Roland R. Rueckert

Neutralization Of Poliovirus By A Monoclonal Antibody: Kinetics And Stoichiometry

First-order kinetics of neutralization have usually been interpreted as evidence that a single antibody, binding at a critical site, neutralizes the infectivity of a virus particle. In such a case, if all the binding sites were critical, an average of one antibody bound per virion would be required to reduce the infectivity of a virus sample to 37% (1/e) of its initial infectivity. However, in the work reported here using a monoclonal antibody to poliovirus which inactivated with first-order kinetics, an average of four bound antibodies were required. These results are consistent with two different models: one in which only one-fourth of the antibody binding sites on the virion are critical for neutralization; the other, in which none of the sites is critical, but neutralization takes place instead in a stepwise fashion in which each bound antibody reduces the infectivity by a factor of 3/4. The maximum binding capacity of the virion for this monoclonal antibody was approximately 30 molecules. Since the 60 protein subunits of the poliovirus capsid are related by 30 twofold axes of symmetry, it is proposed that each monoclonal antibody binds bivalently to two protein subunits related by a twofold axis. Such a binding mode would crosslink pentamers, the basic structures in picornaviral assembly and dissociation. It is proposed that pentamer crosslinking is an important element in neutralization by this monoclonal antibody. Another antibody, which may neutralize by a different mechanism, is also discussed briefly.

Complete sequence of the RNA genome of human rhinovirus 16, a clinically useful common cold virus belonging to the ICAM-1 receptor group

We report here the complete nucleotide sequence and predicted polyprotein sequence of HeLa cell-adapted human rhinovirus 16 (HRV16). This virus is more suitable than human rhinovirus 14 (HRV14) for clinical studies, and its growth and physical properties are favorable for biochemical and crystallographic analysis. The complete message-sense RNA genome of HRV16 is composed of 7124 bases, not including the poly(A) tail. An open reading frame, extending from base 626 to 7084 predicts a polyprotein containing 2152 amino acid residues. Comparison with other rhinovirus sequences shows HRV16 is much more representative of human rhinoviruses than HRV14. No apparent relationship was found between receptor group and amino acid sequence in VP1, the capsid protein bearing the binding site for the intercullular adhesion molecule-1 (ICAM-1) in both HRV14 and HRV16.

[46] Preparation and characterization of encephalornyocarditis (EMC) virus

Publisher Summary This chapter discusses the preparation and characterization of encephalomyocarditis (EMC) virus. Encephalomyocarditis virus is used for two principal purposes in interferon research, which are (1) as a test object for measuring the plaque-suppressing power of an interferon preparation, and (2) as a source of messenger RNA in cell-free translation studies aimed at defining the biochemical mechanism(s) of interferon action. The chapter describes procedures for propagating the virus, measuring its infectivity, and preparing RNA suitable for translation in cell-free systems. The multiplicity of infection is determined by the purpose of the experiment. A low multiplicity, 1-5 plaque-forming units (PFU) per milliliter, is preferable for expanding high-titer virus stocks while a high one, 50-100 PFU per cell, synchronizes and shortens the infection cycle, from 7-8 hours at single multiplicity, to 5-6 hours at 100 PFU per cell. The shorter growth cycle favors efficient incorporation of radiochemical precursors and roughly doubles the final yield of infective virus.

On the structure of rhinovirus 1A.

Abstract Conditions for the propagation, plaque assay, and purification of radioactively labeled rhinovirus 1A are described. Purified virus was free of empty capsids. It sedimented as a single peak at a rate indistinguishable from that of type 1 poliovirus and ME virus on density gradients. Treatment at pH 4, which left ME virus intact, completely disrupted rhinovirus 1A to produce largely insoluble products. Buoyant density measurements in cesium chloride confirmed that rhinovirus 1A is denser (d = 1.386) than ME virus (d = 1.342). Analysis of purified rhinovirus 1A on SDS-polyacrylamide gels revealed five components; of these, four with apparent molecular weights of 33,800, 29,500, 26,000 and 7000, respectively, were present in roughly equimolar proportions. Roughly 1 30 th as much of the fifth component, weighing about 36,700 daltons, was observed. This distribution of polypeptides, which is similar to that of ME virus and of poliovirus, supports the notion that all three viruses represent a common structural archetype. Thus acid lability and high buoyant density which is characteristic of rhinoviruses cannot be attributed to any gross differences from other picornaviruses in size or number of polypeptide chains in the virion. It is proposed that the rhinoviral capsid is comprised of 60 identical four-chain subunits each weighing about 96,000 daltons. Assuming an RNA content of 30–32% this model predicts that the size of the virion is 8.4 × 106 daltons and the size of the RNA is 2.6 ± 0.1 × 106 daltons.

Kinetics of synthesis and cleavage of encephalomyocarditis virus-specific proteins

Abstract A mathematical model is presented which describes the kinetics of synthesis and cleavage of encephalomyocarditis (EMC) virus-specific proteins as observed in pulse-chase and progressive labeling experiments. Although there is evidence that each ribosome completes translation of the entire protein coding region of the viral RNA, at 4 hr postinfection no polypeptide or nascent chain larger than the 100,000 dalton capsid precursor chain was observed. Hence, little or no “polyprotein” (mass 220,000–260,000 daltons) was produced. Rather, each of the three known primary products (polypeptides A, F, and C) was released upon completion of its synthesis. The precursor of the capsid polypeptides, polypeptide A, was always completed before it was cleaved. This resulted in the initial sequential labeling of the capsid polypeptides following the addition of radioactive amino acids to an infected cell suspension, and provided a means for ordering the capsid polypeptides within A. Chain F was stable, confirming previous reports. Polypeptide C cleaves to form D, which in turn cleaves to form E. However, only 15% of the C chains were released in the intact form. Thirty percent of the time, the nascent C chain had undergone one cleavage thus releasing D as the primary product, and 55% of the time both cleavages had occurred during translation, thus releasing E as a stable primary product. The half-life of the A chain was about 6.7 min, that of the C chain was 10 min, and that of the D chain was 12 min.

