Harry Rubin

Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture

Abstract An accurate tissue culture assay for Rous sarcoma virus (RSV) and Rous sarcoma cells is described. The Rous sarcoma virus changes a chick fibroblast into a morphologically new and stable cell type with the same chromosomal complement as ordinary chick embryo cells. One virus particle is enough to change one cell, but at any one time 90% of the cells in a culture are not affected by RSV. The cellular resistance is the same in a clonal population. The physiological state of the cell is of some importance in deciding whether or not it is competent to be infected by RSV but so far attempts to infect all the cells in a chick embryo culture by altering the physiological condition have failed.

A radiological study of cell-virus interaction in the rous sarcoma☆

Abstract Rous sarcoma virus (RSV) and Newcastle disease virus (NDV) are in activated exponentially by X-rays and ultraviolet (UV) light. While both viruses are similar in their sensitivity to X-rays, RSV is about ten times more resistant to UV light than is NDV. The capacity of chick fibroblasts to initiate the growth of RSV is as sensitive to inactivation by both X-rays and UV light as is their ability to divide and form colonies, while their capacity to initiate the growth of NDV is many times more radioresistant. A common radiosensitive target controls the ability of cells to divide and to initiate the production of RSV. Once the cells have started to produce virus, they may upon irradiation lose their ability to divide and yet continue to produce virus. The results suggest that the genome of the Rous sarcoma virus must be integrated with that of the cell before virus production can begin.

Quantitative relations between causative virus and cell in the Rous No. 1 chicken sarcoma

Abstract The quantitative relations between the Rous sarcoma cell and causative virus were studied in tissue culture, with the chorioallantoic membrane of the developing chick embryo for assay purposes. It was shown that 1 virus particle is adequate to initiatie tumor development. Virus from the same clone can initiate either ectodermal or mesodermal tumors, depending on the type of cell infected. Sarcoma cell suspensions under constant conditions released virus at a rate of about 1 particle per 100 cells per hour. Because of rapid thermal inactivation of the virus, the virus concentration tended to an average constant level, reached in 6 hours, of about 1 particle for every 25 cells. This relationship was maintained through thirty-nine serial passages of the tumor, six passages in a second series, and through the second passage of a third series. Washed sarcoma cells contained an average of only 1 virus particle per 230 cells when broken open by freezing and thawing. When intact sarcoma cells were placed on the chorioallantoic membrane, 1 tumor was produced for every 1.9 to 17 cells. It was shown that this represented the fraction of cells releasing virus in little more than a day and was not due to invasive multiplication by the cells themselves. Experiments with single virus-producing cells in suspension showed that 1 cell in 35 to 60 produced virus in a 6-hour period. Each cell produced only small amounts of virus, intermittently, in a manner not significantly different from random. These findings, when taken together, suggest that all the Rous sarcoma cells produce virus during one generation time. Virus is produced at a very slow rate, and the cells survive. It is also suggested that the virus plays a direct and continuing role in perpetuating the cell in its malignant state.

A kinetic study of infection of chick embryo cells in vitro by Rous sarcoma virus

Abstract Results of studies on the early stages of infection of chick embryo cells by Rous sarcoma virus (RSV) in vitro are reported. Progeny infectious virus is first detected at about 12 hours after infection and the final rate of virus release of 1 focus-forming unit (FFU) per cell per 5–10 hours is reached at about 40 hours after infection. The ability of a cell to produce RSV is transmitted to its progeny as an intracellular event, the number of Rous sarcoma cells doubling every 15–20 hours. Direct observation of isolated cells in microdrops shows that a cell can release virus and then divide. The capacity of infected cells to release virus following X-irradiation was studied as a function of time after infection. An increase of 30–100 times in the radioresistance of the capacity to produce virus was found in some cells 5 hours after infection and in all cells by 15–20 hours after infection. The time at which the capacity of a cell to produce virus becomes radioresistant depends on the original multiplicity of infection—the higher the multiplicity, the shorter the time.

The production, purification, and properties of Newcastle disease virus labeled with radiophosphorus

Abstract Tissue culture techniques were used to prepare high titers of Newcastle disease virus labeled with P 32 . Techniques for purifying this radioactive virus are presented, and criteria for purity are described. The purified virus was fractionated by the Schmidt-Thannhauser procedure to determine the chemical distribution of P 32 . The chemical components were investigated in some detail and the results indicate that while some of the small molecular phosphorus containing material in the TCA soluble fraction may not be essential to the virus, the phospholipid fraction is essential. P 32 was consistently found in the Schmidt-Thannhauser ribonucleic acid (RNA) fraction, while very little appeared in the deoxyribonucleic acid (DNA) fraction, suggesting that the virus contains little or no DNA.

On the mechanism of Newcastle disease virus neutralization by immune serum.

Abstract Newcastle disease virus (NDV) is shown to be neutralized by immune serum in an exponential manner, which implies that only one antibody molecule is required for inactivation of an infectious particle. A very small fraction of the neutralized particles can be reactivated upon dilution of the serum virus mixture. Experiments using virus labeled with radioactive phosphorus indicate that while a single antibody molecule can inactivate the virus as far as its ability to produce infective progeny is concerned, the attachment of several such molecules is required to prevent adsorption of the virus to the host cell. However, those inactivated particles which successfully adsorb can be removed with receptor destroying enzyme. It is suggested that the basic mechanism of neutralization is to prevent penetration of the host cell. The implications of this work for the first steps of infection by active virus are discussed.

