stall ' I-'-" - o .....-..---_.- —gm_‘_ .‘.-. -~.,. - ‘-.“\g , . - 0-. on O . l "' ‘ "" ' - ' .. . ’ - on- . . o o g g. c... I O . 0.--... .0. 9 o -00... I o I. THE RATE OF INCORPORAWON OF FELINE LEUKEMIA VlRUS POLYPEPTIDES iNTO WELLULAR VIRIONS Dissertation for the Degree at M. S.” MICHlGAN STATE UNIVERSITY : _ sauce 3. MASON 1975 _ I ‘ -‘ . . . a o . 4. __—.—_-—_~n_fl _.- “aw,” -—___‘: 'I ..Q{--l',*-; ‘5 m 9’ am." , .- ’7 '- r :. (pub, _' W 1, )mast ,. p ‘ - h... ‘ ’ taxfiflf " 2. L4. -‘ it 5,; Um versity ‘0 so“, 9-,“.1‘“ This is to certify that the thesis entitled THE RATE OF INCORPORATION OF FELINE LEUKEMIA VIRUS POLYPEPTIDES INTO EXTRACELLULAR VIRIONS presented by Bruce B. Mason has been accepted towards fulfillment of the requirements for Master's degree in Migrobiology jg/L/I/f Major professor Date May 16, 1975 0-169 AW lNG iii”: 33!: 500K BlNUERf INC LIBRARY BINDERS "WISH!” IlchIIBMl é-A“ I .II IIIIIIIIIIIII I I II III III 3 1293 301067 5381 ABSTRACT THE RATE OF INCORPORATION OF FELINE LEUKEMIA VIRUS POLYPEPTIDES INTO EXTRACELLULAR VIRIONS BY Bruce B. Mason Pulse-chase experiments were done using feline thymus tumor cells (FTTC), chronically infected with Rickard's strain of feline leukemia virus (FeLV), to study the incorporation rate of viral polypeptides into completed virions and to indicate differences in the intracellular processing of the viral components. During the first two hours of chase, viral proteins, pulse—labeled with radioactive amino acids for 10 minutes, were incorporated into extracellular virions very rapidly. The rate of incorporation was slower for the following 14 hours. Approximately 0.4% of the total labeled cellular proteins were assembled into virions at the completion of a 16 hour chase. Labeled material was also found that had a density slightly lower than FeLV and contained polypeptides with a mobility on sodium dodecyl sulfate-polyacrylamide gels that corresponded to the major viral protein, p 30, and the major viral glyc0protein, gp 70. In addition, this material coprecipitated with FeLV when treated with antiserum to detergent disrupted virus, but it sedimented slower than FeLV and contained no RNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pulse-labeled virions obtained at various chase intervals showed that incorporation of p 30 and gp 70 continued after 2 hours of chase and incorporation of the minor proteins, p 15, p 11 and p 10, did not. Larger amounts of labeled p 15 relative to p 30 were found in virus purified from the pulse-labeling supernatant compared to virus obtained from longer chase periods. A protein corresponding to a molecular weight of 38,000 daltons was found in virus at early chase times and decreased in quantity after longer chases. THE RATE OF INCORPORATION 0F FELINE LEUKEMIA VIRUS POEIPEPTIDES INTO EXTRACELLULAR VIRIONS by Bruce B. Mason A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology 1975 M? j". {I 4‘) I 0144*" . I IW‘ x“, DEDIC AT ION To Becky, Mother. and Father 11 ACKNWIEIBEHENTS I would like to thank Dr. Leland F. Velicer for his guidance and support. I would also like to thank the other members of aw guidance comittee, Dr. Loren R. Srwder and Dr. John A. Boezi, for their helpful suggestions. My most sincere appreciations go to the graduate stuients and technicians that I worked with in the laboratory. iii LIST OF TABLES LIST OF FIGURES INTRODUCTION . LITERATURE REVIEW General Characteristics TABLE OF CONTENTS Proteins and Glycoproteins Biochemical and immunological characterization Intracellular synthesis . Kinetics of incorporation MATERIALS AND METHODS Growth of Cells . . Pulse-Chase Techniques . Viral Purification . Sedimentation Velocity . lmmunoprecipitation Anakysis Gel Electrophoresis Rad ioact ivity A ssay Protein Determination RESUETS . Purification and Sedimentation Immunoprecipitation Pulse-Chase Experiments of FeLV Virions SDS-Polyacrylamide Gel Electrophoresis . . DISCUSSION . LIST OF REFERENCES iv 0 O O O O O O O O O O O 0 Page O\\n Pwm K») H 10 10 10 12 13 13 17 18 20 20 25 35 49 58 63 LIST OF TABLES Table Page 1"c Mixture . . . . . - 11 l. NEC-hh5 L-Amino Acid- 2. Percent Recovery of 3H-Uridine Labeled Marker Virus . . a“ 3. Normalization of Data . . . . . . . . 52 LIST OF FIGURES Figure l. Immunoprecipitation curve of anti-FeLV serum against disrupted 1“Cu-amino acid labeled virions . . . 2. Comparison by isopycnic centrifugation analysis of FeLV in 15$an and 20-1mm (wt/wt) sucrose density gradients . 3. Velocity sedimentation anlysis of FeLV . . . . h., Comparison of pulse-labeled FeLV by isopycnic centrifuga- tion and velocity sedimentation analysis . . . 5. Immunoprecipitation analysis of peak fractions from equilibrium gradients . . . . . . . 6. Autoradiograpny of immunoprecipitates electrophoresed on 1% SDS, 9% polyacrylamide gels . . . . . 7. Electrophoretic analysis of low density pulse-labeled material a e e 0 e e e e e ' e 8. Isopycnic centrifugation analysis of double-labeled FeLV obtained from pulse-chase experiments . . . . 9. Isopycnic centrifugation analysis of FeLV obtained from short—term.pulse-chase experiments . . . . . lO. Isopycnic centrifugation analysis of FeLV obtained from long-term pulse-chase experiments . . . . . 11. Composite diagram of results from three pulse-chase experiments 0 e e e e e e e e 12. Logarithmic plot of percent polypeptides remaining in the cell versus time of chase . . . . . . 13. Electrophoretic analysis of polypeptides from pulse-labeled and continlIOUSIy labeled FeLV e e e e e e in. Electrophoretic analysis of polypeptides of FeLV from a pulse-chase experiment . . . . . . . . 15. Incorporation patterns of pulse-labeled polypeptides into extracellular virions . . . . . . . vi Page 16 22 24 27 30 p 32 3a 37 no uz as #8 55 57 INTRODUCT ION One of the purposes of microbiological research is the study of in- fectious agents which is an important step in understanding the disease itself. The importance of studying agents such as feline leukemia virus (FeLV) may go beyond the immediate interest of leukemia in cats. One journal article stated that if amt type of human cancer is caused by a virus, it is probably leukemia (32). More recently, human melogenous leukemia cells have been shown to continuously produce a budding type C particle that has RNA tumor virus properties (18). Because of their similarities FeLV is an excellent model system for studying human leu- kemia. In this study the emphasis is on the production of extracellular. virus. Because a continuous cell line was used to produce the virus rather than its normal host environment, it is assumed that what occurs in this system is similar to the situation that exist in the infected feline host and no information is given concerning the pathogenesis of the virus or its infectivity. The purification of whole virus with the use of sedimentation velo- city and equilibrium density gradients was examined to improve the methods for obtaining FeLV without cellular contaminants. The incorporation rate of viral polypeptides into completed virions was studied by pulse-label- ing feline tlwmus tumor cells (FI‘TC) that were chronically infected with FeLV and purifying the extracellular virus from the culture medium after various chase intervals. The polypeptide composition of virions released from the cell during various chase times was determined by gel electro- phoresis. These experiments should show when the protein and glycoprotein 2 components are incorporated into completed virions and indicate differences in the intracellular processing of the viral canponents. LITERATURE REVIEW General Characteristics FeLV was first described by Jarrett,4gt.gl. (26) and was isolated from about 60 percent of the leukemic cats examined (“5. 