Benjamin Mandel

Studies on the in vitro uncoating of poliovirus. II. Characteristics of the membrane-modified particle.

Abstract Interaction of type I poliovirus with an extract of isolated HeLa cell membranes resulted in a modification of the viral capsid. As a consequence of the modification, the capsid became sensitive to proteases and detergents, and the sedimentation value of the virion was slightly diminished. Treatment of the modified particle with chymotrypsin reduced its sedimentation value and, although the RNA genome was still encapsidated, it was degradable by RNase. Treatment of the membrane-modified particle with detergent (sodium dodecyl sulfate, SDS) disengaged the viral RNA as an intact (i.e., 35 S) molecule. Each of the three serotypes of poliovirus was modified when treated with membrane extract. They differed, however, in the effect of secondary treatment with chymotrypsin, viz., type I was further modified, but types II and III underwent degradation. When secondary treatment was with SDS, all three released 35 S RNA. The above in vitro reactions of the membrane-modified particle are discussed as possible counterparts of the in vivo uncoating phenomenon.

Reversibility of the reaction between poliovirus and neutralizing antibody of rabbit origin

Abstract The reaction between poliovirus and neutralizing antibody (rabbit) has been studied with respect to the reversibility of the reaction. At neutral pH and with either early (5 days after start of immunization) or late (hyperimmune) antiserum, the reaction is irreversible. This conclusion is based on attempts to demonstrate reversibility by dilution of reaction mixtures and by isolation of virus-antibody complexes in an environment devoid of free antibody. For each case there is no evidence of re-equilibration. Neutralized virus can, however, be reactivated quantitatively by reducing the pH of the reaction mixture to between 2.5 and 2.0. That reactivation is the result of dissociation and not of denaturation of antibody is shown by the unimpaired capacity of the reactivated mixture to undergo reneutralization when the pH is readjusted to neutrality. After prolonged contact (4 months at 5°) of virus with antibody, infectious virus can be recovered nearly quantitatively by pH dissociation. This result indicates that the neutralization of virus by antibody is not necessarily accompanied by permanent or irreversible changes in the viral particle. An observation, unrelated to the problem of virus-antibody interaction, on the use of lactalbumin hydrolyzate in culture media is described. It was observed that, although this material in a concentration of 0.1% is beneficial to the growth of HeLa cells, concentrations of 0.3% or greater are either less effective or inhibitory.

Characterization of type 1 poliovirus by electrophoretic analysis

Abstract The electrophoretic characteristics of type 1 poliovirus were studied in 2 different stabilized, free-flowing electrophoresis systems. In one, direct determination of isoelectric point in an electrofocusing system revealed that poliovirus has 2 isoelectric points at pHs of about 7.0 and 4.5. In the other, studies on the variation in mobility with variation in pH revealed a sigmoidal relationship, again indicating that poliovirus is isoelectric at pHs of about 7 and 4.5. These data have been interpreted as evidence for the existence of 2 resonating conformational states (designated A and B, respectively) of the viral capsid protein. Inactivation of poliovirus infectivity by several different treatments resulted in the irreversible stabilization of the capsid in the B state. These treatments included heat, adsorption-elution from cells, and neutralization by specific antibody. At low doses, UV irradiation inactivated without affecting the A-B resonance, but at high doses stabilization in the B state resulted.

Studies on the Interactions of Poliomyelitis Virus, Antibody, and Host Cells in a Tissue Culture System.

Abstract A mixture of poliovirus (type 1) and antiserum contains virus-antibody complexes which, although incapable of causing infection of cells, are capable of adsorbing to HeLa cells. Such adsorbed noninfectious virus has a finite but low probability of undergoing spontaneous reactivation under physiological conditions. This probability is reduced in the presence of antiserum, but is greatly enhanced as the pH of the medium is lowered. At a low pH, antibody dissociates from the adsorbed virus-antibody complex but the cellvirus bond is not adversely affected. One molecule of antibody is sufficient to produce a noninfectious but adsorbing and acid-reactivable virus-antibody complex. As the multiplicity of antibody is increased, the adsorptive capacity and/or the reactivability of the resultant virus-antibody complexes are diminished. Noninfectious, adsorbing virus-antibody complexes are produced with “normal” antibody-containing human sera as well as with rabbit hyperimmune antiviral sera. When about 104 or more plaque units of virus are mixed with antiserum, the surviving fraction decreases with serum concentration until a constant level is reached which is independent of further increases in serum concentration. This is the “persistent fraction” described by Dulbecco et al. (1956). When cultures that had been inoculated with such a mixture are washed and then treated subsequently with viral antiserum, the number of plaques is reduced up to tenfold as compared with cultures that did not receive the secondary serum treatment.

Neutralization of poliovirus: a hypothesis to explain the mechanism and the one-hit character of the neutralization reaction.

Abstract Poliovirus capsid protein undergoes pH-dependent reversible conformational transitions between state A (isoelectric point about 7) and state B (isoelectric point about 4.5). When virus reacts with homotypic antibody, the virion becomes stabilized in state B. In this respect, the multiplicity of antibody per virion is not a variable factor since the smallest amount of antibody, based on the smallest measurable degree of neutralization, induces the same effect on the virion as a saturating amount. No conformational states intermediate between A and B are seen under any conditions of neutralization. The same result was seen with each antiserum from several rabbits, with human antiserum, with antibody obtained from rabbit serum by ammonium sulfate precipitation, with IgG and IgM types of antibody, and with the Fab fragment of rabbit antibody. Although small differences in isoelectric points exist among the different serotypes of poliovirus, they share the basic observations, namely, the property of conformational transitions, and stabilization in the B-state upon reacting with homotypic antibody. Failure to be stabilized in the B-state was found not to be the underlying cause for nonneutralizability. A system has been developed for in vitro uncoating of poliovirus. The essential component of the system is an extract of HeLa cell plasma membrane. It was found that neutralized virus is resistant to uncoating in this system. Based on the above results an attempt has been made to explain both neutralization and its one-hit characteristic in terms of conformational transitions and cooperativity among the capsid subunits.

