W. Wilbur Ackermann

Studies of the biosynthesis of protein and ribonucleic acid in HeLa cells infected with poliovirus

Abstract The amounts of protein, RNA (ribonucleic acid), virus, and the rate of incorporation of P 32 into RNA were determined in various subcellular fractions of HeLa cells at various times during a single sequence of infection with poliovirus. From these data interpretations are drawn concerning the biosynthesis at the cellular level of protein and RNA which are induced by virus infection. Within 1 hour after the initiation of infection, there is a detectable accumulation in the cellular cytoplasm of newly synthesized protein and RNA. The synthesis of protein continues at a constant rate until the seventh hour of infection. RNA in the cytoplasm increases at a constant rate until the fourth hour, at which time the rate is markedly enhanced, and the first virus, as infectious activity, appears in the cytoplasmic fraction. The synthesis of RNA stops by the sixth hour. Virus accumulates at an increasing rate in the cytoplasm between the fourth and seventh hours. At the seventh hour, 99% of the virus formed is present in the intracellular state. From the amounts of nucleic acid and protein produced, their distribution relative to virus among the various subcellular fractions, and from the nucleotide composition of the RNA, it was concluded that the major portion of the newly formed materials was not virus.

SOME PROPERTIES OF NUCLEIC ACIDS EXTRACTED WITH PHENOL

SUMMARY Preparations of nucleic acids obtained by extraction of mouse liver, HeLa cells and cell fractions with phenol and deoxycholate have been characterized with regard to the differential solubility of ribonucleic acid and deoxyribonucleic acid in ethanol, density-gradient centrifugation and the presence of high-molecular-weight contaminants. Ribonucleic acid obtained by this method is less soluble than deoxyribonucleic acid. It was precipitable with 20 % ethanol, nearly free of deoxyribonucleic acid, but containing 4-5 times its weight of polysaccharide which is not removed by repeated fractional precipitation nor entirely by ~-amylase (EC 3.2.1.1) digestion, but is removed by density-gradient centrifugation. Deoxyribonucleic acid could be subsequently precipitated with 50 % ethanol free of ribonucleic acid but contaminated with polysaccharide. The buoyant density of the latter is identical with deoxyribonucleic acid and they are not separated by density-gradient centrifugation. The contaminating polysaccharide appears to be a single entity, the fl-subunit of glycogen granules. Its isolation and some of its properties are described. Its effect upon the properties of the nucleic acids is discussed.

Initial stages of the interaction of HeLa cells with poliovirus.

As a c(~nsequence of the usual interaction of viruses and cells, one detects an initial loss of viral activity, a viral increase and destruction of cells. I n such cases the reaction may be followed in terms of any of the resultant~effects, ttowever, all interactions may not b e productive of virus; nor result in cellular ,destruction and viral activity m a y lend itself .to competing reactions. Td follow the reaction by a single effect may preclude the observation of unexpected phenomena. The present study is centered upon the cytopathogenic effect of the poliovirus and for this reason its reaction with the HeLa cell is followed primarily in terms of cellular destruction. For this purpose a method has been developed for quantitating cellular injury. By the application of the method, the kinetics of two early phases of the interaction of virus and cell have been investigated.

A new host-virus system.

Summary A new host-virus system is described. The WS strain of Type A influenza virus was adapted to culture in the Ehrlich ascitic carcinoma of mice. This adapted strain of virus has been maintained through 38 serial passages in this tissue. The growth characteristics during a single passage have been described. Associated with the multiplication of this virus is a loss of viability of the host cell. The effect of this phenomenon on the course of development of an established ascitic tumor has been followed. The importance of this new host-virus system as a tool for the study of the mechanism of viral multiplication is discussed.

RNA dependent DNA synthesis in cell free preparations of human leukemia cells

Abstract A cell free preparation of human leukemic cells, grown in tissue culture, incorporated H 3 -thymidine phosphate into an acid insoluble product which was rendered acid soluble by the action of DNAase but not KOH or RNAase. RNAase, if added before incubation of the reaction mixture, prevented incorporation of the isotope into the product. For maximum incorporation Mg and all four deoxyribonucleotide triphosphates must be present. The enzyme has the properties of an RNA dependent DNA polymerase.

Effect of DL-ethionine on poliomyelitis virus growth in tissue culture.

