. 1 . . ‘ A EN.” w ”(4.. ’- T ‘ _ q ‘ OI , 1.59. i. “WEUJ . + m 1.. .. 44mm” :0 a 'o" A, ‘ . 5,. (1.? ”ramdxmwwfi in: t 3 . n. “Lunar/9.5x :mef: {.55 : ‘3 :- 3.3 h... I: .r; 1.. .737! dunumfifz .fw- SquWr-wali . r, a lllllllllilllllllllillllllllilllllllllillllllllll “ -. 3 1293 10580 3666 LIBRARY : ' _ e ' = c I f...- 5‘ ixlqrgm which Umvcmty « 43 V t 0 L'IS‘E‘ £5 This is to certify that the thesis entitled The Synthesis of file. Q/Kac fura/ Profe/ns 0f ICE/[716. Lea/40.2713 Karl/s presented by Gregory F. Okasinski has been accepted towards fulfillment of the requirements for Ph.DL degree in Microbiology Maia/7C: 5/0620 Major professor 11-4-76 Date 0-7639 g." BINEING av -? 4 HO“ & SUNS’ ‘ mama! INC. UBRARY muons final-fin..- m- ...- w.» 3,-.. .. . {bfiéé’i-‘J 0 3 66"? 7 ABSTRACT THE SYNTHESIS OF THE STRUCTURAL PROTEINS OF FELINE LEUKEMIA VIRUS BY Gregory F. Okasinski The main structural protein of feline leukemia virus (FeLV) is a 30,000 dalton polypeptide termed p30. The synthesis of FeLV p30 was studied using a permanently infected feline thymus tumor cell line (F-422). Intra- cellular proteins were divided into two subcellular fractions; a cytoplasmic extract (CE) representing cellular material soluble in 0.5% NP-40 and a particulate fraction (PF) insoluble in 0.5% NP-40 but soluble in 0.2% deoxy- cholate and 0.5% NP-40. Intracellular p30 was isolated from these subcellular fractions by immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When cells were labeled for 3 h with 35 S methionine, equal amounts of p30 were found in both subcellular fractions. Immune precipitates from pulse labeled cells contained a 60,000 dalton protein (Pp60) in the PF and FeLV p30 in the CE. When pulse labeled cells were chased the label seen at the 60,000 dalton position was rapidly lost while the , 1 n 2.0 .'IU. .11.”! K“ ‘ .4. an.“ level I; OL do but- TLIEE ll" 1 Gregory F. Okasinski level of radioactivity in the p30 position increased. Examination of the intracellular and extracellular p30 levels during a 0.5-24 h chase period suggested that most of the intracellular p30 was assembled into extracellular virus. Tryptic peptide analysis Of Pp60 and viral p30 taken together with the kinetic data provides strong evi- dence that Pp60 is a precursor of FeLV p30. The rapid loss of radioactivity from Pp60, follow— ing pulse labeling, could be inhibited by the general pro— tease inhibitor, phenyl methyl sulfonyl fluoride (PMSF). This inhibition was found only to occur if PMSF was present during pulse labeling. When cells were grown in the presence of the proline analogue, L-azetidine-Z-carboxylic acid, a 70,000 dalton polypeptide (Pp70) was found in addition to Pp60 upon immune precipitation of pulse labeled cells. Intracellular Pp70 and Pp60, and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. Pp70 and virion p70 were iden- tical under this type of analysis and both contained the tryptic peptides of FeLV p30, p15, p11, and p10, while Pp60 lacked those of p11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered pll-plS-plO-p30. The data indicates that the small structural proteins of FeLV are synthesized as part of a 70,000 dalton precursor Gregory F. Okasinski polypeptide. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypep- tides is proposed. THE SYNTHESIS OF THE STRUCTURAL PROTEINS OF FELINE LEUKEMIA VIRUS BY 'L) Gregory F? Okasinski A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1976 DEDICATION To Chris and Kim ii "~21 Mlcr ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. Leland F. Velicer for his support and guidance during the course of my research. I also wish to express my gratitude to the members of my guidance committee-- Dr. Charles H. Cunningham, Dr. Loren R. Snyder, and Dr. Robert A. Ronzio--for helpful suggestions. I would also like to thank Dr. Robert J. Moon for permitting me the use of his equipment. I was supported through most of my graduate work by a departmental assistantship from the Department of Microbiology and Public Health. iii 0"" fl .ID. 0? Ova” V" \ ion}. or n‘.u.‘ ”IT" \‘M “Jam 'U 0 y““v - .