The virus-induced leakage of host-cell constituents represents a true increase in cellular permeability rather than an unpeeling of cell surface components, since an intracellular enzyme participates in the leakage.
All of the T-system bacteriophages exhibit this leakage. The leakage does not occur with salt concentrations which permit only reversible virus-cell attachment but no penetration. These facts support the idea that the reaction underlying cell leakage is a part of the invasive mechanism.
With increasing multiplicity of T2 infection of young, fresh Escherichia coli B cells, progressively larger molecules leak out of the cell. Acid-soluble P32 appears in large amounts with single infection. Appreciable amounts of galactosidase enzyme and RNA do not leak until multiplicities of 5 to 30 are attained. Cellular DNA is not liberated unless sufficiently high multiplicities are used to cause the extensive cell destruction and clearing of the suspension characteristic of lysis-from-without. This progression is interpreted as an increase with T2 multiplicity in the maximum hole size produced in the cell membrane.
Calculation shows that this increase in hole size must result from a spreading change in the character of the cell wall, rather than the coincidental juxtaposition of 2 or more viruses at adjacent attachment sites.
T1 virus liberates less macromolecular constituents than T2 from E. coli B.
The following experimental results constitute evidence that in the course of normal virus infection, a resealing reaction is rapidly instituted in the cell wall which reverses the effect of the original permeability increase, and renders the cell refractory to a second lytic reaction by a homologous virus: (a) Cell leakage induced by T2 virus in the course of normal infection markedly slows down or stops within a few minutes, even when only a small fraction of the material potentially available for leakage has been released, (b) Superinfection after 8 minutes at 37°C., of a cell previously infected with a homologous virus causes little or no appearance of a second leakage of cell constituents. This experiment also leads to the conclusion that the sealing reaction, like that which causes the leakage, also involves a disturbance which spreads over all or most of the cell wall. (c) If a multiple virus infection is allowed to occur at 0°C. and then the cells are placed in a 37°C. bath after completion of attachment, a much greater cell leakage results than if the entire course had occurred at 37°C., as would be expected if a resealing reaction comes into play at 37°C. within a time less than that required by the completion of attachment. The virus particles attaching secondarily at 37°C. are prevented from exercising their permeability-increasing effect by the sealing reaction of the virus which had penetrated first.
Although a second homologous cell infection with T1 or T2 phages after a 37°C. incubation fails to yield a second leakage, a second heterologous infection always causes exacerbation of new leakage, which, especially if T1 has preceded T2, may be much greater than the sum of those produced individually by each virus in separate cell suspensions. This phenomenon may be the action responsible for the "depressor" effect which occurs when 2 unrelated viruses attack the same cell.
The properties of the sealing phenomenon are such as to make it appear a logical candidate for the mechanism underlying the exclusion of a superinfecting phage from participating in reproductive processes in a cell previously infected with a homologous virus, since the DNA of the second virus would be unable to penetrate the new barrier. Experiments to test this hypothesis revealed that the DNA from such superinfecting virus is completely extractable from cells by washing in dilute buffer, whereas about 40 to 50 per cent of the attached DNA of virus which has invaded virgin cells remains bound to the cells.
Most of the viral DNA which appears in the original supernatant when P32-labelled T2 invades E. coli B in a multiplicity less than one, does not represent inert material but rather virus DNA which has been split, or split and hydrolyzed as a result of its interaction with the cells, as judged by the altered susceptibility to hydrolytic enzyme or to TCA precipitation. This suggests that 25 per cent or more of the virus DNA may be expendable, at least after the penetration stage of the infection cycle.
Mg++ which strongly depresses the amount of cell leakage attending T2 infection, does not prevent T2 penetration nor does it block the appearance of the exclusion reaction. Hence, if the initial leakage does mirror the lytic process by which a hole for the DNA injection is provided, the Mg++ does not function by preventing this hole formation. Its effect would have to lie in prevention of the spreading lysis-potentiating reaction or in augmenting the sealing mechanism.
A large number of independent lines of evidence indicate that the phenomenon of lysis-from-without exhibited by the T-even coliphages is the result of failure of the sealing mechanism to keep pace with the lytic reaction. This can result from an excess of infecting phages or inhibition of the cellular energy-liberating reaction required by the sealing mechanism. The complete parallelism between the development of refractoriness to lysis-from-without and development of refractoriness to the production of a new leakage from a homologous superinfection is especially convincing in this connection.
It is proposed that the early phase of bacteriophage invasion involves the following steps: reversible electrostatic attachment; splitting of the viral DNA from its protein coat; initiation of a lytic reaction in the cell wall at the site of virus attachment; injection of the DNA through the hole so produced; a spreading disturbance over the cell surface which makes it momentarily more susceptible to the lytic reaction; sealing of the hole and a concommittant spread over the cell wall of a reaction making the cell refractory to initiation of a second lytic reaction.
Na+, K+, and Mg++ all behave differently in their effect on the leakage produced in the course of T2 invasion of E. coli.