Conclusions The Lansing strain of poliomyelitis virus requires L-methionine for its growth and multiplication in cultures of human embryonic brain tissue. Its growth can be materially inhibited with DL-ethionine, a structural analogue of the essential amino acid. The inhibitor acts by interfering with the biosynthesis of the poliomyelitis virus just as it suppresses the growth of influenza virus. This technic affords a procedure for studying the growth requirements of poliomyelitis virus and for testing materials which may influence its growth.

MECHANISMS OF PERSISTENT AND MASKED INFECTIONS IN TISSUE CULTURE

I t has been repeatedly observed that certain infected tissues that initially cannot be made to yield virus upon isolation will do so after the tissues have been subcultured i.n aitro (Rowe et al., 1953, and Hull, Minner, and Smith, 1956). This cultivation in vitro subjects the cells to new environmental influences. The cells are transformed from a resting state to one of rapid proliferation; furthermore, they are removed from the humoral influences of the host. Once isolated, the viruses are apparently cytopathogenic to the progeny of the cells that formerly harbored them. If one disregards the possibility that the cultures were repeatedly contaminated after isolation, one must assume that the virus was present in the tissue in some nontransmissible or masked form. The factors involving the length of time the virus had persisted in the tissue and whether it had been undergoing some restricted multiplication present questions of considerable interest. Several years ago we made the observation that poliovirus could be repeatedly isolated from subcultures of HeLa cells in which prolonged and extensive cellular multiplication was occurring (Ackermann and Kurtz, 1955). In this experimental system there was strong evidence that the virus not only persisted, but to some degree replicated. More recently, this type of observation in vitro has been extended to the adenoviruses (Ginsberg and Boyer, 1956). The aim of this presentation is to consider what relation these in vitro systems bear to the phenomenon described by Rowe and Hull and their associates, on the one hand, and to the problem of recurring infections and persisting immunity, on the other. The first efforts a t explanation should be in terms of the known framework and principles of the single infectious sequence, and one should be cautious in postulating new principles and laws. From this viewpoint it may be worth while to note that there is a nontransmissible phase in the normal infectious sequence of animal viruses. Further, if the normal sequence of development is interrupted by the use of metabolic antagonists after infection has been initiated, as has been done with influenza virus (Ackermann and Maassab, 1955), or by the use of deficient medium as applied to psittacosis (Morgan, 1956), the infected cell can remain in a state of virostasis for long periods, even for days. The period of virostasis may be terminated upon change in the environment, and the infectious sequence will then continue and will yield virus. In the case in which virostasis was induced with an inhibitor of protein synthesis, viral development still continued in this noninfectious stage. The fact that virus in the noninfectious state is quite durable, perhaps more so than when in the extracellular infectious form, is further illustrated by recombination experiments in which irradiated influenza virus was found to react

Reaction of Polyoma and Influenza Viruses with Receptors of Erythrocytes and Host-Cells.

Summary Evidence is presented for the existence of 2 types of erythrocyte receptors for the PR-8 strain of influenza virus; one of these is available also to polyoma virus. Modifications of the quality of the hemagglutination reaction, resulting from treatment of either virus or red cell, are described. While a similarity in the receptors for influenza virus on the host-cell and the erythrocyte can be demonstrated, these were found to be distinct in the case of polyoma virus.

STUDIES OF VACCINIA HEMAGGLUTININ OBTAINED FROM VARIOUS VACCINIA INFECTED TISSUES. DENSITY GRADIENT CENTRIFUGATION AND ELECTRON MICROSCOPY.

Summary Serologically specific vaccinial hemagglutinin (VHA) was obtained from vaccinia infected HeLa cells, chorioallantoic membranes of embryonated eggs and mouse fibroblast cell cultures. When concentrated and partially purified preparations were examined by density gradient centrifugation, the preparations varied in density according to the tissue of origin. The host-associated differences in peak density were accompanied by a marked degree of poly-dispersion not resolvable by a second density gradient centrifugation or by prolonged centrifugation. Electron micrographs showed that the particle size was in the range of 45–54 mμ. Substructure was detected in some particles which suggested the coiling or folding of a strand.

Effect of chloride lons on the reaction of diphenylamine with DNA

Abstract The sensitivity of the Dische test (diphenylamine) for DNA is reduced by the presence of chloride ions, which cause formation of a new reaction product whose absorption maximum is at lower wavelength, and also a reduction in the total chromogenic potential. Additionally, the reaction with other sugars (particularly fructose) is intensified by chlorides reducting the specificity of the reaction for DNA.