39). Electron microscopy reveals that FeLV has the typical C-type morphology with a diameter of about 100 nm. It has a complex structure consisting of an outer-lipid containing envelope and an electron dense core which consists of ribonucleic acid (RNA) complexed with.protein. Like many other'RNA tumor viruses, FeLV obtains its outer envelope when the nucleocapsid buds through the host cell membrane. ‘rhe chemical composition of FeLV on a dry weight basis is similar to that of otherIRNA tumor viruses and ins eludes 30.35% lipid, 60.65% protein and 29% RNA. There are also small amounts of carbohydrate moities found in the virus (2, 36). Proteins and Glycoproteins The proteins and glycoproteins of RNA tumor viruses have been investigated in a wide variety of animal virus systems and these studies can be grouped into three categories: 1. Biochemical and immunological characterization 2. Intracellular synthesis and processing 3. Kinetics of incorporation into extracellular virions 'Phe study of RNA tumor virus proteins and glycoproteins has been complica- ted by the fact that there is no shut off of host protein synthesis after -infection as occurs with some other animal viruses, such as adenovirus (#6) and influenza virus (23, 28, 33). The addition of actinomycin D, 3 14. as used before infection with picornavirus (8, 9, 25, 38), paramyxovirus (6, 23) and rhabdovirus (27, 42), is also ineffective since it shuts off viral as well as cellular'RNA synthesis (4, 29). Therefore, a large percentage of non-viral protein synthesis occurs whenever labeling is done and pulse-chase methods are less effective due to contamination by host proteins. Study of intracellular synthesis and processing is a particular problem requiring special techniques which will be discussed later. Biochemical and immunolggical characterization 'When chromatographed on 6% agarose in the presence of 6H quanine hydrochloride, Rickard's strain of FeLV contained two large glycoproteins (gp) and five smaller polypeptides (p) of molecular weights (MW) 100,000 (gp Z 100), 70,000 (gp 70), 30,000 (p 30), 21.000 (p 21), 15.000 (p 15) 11,200 (p 11), and 10,000 (p 10). Afterzall and p 10 were rechromato- graphed on 8% agarose and coelectrophoresed by SDS-PAGE, it was found that they comigrate at a position corresponding to a.MW of 12,000 daltons. ‘When further examined by SDS—PAGE, p 30 and p 21 had lower MWs of 27,000 and 18,000 respectively, while p 15 maintained its 15,000 MW (19). These results are similar to those seen‘with avian (l, 5. l3, l6, 17, Zn) and mammalian (1, 5, 20, 35, 36, #9, 51) RNA tumor viruses. Five proteins, p 10, p 12, p 15, p 19 and p 27, and two glycoproteins, gp 35 and gp 85, were found in avian oncornavirus,'whereas h proteins, p 10, p 12, p 15 and p30, and two glycoproteins, gp b5 and gp 69/71, were found in murine oncornavirus. Other studies on the maturation of extracellular virus (10) have indicated that may more proteins are seen when Rous sarcoma virus is obtained by rapid harvest methods (harvested 5. 10 and 20 minutes after release of the virus). Immature (5 min) virus particles 5 contained 26 readily identifiable polypeptides and mature particles con- tained only 13. Virus particles of an intermediate age contained an intermediate number. No precursor product relationship was found; rather, it appeared that 13 polypeptides were lost during the maturation process. When the antigenic properties of MuLV and FeLV were sttxlied by radio- immunoassay, type (43), group (37), and interspecies (37) specific anti.- gens were found on both p 30 and gp 70 (M4). The results suggested that the majority of the determinants-of the major structural protein were group specific, 5% to 30% were interspecies, and a small fraction were type specific. In the case of gp 70, the chief determinants were type and group specific, and a small fraction were interspecies specific. Intracellular smhesis and processing The second group of experiments involve the use of immumoprecipita- tion to isolate the viral polypeptides that exist inside of the host cell. This method has to be used because cells infected with RNA tumor viruses continue to grow, devoting only a small percentage of cellular protein synthesis to viral proteins. By isolating murine sarcoma-leukemia virus MSV(MLV) proteins with immmt'oprecipitaticn methods and resolving into components with electrophoresis in 1% SDS, 6% polyacrylamide gels, it was determined that the mobilities of intracellular polypeptides were identical to those of the purified virion (41). In addition, the labeled polypeptides having mobilities higher than that of the major polypeptide, p 31 for NSV(m.V), were found in higher proportions in the cell extract than in the intact virion. These polypeptides were thought to be of cel- lular origin since only small amounts were present in the imunoprecipi. tate of viral antiserum that had been absorbed with an extract from unin- fected cells. 6 A large precursor protein was detected within avian ryeloblastosis virus (AMV) infected-cells by the imunoprecipitation method (1+7). This protein (76,000 daltons) was analyzed by tryptic peptide mapping and appears to be a precursor of at least the two major AMV proteins, p 27 and p 15. A maller protein 12,000 (daltons) is the precursor to the 11,000 dalton viral protein. Another protein that was partially glycosy- lated (gp 70) was thought to be the precursor for the high MW glycoprotein (gp 85) of avian sarcoma virus (21). The evidence suggest that a large precursor protein exist for oncorna- virus as is the case 'for polio and encephalonwocarditis virus (9, 25). The precursors that have been found, however, are much smaller than the 200,000 - 300,000 PM polypeptide one would expect if the entire 288 RNA subunit (3 - 1+ X 106 daltons) was translated as one large piece and later cleaved to form the viral proteins. However, the larger precursor could only be found in polio virus when amino acid analogues were used and, unfortunately, these analogues appear tohave no effect on the synthesis of RNA tumor virus proteins (47). Kinet ics of Incomrat ion Initial experiments on the incorporation of radioactive proteins into RNA tumor viruses were done in the avian system (it). Freshly explanted leukemic meloblast were used which produce AMV at a constant rate with- out ary obvious cytopathic effects. Studies with luC-phemrlalamine revealed that some protein synthesis occured at or near the cell membrane immediately prior to maturation and release of virus. This was deduced from the ap- parent rapid incorporation of labeled proteins into extracellular virus following a 15 minute-pulse. The data indicated that 19% of the total 7 labeled proteins and/or glycoproteins (100% being at 7 hours) were incor- porated into the virus after a one-half hour chase. ' A later pulse-chase experiment done with Rauscher leukemia virus (RLV) indicated a difference in the processing of some of the viral structural polypeptides (35). Separation by SDS-PAGE' showed that following a 30 minute pulse, gp 45 and gp 69/71 appeared as a larger percentage of the virus after a one hour chase than after two hours. This suggested a smaller intracellular pool size and/ or a faster turnover in these components com- pared to p 30, p 15, p 12 and p 10. The protein corresponding to p 10 also had a relatively high arginine content which made it a candidate for a histone-like protein. The immunoprecipitat ion experiments on MSV(MLV) discussed previously also dealt with the kinetic incorporation of labeled polypeptides into extracellular virions (1+1). Radioactive proteins were assembled into extracellular virions rapidly for the first four hours after a ten minute labeling period. Approximately 20% of the 7 hour total was incorporated after 30 minutes. A slower rate followed for the next 20 hours, until more than 2% of the total labeled proteins of the cell were incorporated into