RADIOBIOLOGICAL AND BIOCHEMICAL INVESTIGATIONS OF POLYOMA VIRUS - CELL DITERAC'TIONS Thesis by Thomas L. Benjamin In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy California Institute of ?echnology Pasadena, California 1965 DEDICATED TO MY FATHER iii ACKNOWLEIX}D1EN'J.'S To members of the f aculty of the Biology Division I would like to express my sincere appreciation for the knowledee, stimulation, gui dance and friendship which was extended to me. To Dr. Renato D.llbecco I owe special thanks for his expert guida nce and invaluable a dvice . I am also greatly indebted to Dr. Giuseppe Attardi for his generosity, advice and encouragement during the last half of my graduate work . Certain phases of my work have benefitted greatly f r om the sound advice and help of Dr. Jerome Vinograd and his associates, for which I run most srateful. The invaluable technical assistance given by Mrs. Laverne Wenzel, Mrs. Arger Drew and Mrs. Bennetn Keeley is very gratefully acknowledged. A great meas~re of thanks is also due my fellow graduate students, contacts with whom have been most stimulating and rewarding. Finally, I would like t o acknowledge the financial support received f rom the United States Public Health Service (training grant 5 Tl­ GM- 86.) iv ABSTRACT' The cytolytic in-ceraction of Polyoma virus with mouse embryo cells has been st~clied by radiobioloGical methods known to distinrsuish tem- p-erate f'rom virulent bacteriophage . No evidence :for " t.emp e:rate" proper- ties of Polyoma was found. Thlring the course of these studies, it was observed that the curve of inactivation of Polyoma virus by ultraviolet light had. two components - a more sensitive one at low doses, and a less sensitive one at higher doses. Virus which survives a low dose has an eclipse period similar to that of unirracliated virus, while virus surviving higher doses shows a significantly longer eclipse period. If Puromycin is present during the early part of the eclipse period, the survival curve becomes a single exponential with the sensitivity of the less sensitive component. These results suggest a repair mech- anism in mouse cells which operates more effectively if vir us development is delayed. A comparison of the rat es of i nactivation of the cytol ytic and trans forming abilities of Polyoma oy ultraviolet light, X-rays, nitrous - 2 acid treatment, or the decay of incorporated P3 , showed that the trans- forming ability has a target size rougi:lly &:Yfo of that of the plaque- forming abili ty. It is thus concluded that only a fraction of the viral genes are necessary for causing transformation. v The appearance of virus-specific RNA in productively infected mouse kidney cells has been followed by means of hybridization between pulse-labelled RNA from the infected cells and the purified virus DNA . The results show a sharp increase in the amount of virus-specific RNA ·around the time of virus DNA synthesis. The presence of a small amount of virus-specific RNA in virus- free transformed cells has also been shown. This r esult offers strong evidence for the persistence of at least part of the viral genome in transformed cells. vi TABLE OF CONTIDrI'S Part Title GENERAL I NTRODUCT ION 1 A. Results with Rous Sarcoma Vi rus 2 B. Results with Polyoma Virus 3 C. Outline of the Thesis 5 References 7 PART I . A RADIOBIOLOGICAL STUDY OF THE POLYOMA VIRUS - MOUSE EMBRYO CELL INTERACTION 10 Figure 1 . X-ray inactivation of Py and HSV 30 Figure 2 . X-ray capacity and cloning curves 31 Figure 3. UV inactivation of Py and HSY 32 Figure 4. UV capacity and cloning curves 33 Figure 5. Survivals of irradiated virus on irradiated cells Figure 6. Growth curves of UV-irradiated and unirradiated Py 35 Figure 7. Gro•rth curves of UV-irradiated and unirradiated Py with Puromycin between 3rd and 11th hours Figure 8. Growth curves of UV- i rradiated and unirradiated Py with Puromycin between 2oth and 28th hours 37 Figur e 9. UV survival curve of Py on cells exposed to UV or Puromycin vii Part Ti tle PART II. RELATIVE 'l'AllG~ SIZES FOR THE I NACTIVATIONS OF THE Til4NSFORMING AliD REPRODUCTIVE ABILITIES OF POLYOMA VIRUS 39 Figure 1 . Inactivat ion of plaque- forming and t rans- forming abilities of polyoma virus 47 PARI' III. VIRUS- SPECIFIC RNA I N CELLS PRODUCTIVELY INFECTED OR TRANSFORMED BY POLYOMA VIRUS 48 Figure l . Growth curves of virus under normal and low-phosphate conditions Figure 2. Virus- specific fu"'tA in infected mouse kidney cells 66 Figure 3. Kinetics of hybrid formation at 6o 0 c 67 Figure 4. Attempts to exhaust virus -specific RNA f rom ru1A of diffe ren~ periods 68 Figure 5. Attempts