Studies on the in vitro uncoating of poliovirus. I. Characterization of the modifying factor and the modifying reaction

Abstract A method is described for obtaining a membrane-rich fraction from HeLa cells that can induce a modification in poliovirus characterized by (a) the loss of infectivity and (b) the development of sensitivity of the viral RNA to ribonuclease after treatment with chymotrypsin. Physico-chemical characterization of the modifying factor indicates that it is, or is bound to, a high molecular weight component of the plasma membrane. Modifying activity is lost after exposure to ether, deoxycholate, low pH, trypsin, or brief sonication. Treatment of intact cells with trypsin prior to fractionation, yields a final product devoid of activity. Activity is less readily lost after treatment with alkali. About 50% of the activity is lost after several cycles of freeze-thaw, without further reduction on continued treatment. At 5°, activity gradually diminishes to an undetectable level in about 1 week. At higher temperatures, e.g., 25–45°, activity is lost more rapidly. Neither DNAse nor RNAse has any effect on activity. The modifying reaction proceeds optimally at pH 8. The reaction is readily inhibited by nonionic and ionic compounds. Sucrose and glycerol at a concentration of 5% inhibit almost completely. Ionic compounds at a molarity of 0.05 are strongly inhibitory. On a molar basis, divalent cations are 10-fold more inhibitory than monovalent cations. Kinetic studies indicate that at 25° there is no measurable activity, but between 30 and 37° the reaction has an activation energy of 60 kcal/mole. The modifying factor is active on a wild-type and an attenuated type I poliovirus, but inactive on types II and III viruses. Evidence has been obtained that the membrane extract contains two factors, one with modifying activity, and another that can competitively inhibit the modification reaction.

The relationship between penetration and uncoating of poliovirus in HeLa cells

Abstract To investigate the cellular site of poliovirus uncoating, the interaction of adsorbed photosensitized virus with HeLa cells was followed by three measurable transitions: (a) to nonneutralizability, (b) to photoresistance, (c) to nonrecoverability from lysed cells. At 37° the rates of these transitions were similar. At lower temperatures the rates decreased, but the rate of the neutralization transition decreased less than the rates of the other two. At 25° virus penetrated cells so that it could no longer be neutralized, yet it was still photosensitive and could be recovered in infectious condition when the cells were lysed. If virus was allowed to penetrate at 25° and the temperature was then raised to 37° the transition to photoresistance and to nonrecoverability occurred. The capability of HeLa cells to uncoat poliovirus does riot require an induction period. Through the use of inhibitors it was shown that the capability to uncoat is independent of cellular protein synthesis. These observations indicate that the uncoating system is a normal component of HeLa cells.

Interaction of Viruses with Neutralizing Antibodies

Possibly the earliest published expectation and demonstration of the neutralizing capacity of humoral antibody is contained in the report of Sternberg (1892). He suspected that the blood of an individual recently recovered from a viral infection contained an “antitoxine” that could nullify the infectious capability of the causative agent. He then demonstrated that the serum of a calf recently vaccinated with cowpox (?) virus neutralized the infectivity of this virus when the two were mixed prior to inoculation. The validity of this result was corroborated, and extended, by Beclere et al. (1898) in their studies involving a different host, man, and a different virus, variola. Worthy of note is the empirical aspect of these speculations and experimentations since the nature of antibody as well as virus was at that time unknown.

The interaction of neutralized poliovirus with HeLa cells I. Adsorption

Abstract Adsorption of neutralized poliovirus to HeLa cells was studied using purified virus labeled with either 32 P (an RNA label) or 14 C (a protein label). The effect of three variables on the adsorptive capacity of virus-antibody complexes was investigated: ratio of antibody to virus, duration of virus-antibody interaction, and type of antibody, i.e., 7 S and 19 S. Virus that has reacted with 7 S antibody at a low ratio adsorbs almost as well as, or better than, the unneutralized control depending on how long virus and antibody interact. If the ratio of antibody to virus is low and the virus-antiserum mixture is held at 5°, there is a gradual enhancement in capacity to adsorb which may result in a 2- to 3-fold improvement over the control. At high ratios, enhancement with storage at 5° is correspondingly curtailed. Virus that has reacted with 19 S antibody shows a reduced capacity to adsorb and, irrespective of ratio of antibody to virus, no enhancement occurs with storage at 5°.

The use of sodium dodecyl sulfate in studies on the interaction of poliovirus and HeLa cells

Abstract Poliovirus (type 1) is inactivated by sodium dodecyl sulfate (SDS) only at pHs below 4.7. When virus adsorbs to cells at 2°, only about 2% of the cell-associated virus can be recovered by freezing and thawing the infected cells. However, at least 50% is recovered by lysing the cells with SDS. If the debris derived from the freeze-thaw-disrupted cells is treated with SDS, the titer of infectious virus increases to the same level obtained by direct SDS treatment. At 37° the amount of cell-associated virus that can be recovered with SDS decreases to about 30% of the total in 2–3 hours. Variations in multiplicity of infection affect neither the amount of virus that becomes irrecoverable (possibly because of true eclipsing) nor the earliest time at which newly synthesized virus can be detected.