virions. The high molecular weight polypeptides (18,000, 83,000 and 150,000 daltons) in virus released during short chase periods had the same electrophoretic mobilities as those found in imunoprecipitates of cell extracts. A larger prOportion of high molecular weight proteins was detected in the virions that were released during short chase periods (30 - 120 minutes) than after longer chase periods (6.2“ hours). As stated before these results might indicate that the glycoproteins had either a faster turnover or a difference in pool size. Another alter- native suggested for MSVOILV) was a proteclytic cleavage may occur during longer chase times (41). A third possibility is that the other proteins, 8 p 30, p 15, p 12 and p 10, may be betmd to viral RNA during the period before virus assembly and subsequent release (35). Viral.RNA is supposedly incorporated following a lag period of 75 minutes after synthesis (3, 4, 48). This would decrease the rate of appearance of any labeled proteins that were bound to the viral RNA if the proteins were bound during an early portion of the intracellular processing of the RNA. Results obtained by East (14) using MSV indicated a shorter lag period with labeled 50 3 RNA being found in the virus after 30 minutes. Dif. ficulty arises in comparing the data produced by East to that obtained for ARV and RLV because the MSV system produced more virus, higher concentra— tions of labeled uridine,and continuous labeling was used instead of pulse labeling. Using pulse-chase labeling, Brian found that uridine_ labeled FeLV was obtained during a 30 minute chase (7). It should also be noted that there is a difference between.AMV and RLV in the rate of incorporation of viral polypeptides. There is an increase in the rate of appearance in extracellular virions after 1 hour of chase for RLV and there is no increase for AMV (4) although the data for continuous label- ing of AMV with lI‘m-phenylalanine indicates a slower rate of incorpora- tion during the first hour. In a more definitive study, the polypeptides of MSV(MLV) were examined by pulse-chase techniques and the use of 1%- SDS, 4, 7, 10 and 14% poly- acrylamide gels (49). Ten minute pulses were followed ‘3’ 30 minute chase intervals that were continued for 3 hours. In order to follow the incor. poration of 16 different polypeptides, the gels containing the high per. centage and the low percentage acrylamide were used to resolve the high molecular weight proteins and glycoproteins and the low molecular weight proteins, respectively. 'Nost of the proteins followed the basic incorpora- tion pattern of the major protein p 30. Either the rate decreased slowly 9 for the 3 hour chase period or it was steady for the first hour and then dropped at the same rate as p 30. There was a slight increase in the incorporation of p 10 during the first hour. This protein also has a high basic to neutral amino acid ratio, but contains:tryptophan‘which may eliminate it as a potential histone—like protein. However, there is still a possibility that two proteins migrate in the same region. The glycopro— teins most highly labeled with glucosamine, corresponding to 73,000 and 75,000 daltons, increased for the first 2 hours before the rate decreased a third unique incorporation pattern was observed for one low molecular weight polypeptide (18,000 daltons). This protein did not appear during the first 30 minutes of the chase, but appeared in increasing amounts during chase intervals of 30-60 minutes and 60—120 minutes. MATERIALS AND METHODS Growth of Cells Rickard's strain of FeLV was produced by chronically infected cat thy- mus tumor cells F-422 (39) maintained in suspension culture in 60% Leibowitz and 40% McCoy's media supplemented with 15% fetal calf serum (GIBCO, Grand Island Biological Co., Grand Island, N.Y.). The cell concentration doubled every 22 hours to an optimum of 2 X 106 cells/ml and then was cut back to 2 x 105 or 1 x 105 cells/ml in fresh media. Cell viability was determined by diluting cells with trypan blue dye (GIBCO) to a final dye concentra- tion of 0.08% and counting in a hemocytometer. Pulse-Chase Techniques Thymus tumor cells grown to a concentration of 1-2 X 106 cells/ml were washed twice with HBSS and resuspended in fresh media deficient in bacto. 1[IO-amino acid labeling peptone and the same amino acids contained in the mixture (Table 1). Cells were incubated for 1 hour to deplete the amino acid pools. The cells were then centrifuged and resuspended at 5-10 X 107 “Canine cells/ml in prewarmed media which contained 10 p curiesof mixed 1 acids moms, New England Nuclear (NEN)] per ml (pH was adjusted with 0.2M NaOH before the cells were added). After a 10.15 minute pulse, chilled normal media was added to stop amino acid incorporation and the cells were pelleted, washed twice in cold media and resuspended in pre- warmed normal media for subsequent chases. At the end of each chase period the desired volume of supernatant fluid was prepared for virus purification as described. Each cell pellet was washed once with Hank's buffered saline solution (HESS) , resuspended in phosphate buffered saline (PBS), and saved for protein determination and counting of the radioactivity content in the cellular material. 10 11 um; Lune-M5 L-Amino Acid-Cl“ Mixture W Amino Acid Clu(u) millicures/ no amino acid millimole per mc mixture L-Alanine 129 80 L-Arginine 256 70 L-Aspartic acid 172 80‘ LaGlutanic acid 215 125 Glycine 93 “0 L-Hist id ine 258 15 L-Isoleucine 258 50 L-Leucine 280 1&0 L-Lysine 258 60 L-Phenylalanine hlh 80 L-Proline 210 50 L-Serine 129 “O L-Threonine 172 50 L-Tyrosine too 1 ho L-Valine 215 80 12 The supernatant samples were stored at h° C overnight or frozen at .2000 when frozen for 2.3 days. Marker FeLV labeled with BH-uridine (42.2 Ci/nmoles. mm) and 0.1 mg of FeLV carrier were added to the super. natant before purification by pelleting. With this method. between 25-50% of the marker could be recovered from the final gradient. The rest of the procedure will be discussed in the section on viral purification. Viral Purification Purification of FeLV was done by two methods. First, for purifica- tion of carrier virus and marker virus, polyethylene glycol (PEG) preci- pitation was used (52). Cells were removed by centrifuging at 1,000 rev/min for 10 minutes. The supernatant was clarified by centrifuging again in a Sorvall 03‘ or 88-314 rotor at 16,000 X g for 10 minutes to remove cell- ular debris. Then 50% PEI} was added to the clarified supernatant to a final concentration of 9%. This mixture was stirred slowly at no C over- night and the virus precipitate was pelleted by centrifuging at 16,000 X g for 30 minutes in a Sorvall rotor. The pellet was resuspended in TNE buffer [0.01M Tris (Trisrnvdroxymetrwl aminomethane), pH 7.u, 0.1M NaCl, 0.001M ED’I‘A (ethylene diaminetetraacetatefl . The virus suspension was purified according to a modificat ion of the sucrose gradient ultracentri- fugation method used by Duesberg (13). Briefly, the virus was ultracentri- fuged on a'discontinuous gradient consisting of 8 ml of #01? (wt/wt) sucrose and 10 ml of 20% (wt/wt) sucrose. The viral band was collected from the top of the #076 layer and centrifuged on two consecutive 20-h0% (wt/wt) sucrose continuous gradients. The virus was collected from the second continuous gradient and dialyzed against TNE buffer. The second method was used in the kinetic experiments and is similar to the one used by Hung, _e_t_ 5.1 (21+). The virus was pelleted from clarified 13 media at 25,000 rev/min for 2 hours using a SW 27 rotor. In later experi- ments the virus was pelleted through an 8 ml layer of 20% (wt/wt) sucrose. The pellet was resuspended in.TNE and sonicated for 30 seconds to a homo- genous suspension with a 150 watt Bronson ultrasonic cleaner (Bronson Instruments, Stanford