A neutralizing monoclonal antibody against poliovirus and its reaction with related antigens

Abstract A neutralizing monoclonal antibody against type 1 poliovirus reacted, not only with native virions (N-antigen) as expected, but also with naturally occurring RNA-free proteins shells (70 S) and with 14 S assembly subunits previously thought to lack N-antigenic sites. The latter finding has important implications for development of subunit vaccines for poliovirus and probably for other picornaviruses as well.

Fragments generated by pH dissociation of ME-virus and their relation to the structure of the virion☆

Abstract ME-virus, in the presence of 0.1 m -chloride ions at pH 5.7, can be thermally dissociated into 14 s subunits, free RNA and an insoluble precipitate (I-protein). The 14 s is a pentamer that can be further dissociated with 1 to 2 m -urea into five 5 s subunits. By sedimentation equilibrium measurements the 14 s subunits weighed about 425,000 daltons; the 5 s subunit weighed about 86,000 daltons. The latter subunit is a protein molecule (protomer) composed of one each of three non-identical polypeptide chains α, β and γ and is believed to be generated by dissociation of a δ chain from each of 60 structural subunits in the capsid. The I-protein contains the two minor components δ and e which are always found in virus preparations; it also contains a definite proportion of the α, β and γ chains. The stoichiometry of the non-δ portion of the I-protein is consistent with that expected for a structure (e-pentamer) analogous to a 14 s pentamer containing one e chain in place of one of the five β chains; the amount of precipitate implies that there are two such e-pentamers per average virion. Thus I-protein is believed to consist of two separate components (a) e-pentamers and (b) δ chains. The requirement for 12 fivefold vertices in an icosahedral particle and the five-co-ordinated nature of the pentamers suggest that the ME-virion is composed of 12 × 5 or 60 protomers; the composition of the I-protein is consistent with the hypothesis that there are 58 (α, β, γ, δ) protomers for every 2 (α, e, γ) protomers. The ME-virion behaves as if its 60 subunits were held together by two sets of bonding sites; (a) type 1 sites binding 12 pentamers together to form the 60 subunit shell and (b) type 2 sites binding 5 protomers together to form pentamers. It is proposed that mild acid dissociation of ME-virus is due to specific rupture of type 1 bonding sites and that dissociation of pentamers into protomers by molar concentrations of amide is due to dissociation of type 2 bonding sites.

Synthesis and Processing of Picornaviral Polyprotein

Under properly defined conditions picornaviruses interrupt host RNA and protein synthesis (1) and subvert the cellular machinery to production of viral protein and RNA. By feeding radiolabeled amino acids to virus-infected cells after cessation of host-protein synthesis, viral protein can be selectively labeled. In a pioneering study, which introduced the now widely used SDS-polyacrylamide gel electrophoresis technique, Summers et al. (2) identified some 14 different virus-specified polypeptides in extracts of poliovirus infected HeLa cells. The net mass of these polypeptides exceeded two-fold or more the known coding capacity of the viral genome. The explanation was later traced to cleavages which took pla.ce during and after synthesis of the viral protein.

Infection of mouse fibroblasts by cardioviruses: Premature uncoating and its prevention by elevated pH and magnesium chloride

Abstract The plating efficiencies of ME-, EMC-, MM-, Columbia SK-, and mengoviruses, collectively called “cardioviruses,” were enhanced 2- to 5-fold by conducting the virus attachment step at 22° rather than at 37°. This behavior was traced to a temperature-dependent loss of infectivity (abortive infection) following attachment of the virion to the cell. Abortive infection was virus specific; it was detectable with cardioviruses but not with poliovirus type 1 or coxsackievirus B1 and took place on both HeLa and L cells. Studies with purified, isotopically labeled ME-virus showed that loss of infectivity was accompanied by release of the RNA genome from cell-attached virions into the extracellular fluid. The coat protein initially remained attached to the cell after RNA release but subsequently dissociated from the cell in the form of 14 S subunits. Premature uncoating was inhibited by alkaline pH and by hypertonic salt solutions.

Three-dimensional structures of drug-resistant mutants of human rhinovirus 14.

Mutants of human rhinovirus 14 were isolated and characterized by searching for resistance to compounds that inhibit viral uncoating. The portions of the RNA that code for amino acids that surround the antiviral compound binding site were sequenced. X-ray analysis of two of these mutants, 1188 Val----Leu and 1199 Cys----Tyr, shows that these were single-site substitutions which would sterically hinder drug binding. Differences in the resistance of mutant viruses to various antiviral compounds may be rationalized in terms of the three-dimensional structures of these mutants. Predictions of the structures of mutant rhinovirus 14 with the substitutions 1188 Val----Leu, 1199 Cys----Tyr and 1199 Cys----Trp in VP1 were made using a molecular dynamics technique. The predicted structure of the 1199 Cys----Tyr mutant was consistent with the electron density map, while the 1188 Val----Leu prediction was not. Large (up to 1.4 A) conformational differences between native rhinovirus 14 and the 1199 Cys----Tyr mutant occurred in main-chain atoms near the mutation site. These changes, as well as the orientation of the 1199 tyrosine side-chain, were correctly predicted by the molecular dynamics calculation. The structure of the predicted 1199 Cys----Trp mutation is consistent with the drug-resistant properties of this virus.