Infection and growth of Newcastle disease virus (NDV) in cultures of chick embryo lung epithelium.

Abstract The present report constitutes a quantitative study of various stages of infection of chick embryo lung epithelium in vitro with Newcastle disease virus (NDV). The virus was assayed throughout these experiments by the plaque technique. The velocity constant for adsorption was determined as well as the true multiplicity of infection. One-step growth curves were carried out in which both the virus liberated into the medium, and that remaining associated with the cells (cell-associated virus or CAV) were measured. It was found that extracellular virus and CAV were equal in amount during the period of exponential virus increase, and the apparent release time (i.e. the time required for release of a newly formed virus particle into the medium after it has gained the property of infectivity) was calculated as about 80 minutes. Further study showed that practically all of this time was spent at the cell surface. In fact, there was no compelling evidence for infective virus at any place other than the cell surface. X-ray inactivation studies showed that both CAV and released virus occur as independent particles rather than in clumps. The small fraction of inoculated virus which retains its infectivity upon coming in contact with susceptible cells and which can be found associated with these cells during the latent period is largely resistant to serum inactivation. These particles are somehow protected from serum neutralization by their association with subcellular particulates, but do not participate in the infectious cycle unless liberated by homogenization and plated on another cell layer.

Interactions between newcastle disease virus (NDV), antibody and cell

Abstract The present work is a quantitative study of the inactivation by antibody of two measurable properties of Newcastle disease virus—infectivity and enzymatic activity—and the role played by cells in this inactivation. By use of P32-labeled virus, the enzyme, measured by the capacity of virus to elute from red blood cells (RBC), is shown to be inactivated according to first order kinetics. Repetition of the adsorption-elution cycles shows that there are multiple enzyme sites on the virus. A method is introduced for determining the number of enzyme sites per virus particle. The fraction of virus which remains infective after antibody treatment largely retains its infectivity when firmly adsorbed to cells. On the other hand fully infective untreated virus loses its infectivity upon firm adsorption. This observation leads to the conclusion that there is more than one site per virus particle at which inactivation by antibody can occur, as shown in the text. None of the sites is critical in the sense that inactivation of no single site determines, a priori, that the particle is inactive. Calculation from these data of the number of “infectivity” sites agrees in magnitude with the calculated number of enzyme sites which was derived by an independent technique. Evidence is presented for the reactivation of virus-antibody complexes on the surface of the cell. This reactivation can be blocked if serum is present in the medium. The relevance of these findings to various theories of antibody inactivation, and cell penetration is discussed.

THE PRODUCTION OF VIRUS BY ROUS SARCOMA CELLS

Our understanding of the quantitative interrelationships between cells and certain viruses has increased spectacularly in the last fifteen years. This progress has been made possible through a combination of technical developments, such as precise assays in vitro and the one-step growth curve, which have permitted accurate measurement of the dynamic aspects of infection. Such precise information about the multiplication of tumor viruses has been markedly absent, however, a fact that can be attributed largely to the inadequacy of the virus-assay techniques. An assay technique meeting some of the basic requirements was developed for the Rous sarcoma by Keogh (1938). Virus concentration in a sample was estimated by the number of tumors produced by plating the sample on the chorioallantoic membrane (CAM) of the developing chick embryo. Although some workers have found this technique to be erratic with the strains of embryos and virus used in their laboratories, the virus strain that we used produced tumors on the CAM of our embryos with great efficiency. We have combined this technique with modern tissue-culture and cell-counting methods in an effort to define some of the parameters of cell infection induced by the Rous sarcoma virus. Particular stress has been placed on finding out the fraction of cells in a tumor that produces virus, and the amount of virus produced by the individual cells. Much of this investigation has been ful.ly reported elsewhere (Rubin, 1955, 1956), and these papers should be consulted for detailed information. I shall review briefly some of this published work, and also describe a t greater length more recent unpublished work.

An analysis of the apparent neutralization of rous sarcoma virus with antiserum to normal chick tissues

Abstract The effect of antiserum to normal chick embryo tissues on the infectivity assay of the Rous sarcoma virus was investigated. It was found that such serum, when mixed with the virus and inoculated on the chorioallantoic membrane (CAM) of the developing chick embryo, caused a sharp reduction in the number of virus-induced tumors when compared with a control containing normal serum. In contrast to neutralization with specific antiviral serum, however, the reaction required complement and was completely reversible on dilution of the serum-virus mixture. Furthermore, the anticell serum suppressed tumor formation even when added after virus had adsorbed to susceptible cells, whereas the antiviral serum was effective only before adsorption. Most of the tumor-inhibiting activity of the antichick serum could be removed by absorption with normal chick cells and to a lesser degree by absorption with sheep red blood cells. Moreover, antiserum to boiled sheep red blood cell stroma was also capable of reducing the number of tumors. The antichick serum had no significant effect on the assay of vaccinia virus on the chorioallantoic membrane. It was concluded that the apparent neutralization of Rous sarcoma virus by antichick serum was due to impairment of the ability of the infected cells to multiply rather than to a direct neutralization of virus itself. This contradicts some earlier reports which indicated that unaltered normal cell protein forms an integral part of the functional surface of the Rous sarcoma virus.