Conn.). Then a 0.5 ml sample of resuspended virus was placed on a the ml 25.4071. (wt/wt) continuous gradient and centrifuged for 12.18 hrs at h5,000 rev/min in;a SW 50.1 rotor to ensure that equili- brium.was reached. Gradients were fractionated by either penetrating the bottom of the tube with a needle and dripping or by using a density gra- dient fractionator [model #600, Instrument Specialty Company (ISCO), Lincoln.Nebraska]. Fractions were either spotted on filter disks or pooled for further experiments. Sedimentation Velocigy FeLV suspensions were usually sonicated for 30 seconds and used in the lowest concentration possible to prevent aggregation. Preparations with higher concentrations (greater than 200 pg/O.l ml) of FeLV resulted in a virus peak that sedimented ahead of the normal virus peak. Bacterio— phage Tu (S = 1025) was used as a sedimentation marker (12) and its position in the gradient was determined by absorbance at 250 nm. Large samples of FeLV were usually pelleted and resuspended in 0.1 to 0.2ml TNE buffer. Then the sample and marker virus were placed on 5.20% (wt/vol) sucrose density gradients and centrifuged for 20 minutes at 25,000 rev/min, lPCina SW 50.1 rotor. Immungprecipitation Analysis, Direct immune coprecipitation was done with rabbit antiserum against Triton X-100 (11) disrupted FeLV (Serum provided by Dr. Leland Velicer). 14 This technique was used because large quantities of purified FeLV were available and direct coprecipitation does not require antiviral antibody of as high a specificity as does the indirect technique. 'When this tech. nique is used, it is necessary to quantitate the optimum antibody and antigen concentrations. Unlabeled, purified FeLV was used to adjust the concentration of antigen and small quantities of 1uC-amino acid labeled FeLV (much less than 50 pg) were used to determine the amount of precipi- tation that occured with different concentrations of viral antiserum (#1). Triton X-100 was added to a concentration of 0.8% in 0.“ ml samples that contained a specified amount of labeled and unlabeled FeLV. They were incubated 10 minutes at 37° C to allow the detergent time to disrupt the virus. Then dilutions of serum were added to make the final volume 0.6 mls. and 0.1 ml aliquots were immediately spotted on filter disks. After incubation, 37°C for 2 more hours, samples were centrifuged at 1,000 X g for 10 minutes and 0.1 ml aliquotes of the supernatant fluid were spotted again. Controls were done with normal rabbit serum. The nonspecific precipitation was between 10 and 15%. Percent cpm precipitated was calcu. lated by the following formula: 5% cpm precipitated=(Aa/Ab - c,/cb) x 100 where Aa = 14C/0.1 ml after precipitation with antiserum Ab = 1("C/0.1 ml before precipitation with antiserum Ca = 1“Ci/0.1 m1 after precipitation with control serum Cb = 1uC/O.l ml before precipitation.with control serum An optimum concentration of 100-200 pl antiserum (Fig. l) was found for precipitation of 50—100 pg FeLV. The concentration of antiserum used in immunoprecipitation was 100 pl. Samples that were to be electrophoresed Figure 1. 15 Immunoprecizitation curve of anti-FeLV serum against disrupted l C-amino acid labeled virions. Increasing concentrations of antiserum were added to a constant amount of 14C-amino acid labeled FeLV (2,000 cpm and either 50 or 100 pg FeLV). Percent precipitation was determined by the procedure discussed in materials amd methods. Nonspecific precipitation was 10% and 152 for 50 and 100 pg FeLV, respectively. l6 80v- % 40" I 50u9 lOOpg I l J T I 100 200 ANTISERUM (m) A.— 17 'were also incubated overnight at #0 C. Then they were washed three times with PBS and used for gel electrophoresis. Gel Electrophoresis Gel electrOphoresis was done according to a modification of the method of Fairbanks,‘gtlgl (15). The primary modification was the silanization of the gel tubes prior to forming gels. Briefly, the tubes were washed, vacuum dried, dipped in dimethyl-dichlorosilane, and allowed to set for two hours before being washed and dried again. This procedure reduces band curvature and makes the gels easier to remove from the gel tube. Gels made with 1% SDS and 9% polyacrylamide were used to obtain better resolution of the lower MW viral proteins. Solid samples, such as pel. leted virus and immunoprecipitates, were dissolved in protein sample buf. fer consisting of 10 millimolar'Tris-HCI, pH 8.0, l millimolar EDTA, l% SDS (wt/vol), 10 to 20 pg/ml Pyronin Y, and 3% (wt/wt) sucrose. 2.mercapto. ethanol was added to samples and they were boiled for 5 minutes. 25 to 50 pl of sample was applied to each gel and they were electrophresed at a constant voltage of 70 V for 3.4 hours. After electrophoresis, the gels were either scanned at 280 nm and fractionated or stained with Coomssie blue, dried, and placed on X-ray film for autoradiography (Kodak, Medical X-ray film, NS5¢T). Gels were scanned with a Gilford spectropholometer (model #Zho, Gilford Instrument Laboratories, Oberlin, Ohio) and fractionated into 2 mm fractions with a Gilson gel fractionator (Gilson Medical Electronics, Inc.). Prior to staining, gels were fixed in 100 ml/gel of 7% HOAc and 25% MeOH. They were fixed by stirring for 3 hours at 37° C, changing the fix, and then fixing overnight at room temperature. Staining was done with 0.0133% 18 coomasie blue in 7% HOAc and 3.3% MeOH for 2 hours at 56° C. Destaining was performed in 7% HOAc at 56° until polypeptide bands appeared distinct. Radioactivity Assgy Radioactive samples from fractionated gradients were spotted on 2.3 m Whatman paper filter disks. The disks were then dried, precipitated 20 minutes in 5% TCA at u° c. washed for two minutes in more 5% TCA, and washed once with acetone. After drying,the disks were counted in vials containing 5 ml of phosphor scintillation fluid. Phosphor Scintillation Fluid PPO 22.7 gms POPOP 1.9 gms Tolulene 8 pints PPO - 2,5 - Diphenyloxazole POPOP - 1,“ bis. 2.(h-Methyl-S—Phenoxazolyl) -Benzene Gel fractions were counted in 5 ml of Aquasol (New England Nuclear). Counting of all 3H and 1"0 labeled samples was carried out in a Packard Tricarb liquid scintillation counter (Packard Instruments Co., Downers Grove, 111.). Protein Determination Protein concentration was determined by the modified tannic acid- turbidometric method of Majbaum - Katzenellenbogen and Dobryazycka (20). Tannic Acid Solution Gum Arabic Solution 98 ml 1 N HCl 0.100 gm Gum Arabic 2 m1 Phenol 10 gm Tannic Acid 100 ml water It was usually necessary to filter the tannic acid solution before using it. Both solutions were stored at no C and warmed to room temperature 19 0.50 ml of tannic acid solution was added to 0.50 ml of protein solution containing 5 to 50 pg of protein. This mixture was before being used. stirred vigorously and allowed to stand at room temperature for 20 minutes. After this time, 1.00 ml of gum arabic solution was added and the mixture was stirred. The absorbance was read at 650 nm after 5—10 minutes. A standard curve using Bovine Serum Albumin (BSA) was made for each set of protein determinations. RESULTS Purification and Sedimentation of few Virions In preliminary experiments, when pulse-labeled virus was purified on sucrose density gradients, a shoulder appeared onthe low density side of the peak that contained FeLV virions. Therefore, zero time and 1 hour samples from a pulse-chase experiment were run on two different gradients. A more shallow gradient, 20-40% (wt/wt) compared to 15-1401, (wt/wt) sucrose, was used to provide better resolution of the differences in density and eliminate possible contaminants in the virus peak. When comparing figures 2A and 2C to figures 2B and 2D, the more shallow gradients show a greater difference between virus and the low density shoulder. In the pulse ‘ chase experiments that were done later.25-l+0% (wt/wt) sucrose density gradients were used to take further advantage of this effect. Sedimentation velocity experiments were done with pulse-labeled virus to differentiate between the lower density and higher density components. When FeLV and E. 3313 bacteriophage T1, were sedimented together (Fig. 3A), the sedimentation coefficient of FeLV was calculated as 5&0 S by the method of'Martin and Ames (31). This is a little lower than the 600 S value that has been reported for other RNA tumor viruses (’40). In another similar experiment (Fig. BB), the sedimentation coefficient of FeLV was calculated as 510 s. Figure 3B also illustrates that lac-amino acid labeled FeLV and 3n-uridine labeled FeLV cosediment. Figure 3A shows a large amount of virus sedimenting ahead of FeLV, which can usually be caused by large sample volumes (30) or aggregated virus (51). 