to saturate viral DNA '-Tith RNA of different periods Figure 6. Relative amounts of virus-specific RNA a~ different times af'ter infection 70 Figure 7. Attempts to saturate viral DNA with early and l ate IBiA 71 Figure 8. Hyb riciization of infected and uninfected cell RJ."'ffi. '-Tith mouse DNA 72 Figure 9. Test for viru ~ -s p ecific RNA in normal and transformed cells 73 viii Part Title RECAPITlJLATION AND COKC LUSIONS A. Brief Recapitulation of Results 77 B. General Conclusions 77 l GENERAL INTRODUCTION The phenomenon of virus-induced neoplastic transformation of animal cells in culture (referred to hereafter simply as transformation) has become the object of an increased amount of attention and inves- tigation in recent years. The term "transformation" is broadly used to describe the acquisition of certain heritable characteristics referring both to cellular morpholoey and, more frequently, to patterns of cell growth and interaction - specificall y, multilayer ed and criss­ cross growth pattern, and loss of contact inhibition(l). Cells trans- formed i n vitro by viruses have been shown to give rise to tumors when injected into animals, and primary virus-induced tumors transplanted into culture show growth properties sirr~ lar to those of transformed cells . On the basis of such observations , transformation is generally regarded as the in vitro com1terpart of tumor -induction. Si gnificant proeress in understanding this phenomenon has been made during t he l a st few years , due in part to improvements in tech- niques for studyi ng virus-cell interactions in tissue culture, and also to rapidly expanding knowledge in fundamentally related fields , such as biochernistrJ, physiological phage genetics, etc. Studies of two viruses in particular nave been fruitful in defining the pos sible roles of virus in transformation. One is the Rous Sar co~B Virus (RSV), repre­ 2 sentative of the group of RNA- containing Avian l eukosis vi ruses( ), and the other is polyoma virus (Py), a member of a group of smal l DNA-con- taining Mammalian tumor viruses which also includes rabbit papilloma 2 virus, human warts virus, and SV-40 virus. A brief description and sUlilillary of the important findings with these two viruses will be given, followed by an explanation of the plan of the thesis and the purposes behind the experiments . A. Results with Rous Sarcoma Virus Particles of RSV are roughly spherical, about 100 mµ in diameter, 4 and contain protein and lipid in a ddition to RNA ( 3, ). Release of virus f rora infected fowl cells in culture occurs continuously and without associated cell death(5). When infected at high multiplicity, most or 6 all of the cells become transformed( ) and release virus; however, at low multiplicities of infection, non-virus - producing transformed cells can be isolated. The production of P.SV in transformed cells is linked to the p resence of "helper" virus normally present i n RSV stocks. The helper virus can be any of several viruses in the Avian leukosis group . Non-virus-producini:s transformed cells, grown f or many cell generations, can be made to release RSV by reinfection with one of the helper viruses . The antigenic specificity of the nSV t hus produced is the same as that of the helper virus. (7, 8,9,10 ) . These f indings have shown that RSV is a defective virus, al tered in some late fUnction involved in the synthesis of the protein coat, and that the defective genome persists in t he trans- fanned cells. Furthermore, a direct role of the RSV genome in determin- ing properties of the transformed cells is shown by the f act that dif- ferent virus mutants induce different morphological tY})es of transformed cells (ll). 3 B. Results with Polyoma Virus Py is a small icosaheciral virus, npproximately 43 mµ in diameter(l2 ), and consists of protein and a double- stranded circular DNA of molecular 6 14 weiG. ht 3 x 10. (l3, ) . T'ne vir. us is. capab- 1 e o f in. ducing . a wide. variety. .of tumors in rodents (l5). Both the cytolytic interaction with embryonic 6 mouse Cells(l ,l7) and the non-c yt o lyti~ c (t rans ""~o rma t ion' ) in· t eraction· with hamster cells(lB,l9) can be obtained wi~h the viral Tu'tA alone(20,2l). - 4 The effeciency of t he virus for transformation i s low, roughly 10 of that for plaque formation . In a ddition, a maximum of only a f ew per cent of infected cells become transformed a~er a single exposure . (22) to a high concentration of VJ..rus . Efforts to i nduce the development of infectious virus from trans- formed cells by a variety of treatments knmm to induce the development 2 3 24 o f proph age 'nave give.