovirus at antigenic site 1 (J. P. Icenogle, P. D. Minor, M. Ferguson, and J. M. Hogle, J. Virol. 60:297-301, 1986). We found that incubation of purified or crude type 3 poliovirus preparations with specimens of human intestinal fluid brings about a similar change in the virion structure. Sera from children immunized solely with the regular inactivated poliovirus vaccine (IPV) neutralized trypsin-cleaved Sabin 3 virus poorly, if at all, despite moderate levels of antibodies to the corresponding intact virus. Sera containing very high titers of the intact virus also neutralized the trypsin-cleaved virus but at a relatively weaker capacity. Most sera from older persons who may have been exposed to a natural poliovirus infection before the introduction of the poliovirus vaccines as well as sera from children infected with type 3 poliovirus during the recent outbreak in Finland were able to neutralize the trypsin-cleaved type 3 polioviruses. Serum specimens collected 1 month after a single dose of live poliovirus vaccine from children previously immunized with IPV were able to neutralize the trypsin-cleaved virus as well. During natural infection and after live poliovirus vaccine administration polioviruses are exposed to proteolytic enzymes in the gut. Our results may offer an alternative explanation for the relatively weak mucosal immunity obtained with IPV. Improvement of IPV preparations by incorporation of trypsin-treated type 3 polioviruses in the vaccine should be studied.     

Bivalent attachment of antibody onto poliovirus leads to conformational alteration and neutralization.

The treatment of nonsaturating, neutralizing antibody-poliovirus complexes with papain generally led to the loss of viral neutralization and to the loss of the neutralization-associated change in the isoelectric point (pI) of the virion. Subsequent treatment with anti-immunoglobulin G antibodies restored the neutralization of the virus and the alteration of the viral pI. It appears that, under nonsaturating conditions, poliovirus neutralization by an antibody is dependent upon the ability of the antibody to bivalently attach to the virion. Exceptions are monospecific neutralizing antibodies with an affinity for capsid protein VP3.     

Determination of the length distribution of poly(A) at the 3'' terminus of the virion RNAs of EMC virus, poliovirus, rhinovirus, RAV-61 and CPMV and of mouse globin mRNA.

Rapid, detailed, and accurate analysis of the length spectrum of 3' terminal poly(A) in an RNA population can be obtained by 3'-terminal 32P-labelling of RNA with T4 RNA ligase, digestion with ribonucleases T1 and A, and use of gel sequencing methods. Length distributions of 3'-terminal poly(A) of EMC virus, poliovirus, rhinovirus, RAV-61, and CPMV virion RNAs as well as mouse globin mRNA are presented.     

Altered toxicities of fatty acid salts in green paramecia cultured in different waters

Detergents including fatty acid salts act as surface-active agents and thus possibly damage the plasma membrane structures of aquatic organisms. Therefore, when excess, the house-used and industrial outflows of such detergents into aquatic environments may have considerable impacts on the ecosystem. In this study, we propose the use of green paramecia (Paramecium bursaria) for assessing the acute toxicity of eight fatty acid salts (Na and K salts of oleate, palmitate, laurate and myristate) under various water conditions. The Paramecium in the stationary phase were used for a toxicity assay carried out on 12-well microplates and the median lethal concentration (LC50) was determined for each fatty acid salt. In the low mineral culture medium prepared with ultra-pure water, the LC50 for each fatty acid ranged from 5.8 to 144 ppm (w/v). The toxic levels of fatty acid salts differed in the following order: laurate, myristate > or = oleate, palmitate. The toxic levels of oleate and palmitate salts were ca. 10-fold lower than those of laurate and myristate salts. When river water and local tap water instead of ultra-pure water were used for culturing, the toxic levels of all fatty acid salts were drastically lowered compared to the low mineral condition by 30- to 100-fold (198-660 ppm, w/v). Similar detoxification effect was observed when Ca or Mg was added to the low mineral culture media, indicating that the toxicity of fatty acid salts can be notably lowered as the mineral content increases. As we demonstrated that toxicities of fatty acid salts can be lowered in river water and tap water compared to the low mineral condition, some chemical substances behave differently in the different water conditions. Therefore, the use of natural waters reflecting the real environmental conditions in further collection of data on the ecotoxicity impacts of variety of chemicals is highly encouraged.     

Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells.

An in vitro poliovirus RNA-synthesizing system derived from a crude membrane fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing nucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T1-resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. Our data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor.     

Preservation of Enteroviruses by Freeze-Drying

A method was developed for freeze-dry stabilization of poliovirus type 3. An ultrafiltration procedure was used to remove salts from infected tissue culture fluid, and the virus was freeze-dried after suspension in an alkaline organic buffer. This method was further tested with other picornaviruses including poliovirus types 1 and 2, coxsackieviruses A9, A20, B2, and B5, echovirus 11, and the encephalomyocarditis virus. Freeze-dried preparations of the poliovirus could be shipped to distant laboratories at ambient temperature with excellent retention of infectivity. Data are presented showing effects of freeze-drying as well as results of exposure to temperatures to 37 C.