20 Figure 2. 21 Comparison of isopycnic centrifugation analysis of FeLV in 15-402 and 20-402 (wt/wt) sucrose density radients. 400x106 cells were pulsed with 40 pc of 4C-amino acids for 15 minutes and chased according to the procedure discussed in materials and methods. Virus from 0 time and one hour samples was obtained by pelleting thru 202 (wt/wt) sucrose. Viral pellets were resuspended in 0.5 m1 TNE and centrifuged on either 15-402 or 20-40% gradients. 22 2 (F GRADIENT Figure 3. 23 Velocity sedimentation analysis of FeLV. (A.) 0.1 m1 of 3H-Uridine labeled FeLV and 0.1m1 of bacteriophage T4 marker were sedimented together on a 5.1 m1 5-202 (wt/vol) sucrose density gradient according to the procedure discussed in material and methods. (3.) FeLV pulse- labeled with 14C-amino acids was obtained by the method discussed in figure 2. After isopycnic centrifugation in a 25-40% (wt/wt) sucrose density gradient, peak fractions were pooled along with 3H-Uridine labeled FeLV marker virus and pelleted at 45,000 rpm in a SW 50.1 rotor for one hour. Virus was resuspended in TNE and velocity sedimentation analysis was done according to the method discussed in figure 3A. T4 was sedimented in a parallel gradient. 24 2 J m 0 0 0 u n .r . v I... 4 a a. ems! .1 \ V a r F m e F a.- P L L + b n J a. d + q. d m 8 .b h. 7. n... i: x 535 all 80 1mm do 56 ZWGRADIENT i0 TOP 25 Pulse-labeled virus released during the first hour of chase was studied by pelleting virus from clarified supernatant and dividing the sample for equilibrium centrifugation and velocity sedimentation. AFigure “B shows that a large amount of the pelleted material sediments slower than the marker virus. This large trailing peak found in the velocity sedi- mentation gradient might account for the low density peak that occurs in the equilibrium gradient (4A). To further determine the sedimentation characteristics of one hour virus and show a possible relationship between low density and slow sedi- menting material, virus was pelleted from clarified supernatant and the entire sample was centrifuged to equilibrium in a 25.00% (wt/wt) sucrose gradient (Fig. UC). Half of each fraction was counted and the remaining material from each of the two peaks was pooled, pelleted, and sedimented through velocity density gradients (Fig. 0D). The lower density peak sedi- mented much Slower than the higher density peak that cosedimented with the 3H-uridine labeled marker virus. The peaks in this sedimentation velocity gradient are very wide due to the addition of unlabeled carrier virus, which was added to insure good recovery from the pelleting procedure. The peaks, although not as sharp as possible, indicate the slow sedimenting charac- teristics (approximately 1008) of the low density band. Immunoprecipitation Direct coprecipitation was done on peak fractions from.equilibrium gradients to determine whether the low density material had antigenic properties similar to those of purified FeLV. In order to obtain as many counts as possible in the immunoprecipitate, no controls were done with normal rabbit serum. Instead, the amount of nonspecific precipitation 'with normal serum obtained in the quantitation experiments when 50 pg of Figure 4. 26 Comparison of pulse-labeled FeLV by isopycnic centrifu- gation and velocity sedimentation analysis. FeLV pulse- labeled with 14C-amino acids was obtained by the method discussed in figure 2, except the sucrose layer was eliminated from the pelleting procedure. (A.) Half of a one hour chase sample was analyzed by isopycnic centrifugation in a 25—402 (wt/wt) sucrose density gradient and (B.) the other half was analyzed by velocity sedimentation as described in figure 3A. (6.) FeLV from a one hour chase was analyzed by isopycnic centrifugation and peak fractions were pooled and pelleted as described in figure 33. (D.) Low density material and high density material were analyzed by velocity sedimentation analysis on parallel gradients as described in figure BB. 27 NwO— x zLUl—Ln .III. 6 4 2 n a L D._ 6-4 4-“- fib «00— X ICUIIU 7e OF GRADIENT 28 unlabeled virus was used for coprecipit at ion was subtracted from the per- cent precipitated fran each fraction (see Fig. 1). The results for one hour virus (Fig. 5A) indicate that viral proteins were found in the low density peak but they were slightly less concentrated than the proteins in the peak that corresponded to the marker virus. This could indicate that the low density peak consist of virus that is incomplete or cell vescicles that contain a high percentage of at least some of the viral proteins. The data for 18 hour virus (Fig. SB) indicates that the low density shoulder contains very little viral protein and the viral peak it self probably contains a large amount of labeled background material that is not precipitated by antiserum to FeLV. Irmunoprecipitates were further analyzed by gel electrophoresisand autoradiography. Although the gels do not show good flat bands (Fig. 6) due to the high concentration of protein that was electrophoresed, they do illustrate several points. First, there are no precipitated proteins found in the low density shoulder that are not in the viral peak. The proteins in the shoulder correspond to gp 70 and p 30 found in the virus. Next, there appears to be no p 15 in the low density shoulder, althotgh p 15 can be found in the precipitate from the peak that coincided with the marker virus. Last, there are no observable precipitated viral pro- teins in the shoulder of the 18 hour viral peak. Another experiment was done which supported these result s. When the labeled, low density material (Fig. 7A) from the pulse supernatant of a later experiment (Fig. 1h) was electrophoresed, two peaks corresponding to gp 70 and p 30 were found. This material was eliminated from most of the other gradients but was found in larger quantities in the pulse super- natant (Fig. 7B). Figure 5. 29 Immunoprecipitation analysis og peak.fractions from equi- librium gradients. 500-600x10 cells were pulse-labeled with 60 pc 1 C-amino acids and virions were obtained after a one hour chase by the same method described in figure 2. After isopycnic centrifugation, percent precipitation was determined by the same method described in figure 1. 30 (ElVlIdII-Rld 2 H - ul- III- we I -r- qp ‘b ‘- - “ \ ~§ \‘\ we .' .I \‘ I 4 5 § § : i 3 § LO :1- o co to . H H Pl ..., =r N x 3 H I_(JI kD-lfl H...” Z (F G'WJIEM Figure 6. 31 Autoradiography of immunoprecipitates electrophoresed on 1% SDS, 9% polyacrylamide gels. Precipitates were obtained by pooling fractions seen in figure 5. SDS- PAGE was done as described in materials and methods. X—ray film was exposed to longitudinally cut gels for one week. . I ' . -. .- “I J) a A ‘ ' HIGH DENSITY LOW DENSITY HIGH DENSITY LOW DENSITY 32 u" a D' ' I' ‘ ‘ , 9" a ""e ‘ f .- " .. . i’ 'Q.. . _ ~ _ h. u . b . a; t;"' . ' - " 1 O .— . Figure 7. 33 Electrophoretic analysis of low density pulse—labeled material. (A.) Pulse supernatant from the experiment described in figure 14 was analyzed by isopycnic centri- fugation in 25-40% (wt/wt) sucrose density gradients. (B.) Fractions with a density lower than the virus peak were pooled and processed according to the procedure described in materials and methods. Electrophoresis was performed at the same time as the other samples described in figure 14. 34 HIGH DENSITY 1 "F" LOW DENS ITY e. TOP 20 no 60 80 ITfiT01 fi % r1= GRADIENT E 30 . 1 "’ GP 70 P EL 10 26 36 m3 FRACTIFN I‘ll-“133R 35 Pulse-chase experiments An initial pulse-chase experiment was done which by double labeling with 3H-uridine and 1MC-amino acids compared the incorporation rate of RNA and proteins into virions. This data was obtained by pelleting the cells and resuspending them in new media after different chase intervals. The results (Fig.E3) show a lag for the first hour of the chase in the incorpora- tion of RNA and no lag in the incorporation of proteins. There is a con- stant incorporation of labeled RNA into FeLV during the next 3 hours and a decrease in labeled proteins incorporated compared to the first hour. The data for incorporation of labeled.RNA agrees with that obtained by Brian (7), who showed that this cell pelleting procedure was effective for studying viral RNA. It also shows that there is no RNA in the low density peak that is found in clarified supernatant after pulse-labeling with Inc-amino acid, indicating that only the amino acid label that coincides with the 3H.uridine labeled FeLV can be in proteins of complete virions. It should be noted that the 1 hour sample had a luC-amino acid peak that may have appeared especially wide due to several drops being.lost from some fractions after the dripping of the gradient. Although the low density material contains viral proteins, it does not appear to be viral particles due to its low sedimentation coefficient and its lack of viral RNA. Therefore, in the experiments that followed, efforts were made to decrease the amount of labeled material that had a density lower than the virus. Pelleting through 20% (wt/wt) sucrose selected for the faster sedimenting virions and pulsing the cells at low- er concentrations (50 X 106 cells/ml rather than 100 X 106 cells/ml) also seemed to decrease the amount of low density material. In addition,' Figure 8. 36 Isopycnic centrifugation analysis of double-labeled FeLV obtained from pulse-chase experiments. 200x106 cells were pulsed in 2.0 ml which contained 20 pc 14C-amino acids and 200 pc 3H-Uridine. Purification was done as described in materials and methods, except the sucrose layer was eliminated from the pelleting procedure. 37 «.3 x Salim did 6 h... 2 U- 2 x n it a. t. . a u a a W 1 A an \a\ \\A\\\1- a hull 1. .4. ar\ .. A.“ ‘ull: u 151‘! l -a. a, [title/fit m a e w 1 l a. K. 41:: n A. a a. . 2. . .... S x ENE all. m. i I (F GRADIENT 38 shorter pulse times (10 minutes instead of 15 minutes) were also used which had the second advantage of looking at earlier times of the processing and. assembly of polypeptides into virions. A long tem pulse-chase experiment was done to indicate the amount of time required to incorporate most of the labeled viral proteins into can. pleted virions. The 16 hour sample shown in figure 9 has a total of 2,770 counts/min. After subtracting background, correcting for reduced counting efficiency, and dividing by the amount of label in the cells after a 16 hour chase period, the total viral proteins were calculated as 0.4% of the labeled cellular protein. The ability to quantitatively purify virus was determined by adding 3H--uridine labeled FeLV to the clarified supernatant before pelleting. This marker indicated the density or the virus-on the gradients and the slow recovery. The recovery was usually 25—50% and was probably more dependent upon the age of the marker virus and the amount of degradation of the viral RNA that had occurred. Recoverywas usually 50-60% for marker virus pelleted in independent experiments to determine 1: recovery. The percent recovery from each sample of chase supernatant deviated from the average of the first five samples by no more than 1- 12%. The 16 hour sample deviated from the average by 30%, making the 16 hour figure arti- ficially high. This increase may be due to a larger amount of virus being produced in that sample which would make the virus pellet more visible and easier to quantitatively resuspend. To determine how soon labeled viral proteins are incorporated into canpleted virions, a short term pulse-chase experiment was performed (Fig.10), Aten minute pulse was followed by 0, 15, 30, 60, 120 and 240 minute chase intervals. Recovery of 3I-I--uridine labeled virus showed a deviation from the average of no more than 1 9%, except for the zero time sample which was Figure 9. 39 Isopycnic centrifugation analysis of FeLV obtained from short-term pulse-chase experimnets. Experimental condi- tions were as described in materials and methods. 50 ml samples were taken after 0, 15, 30, 60, 120, and 240 minutes of chase. 1,600 cpm of 3H-Uridine labeled FeLV was added to each sample before pelleting. 25-402 (wt/wt) sucrose density gradients were used for isopycnic centrifugation analysis. 40 15 MIN 30 VIIN .. 4+ r- ‘T‘ C) III- H 1 an. X / ’0’, E: i I ‘t ' ' Le x 1 a.) / ‘. I' ) If one." ‘ - I 0 ’. H V I ' - I W I ‘ l 4*- 1- % (F GRADIENT 41 Figure 10. Isopycnic centrifugation analysis of FeLV obtained from long-term pulse-chase experiments. Experimental condi- tions were as described in materials and methods. 50 ml samples were taken after 0, l, 2, 4, 8, and 16 hours of chase. 5,000 cpm 3H-Uridine labeled FeLV was added to each sample before pelleting. Purification was done according to the procedure described in materials and figure 9. 42 a. o. x 23-x... i--. q. 4HR “ 0 TIME «.2 x 2.66: ..l OF GRADIENT % 43 + 12%. Amino acid labeled virus was obtained within 15 minutes after the start of the chase. In another experiment, virus containing labeled pro- teins was also found in the supernatant from a 10 minute pulse. This will be discussed in a later section. Table 2 contains the percent recovery data for these two pulse-chase experiments. The data for the double labeling, long term and short term pulse- chase experiments were normalized and combined to provide a composite curve, (Fig. 11A). Both the 16 hour sample from the long term pulse-chase experiment and the 4 hour sample from the short term pulse-chase experi- ment were abnormally high. The 16 hour sample was discussed previously, but the 4 hour sample might be high due to settling of viral and cellular material that may occur in the flask during the chase. If settling occurs the 4 hour sample may be unequal to the others. In both the long term and short term experiments, the cells were washed after the pulse with cold media containing 5 times the normal concentration of amino acids to reduce the specific activiby of the labeled amino acids in the cell. Figure 118 and 11C contain data from two experi- ments that show no further incorporation occured during chase intervals following a 10-15 minute pulse. The drop of counts/mg protein can only be explained by contimuous cell growth. The normalized data shown in Figure 11A can be plotted according to the percent of viral proteins remaining in the cell by subtracting the percent of virus released from 100%. ‘When these values were plotted on a logarithmic scale versus time, a straight line relationship was obtained from the four first hour time points and also from the four second to eighth hour points (Fig. 12). This was interpreted to mean that the release of FeLV proteins and glycoproteins from the cell is a first order biphasic reaction. Two different incorporation rates are occuring during 44 TABLE 2.-- Percent Recovery of 3H-Uridine Labeled Marker Virus m Long Term Sample 3H-cpm f Deviation from Average 0 Time 2295 -7.8 1 Hour 2640 +7.3 2 Hours 2760 +12.0 4 Hours 2185 -ll.2 8 Hours 21525 -105 16 Hours 3190 +30.0 Short Term Sample 3H-cpm % Deviation from Average 0 Time 500 +14.0- 15 Minutes 480 -+9.1 30 Minutes 410 -7.8 60 Minutes 440 0.0 120 Minutes 410 -7.8 240 Minutes 430 -2.3 Figure 11. 45 Composite diagram of results from three pulse-chase experiments. (A.) Data from figures 8,9, and 10 were normalized based on 25.6% as virus released after one hour and 1002 at 16 hours. Quantity of virus at each time point was determined by calculating the area of the virus peak above background. (B.) Radioactivity per mg cellular protein in pulse labeled cells. Cells were from the pulse-chase experiment shown in figure 10. Processing of the cells was described in the materials and methods sectioia (C.) Same as (B.). Cells were pulsed with 60 pc C-amino acids for 15 minutes at 100x106 cells/ml and were chased at 1x106 cells/m1. Processing was discussed in materials and methods section. x 10’7 CP1/MG PPOTEI N 46 it 0 SHORT TERM + DOUBLE LABEL A LONG TERM 0 3H—URIDINE 6 8 Br *0 [yr 2.... 8 12 16 K? C... 12.... fl... get u... 2 3 u‘ Figure 12. 47 Logarithmic plot of percent polypeptides remaining in the cell versus time of chase. Percent of viral poly- peptides in the cell was determined by subtracting the percent of incorporation seen in figure 10 from 1002. Results show that incorporation is a first order- biphasic reaction. 48 4o» «r- L db .5 0 O4 49 the assembly of the virus. This may indicate that there are differences in the intracellular pool sizes of the viral proteins or glycoproteins or some of them undergo further processing before becoming part of the virus. SDSnglyacrylamidgflEgl_§lggtrgphoresis SDS-PAGE was done on FeLV virions obtained from pulse—chase experi- ments in order to determine whether differences exist in the rate of incor- poration of the various viral polypeptide components. lLia-amino acid labeled virus was obtained in the initial experiment by a method similar to the double labeling experiment discussed earlier. Cells were pulse- labeled for 15 minutes, washed and resuspended in fresh media for one hour. Then the cells were pelleted, the supernatant was saved and the cells were resuspended for labeling overnight in fresh media with the addition of the pulse supernatant which contained unused labeled amino acids. Consequently, virus released during the first hour of the chase and from continuous labeling with amino acids was obtained (Fig. 13). One obvious difference between one hour and continuously labeled virus is the presence of a polypeptide in the one hour virus corresponding to 38,000 daltons (p 38) relative to the MWs of the other viral proteins. The absence of this peak from the continuously labeled virus could mean that the protein is incorporated early in small quantities or that it is cleaved off during extracellular maturation of the virions. Another difference is the larger amount of gp 70 relative to the amount of p 30 in the early virus. By doing a pulse-chase experiment in larger volumes with more cells and a larger quantity of labeled amino acids, sufficient labeled virus was obtained for SDS—PAGE analysis of virions released during short chase intervals. The data from this experiment were normalized (Table 3) to Figure 13. 50 Electrophoretic analysis of polypeptides from pulse- labeled and continuously labeled FeLV. Ce ls were pulsed with 80 pg 14C-amino acids at 60x10 cells/ml and chased at 3.3x106 cells/ml in 200 ml for one hour. These cells were resuspended for continuous labeling at 5.0x106 cells/ml. Virus was purified, pooled, and pelleted as described in figure 3B. Electrophoresis was done as described in materials and methods. 51 «32:2 . 20:05: on o_ on o— n... on no 1v ol< Gnu 1- +ena .. m :33 .. _ e-Ol x was 52 TABLE 3.--Normalization of Data W cm i of 1 Hr fl Polypeptides f of 1 Hr Normalization incorporated factor (F) (Fig. 11A) 0 Time 1,U50 28.7 _ ..... _ 15 min 2,930 58.1 6.0 23.11 .403 30 min 2,750 54.14 15.8 61.7 1.13 60 min 5,050 100% 25.6 100% 1.00 2 hr 2,830 56.0 32.6 127% 2.27 h hr 8,1120 167% “1.6 162.5 .973 ‘ P12 P15 P85 P30 P38 P21 gp7o ' Time xF xF xF xF xF xF xF c3P1“ (313"1 cpm Opm cpm 0 cpm 15 min 195 78 300 121 0 0 #90 19? 110 M 20 8 120 1&8 30 min 185 209 200 226 20 22_ 510 576 120 135 20 22 100 113 60 min LL00 l+00 670 670 — -_- 890 890 160 160 ’40 [#0 1’40 1’40 i 2 hr 250 568 1150 1020 30 68 630111130 00 91) 20 us 120 272 0 hr 550 535 1000 973 110 107 1690 1640 70 68i70i68 350 3&0 53 agree with the curve established in the previous pulse-chase experiments (Fig.110. Figure 1“ contains the normalized electrophorograms and figure summarizes the data obtained from all the major peaks. Three patterns of incorporation were observed. The first pattern is a large increase of label at early times and a distinct leveling off between 2 and h hours of chase, as seen for p 15 as well as p 10 and p 11. The second pattern is the continued incorporation after 2 hours of more labeled polypeptides that occurs with p 30 and gp 70. The final pattern is the decrease of a labeled polypeptide in extracellular virions exemplified by p 38. Other pulse-labeled polypeptides show a decrease at a hours, but only p 38 decreased after only one hour and by a large percentage (“#fi). Little can be said about the minor polypeptides seen in the electrophero- grams because they appear in such small quantities. The large amount of label used in this experiment made possible the examination of virus released during the 10 minute pulse. The electro- pherogram of this sample (Fig.2L5A)indicates that all the pulse-labeled viral pOlypeptides are incorporated into extracellular virus during the 10 minute pulse. A particularly large peak corresponding to p 15 is found at this early time indicating that this protein may be one of the first to be incorporated into the virus. A substantial peak also exist that has a mobility greater than p 10 and p 11. This peak was seen in other gels and was thought to be a degradation product. Figure 14. Electrophoretic analysis of polypeptides of FeLV from a pulse-chase experiment. 2x10 cells were pulsed in 40 ml with 500 pc of 14c-am1no acids for 10 minutes. Virus was concentrated from clarified supernatant by PEG precipitation as described is the materials and methods section. Resuspended virions were pelleted thru 20% (wt/wt) sucrose before isopycnic centrifugation. Peak fractions were pooled, dialyzed against 0.02 M NH4HC03 and lyophilized. Dried samples were resuspended in protein sample buffer and electrophoresed as described in materials and methods. Results were normalized based on figure 11 (see Table 3). cm x 10"2 8.1 1|" 0 TIME1 15 MINJ. 2 ‘F 1 ._.A _a . .11 up fl '1' a 30 MIN. 1 HOUR db GP P f .1 2 HOUde 4 HOURS" .- “1.8 T 1 .. .0- 1.5 .0. qbu D ~- 4» #‘b -- J1 1'2 D D 1“ 10 30 10 30 FRACT If)?! NU‘IBER Figure 15. 56 Incorporation patterns of pulse-labeled polypeptides into extracellular virions. Polypeptides were resolved by SDS-PAGE and the total label in each normalized peak was calculated for 15, 30, 60, 120, and 240 minutes of chase. 57 16L. p30 12-_ p15 0.: .. O x 2 8-1- a. U -y- n_ p 10, H 4db 9970 " /. 38 A ‘1 p M" I J l a 1 2 3 DISCUSSION Particular attention was paid to the purification of viral particles in order to generate quantitative data that could be used in later experi- ments. It was also necessary to quickly purify the virus to avoid degra— dation that might occur. Pelleting of the virus through a sucrose solution was a very rapid method that took advantage of the virions sedimentation characteristics, and subsequent isopycnic banding on shallow gradients was particularly effective in separating complete virions from particles with a lower density. Early experiments on W indicated that the infec- tivity of the virus was greatest on the high density side of the viral peak (’40). Although no tests for infectivity were done,these experiments suggest that the high density amino acid labeled virus that cosedimented with the marker virus might have a higher percentage of infectious particles. One of the possibilities that was considered to explain the origin of the low density material was that precursors to mature particles or defective particles were formed with little or no RNA. Since the low density material was not seen after continuous labeling with 1“Cuamino acids, viral degradation was eliminated. It was thought that empby capsid structures might exist inside the cell as has been seen for poliovirus (38) and these structures could bud through the cell membrane. However, no precursor-product relationship seemed to exist between the low density material and complete virions, because the low density material appeared in quantities equal to the virus during pulse-chase experiments (Fig. 8). In addition, no intracellular particles have been seen by electron micro- scopf (35). Particles that form and bud simutaneously without a complete viral genome may still be possible. Since RNA is only 2% of the dry ‘weight of FeLV, a density difference of 0.02 gm/ml would occur between 58 59 virus with RNA and particles without RNA. There is one precedent for an RNA tumor virus that contains no RNA. Experiments were done in which cells were treated with ActionOnycin D and production of viral particles that contained primarily as RNA continued for several hours ( . ). This suggests that under normal conditions virus may also be produced which contains little viral RNA or some cellular RNA. Another possibility is that the low density material may be cell vesicles that have‘ been seen by electron microscopy in preparations of purified RNA tumor viruses (36). However, these vesicles have properties similar to FeLV, i.e.. Polypeptide pool sizes (Fig. 8), antigens, p 30 and gp 70, indicating that there is a selection for vesicles containing FeLV structural components. This could be caused by a mechanism similar to the particle-particle aggregation phenomenon observed for MSV(MLV) virions (50). If these vesicles contained a high percentage of viral surface gly- coproteins, they may be capable of similar interactions with virions or other vesicular material. The density of these vesicles may also be similar to virions or membrane fractions observed in cell fractionation experiments performed in other virus systems. Similarly, membrane fractions from cells infected with influenza virus had properties like those of completed virions, such as the ability to hemagglutinate red blood cells (22). Rough and smooth endoplasmic reticulum fractions had densities of 1.12 - 1.1a gm/ml and a high concentration of viral proteins. The pulse-chase experiments show that when the cells are labeled for 10-15 minutes, pools of viral proteins and glycoproteins are made which are incorporated rapidly into virions during the first 2 hours and more slowly in the next 1’4 hours. The results are similar to those obtained for AMV (’4) and HSVOILV) (#1). The kinetics of FeLV and MSVOdLV) are 60 particularly alike except for a difference in the percentage of total pulse- labeled polypeptides that are incorporated into extracellular virions. In the MSV(MLV) system, 23 of the total labeled cellular proteins were incorporated after a Zn hour chase. A similar percentage was found for AMV only when continuous labeling was done for 16 hours. After a 16 hour chase in the FeLV system, 0.0% of the total labeled cellular proteins were incorporated into virus. The difference may be due to higher viral pro- duction in the MSV(MLV) system or a higher amount of cell vesicles obtained in the virus purification. This second alternative is possible because pulse-labeled MSV(MLV) was purified by sedimenting the virus to a pellet at 60,000 x g for 30 minutes and not by isopycnic banding on equdlibrium gradients. However, w% of the cellular proteins were precipitated by antiserum to detergent disrupted.MSV(MLV). One high.molecular weight protein ( p 38) was found at early chase times and seemed to decrease in quantity during longer chase periods. This suggest that it may be cleaved during the chase as proposed by Shanmugam (#1). A protein with a similar electrophoretic mobility has also been non-specifically precipitated from.FeLV infected-cell extracts with normal rabbit serum (Okazinski, unpublished data) indicating that it is a ubiquitous non-viral protein that may be associated with the cell membrane. The kinetics data indicated that two different processes may occur during the incorporation of labeled polypeptides into completed virions. The first process occurs very rapidly and the second is a slower mechanism. This is supported by the results obtained for pulse-1abeled virions examined by SDS-PAGE. The major protein, p 30, and the major glycoprotein gp 70 were incorporated during longer chase times than the smaller struc- tural proteins. In addition, the composition of virus from the pulse 61 supernatant indicates that p 15 is incorporated more rapidly than the other components. _By comparison Shanmugam found much more high.MW protein and glycoprotein at early chase times after a 10 minute pulse (#1), but 'Witte (#9) using the same system.MSV(MLV) found that gp 70 was in super- natant virions at lower levels in early times than in later chase inter- vals. The high degree of resolution obtained the gels used by Witte may have separated components that were released early in the chase from those released later. Also, the viral purification methods used by Shanmugan may have allowed for a large amount of cellular contamination. Immunoprecipitation methods used by Shanmugam indicated that the pro- cessing of p 30 was different from the minor proteins. Analysis of pre- cipitates showed that twice as much p 30 is associated with the cell membranes than is found in membrane free cellular extracts, but the smaller proteins, p 12 and p 15 were found in almost equal amounts from the membrane fraction and the cellular extracts. The rapid incorporation of the small viral proteins, particularly p 15, indicates that some proteins may have a smaller pool size or are directly incorporated into completed virions with little or no further processing. Experiments by'Witte showed no difference in the incorporation rate of these components, but the chase was only for 3 hours. A longer chase should be done to see if differences occur after 3 hours. Although the data from different laboratories conflict to some extent, in general, the association of a viral polypeptide with the cell membrane seems to result in delayed or prolonged incorporation into completed virions. This would be expected for glycoproteins which must undergo further processing to be glycosylated, but further processing of p 30 must be explained. 'There may be a larger pool of p 30 or the incorporation of 62 p 30 may be a rate limiting reaction. Preliminary data (Okazinski, un- published) has indicated that at least part of the p 30 pool decreases very slowly. This may be similar to the influenza virus matrix protein that is incorporated very rapidly into virions but the intracellular pool size does not appear to decrease after short chase intervals (22). The pool size of p 30 can be determined by further experiments using longer chase times. In addition, experiments should be done in.which the intra- cellular processing of viral polypeptides is studied after short chase intervals by immunoprecipitation of specific pulse-lalbeled viral proteins and glycoproteins from cellular membrane fractions and cell free extracts followed by short chase intervals. 5. 7. 9. 10. 12. 13. 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