‘V‘VSCE TABLE LIST OF TABLES I O O O O 0 LIST OF FIGURES . . . . . INTRODUCTION . . . . . . . LITERATURE REVIEW . . . . Post-Translational Cleavage of Proteins . . . . . . Post-Translational Cleavage of Proteins . . . . . Post-Translational Cleavage of Proteins . . . OF CONTENTS Post-Translational Cleavage of DNA Proteins . . . . . . Post-Translational Cleavage of RNA Proteins . . . . . . Oncornavirus Structure Oncornavirus Replication . . . . . Translation of Oncornavirus mRNA . REFERENCES . . . . . . . . MANUSCRIPT I - Analysis of Intracellular Leukemia Virus Proteins. Eukaryotic Alphavirus Virus Tumor I. Identification of a 60,000 Dalton Precursor of FeLV p30 Picornavirus Feline MANUSCRIPT II - Analysis of Intracellular Feline Leukemia Virus Proteins. II. The Generation of FeLV Structural Proteins from Precursor Polypep- tides . . . iv Page vi l3 l6 17 18 19 20 24 32 81 .ahle LI ST OF TABLES Table Page 1. The effect of pactamycin on the relative labeling of FeLV virion proteins . . . . . . . ll4 LIST OF FIGURES Figure Page MANUSCRIPT I - Analysis of Intracellular Feline Leukemia Virus Proteins. I. Identification of a 60,000 Dalton Precursor of FeLV p30 1. SDS-PAGE of immune precipitated p30 from NP-40 disrupted FeLV . . . . . . . . . . . . . 47 2. Immunodiffusion of the cytoplasmic extract (CE), particulate fraction (PF), and NP-40 disrupted FeLV with anti-p30 . . . . . . 50 3. Maximal immune precipitation of intra- cellular p30 . . . . . . . . . . . . . . . . . 53 4. SDS-PAGE of immune precipitates from the cytoplasmic extract and the particulate fraction of long term labeled cells . . . . . 55 5. ‘SDS-PAGE of immune precipitates from particulate fractions of pulse-chase labeled cells . . . . . . . . . . . . . . . . 58 6. SDS-PAGE of immune precipitates from cytoplasmic extracts of pulse chase labeled cells . . . . . . . . . . . . . . . . 6O 7. Analysis of intracellular and extracellular p30 levels during a 0.5-24 h chase period following a 2.5 min pulse . . . . . . . . . . 63 8. Tryptic peptide analysis of Pp60 and FeLV p30 eluted from SDS polyacrylamide gels . . . 68 vi :"V'P _,u.h.v J 1,.ovvflf‘ u~ ‘ . ‘0‘va I s. E g 4. E LO Figure Page MANUSCRIPT II - Analysis of Intracellular Feline Leukemia Virus Proteins. II. The Generation of FeLV Structural Proteins from Precursor Polypep- tides ' 1. The effect of phenyl methyl sulfonyl fluoride (PMSF) on cell viability and protein synthesis . . . . . . . . . . . . . . 94 2. SDS-PAGE of immune precipitates from particulate fractions and cytoplasmic extracts of cells incubated with and without (PMSF) following pulse labeling . . . . . . . . . . . . . . . . . . . 96 3. SDS-PAGE of immune precipitates from particulate fractions and cytoplasmic extracts of cells pulse labeled with 58 methionine in the presence and absence of PMSF . . . . . . . . . . . . . . . 99 4. SDS-PAGE of immune precipitates from the cytoplasmic extract and particulate fraction of cells pulse labeled with 355 methionine in the presence of L— azetidine—Z-carboxylic acid . . . . . . . . . 102 5. SDS-PAGE of immune precipitated p30 and p70 from detergent disrupted FeLV . . . . . . . . 105 6. Tryptic peptide analysis of Pp70 and Pp60 eluted from SDS polyacrylamide gels . . . . . 108 7. Tryptic peptide analysis of FeLV virion p70, p30, p15, pll, and p10 . . . . . . . . . 110 8. Proposed scheme for the generation of the non-glycosylated structural proteins of FeLV . . . . . . . . . . . . . . . . . . . . . 121 vii INTRODUCTION Feline leukamia virus (FeLV) contains five nongly- cosylated proteins, four of which have been purified by gel filtration in guanidine hydrochloride. The most abundant of these proteins has a molecular weight of 30,000 daltons (p30). Antiserum to p30 previously prepared in this laboratory is monospecific for this particular viral protein. A permanently infected feline thymus tumor cell suspension was used in these experiments. In these cells synthesis of viral specific proteins occurs in addition to the synthesis of host cell macromolecules. This bio- logical relationship necessitates the availability of a specific immune probe for the isolation of intracellular viral proteins. The general approach of this work was to employ antiserum to p30 as a specific immune probe and pulse-chase radioactive labeling to provide a population of readily identifiable newly synthesized and mature intracellular proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the resulting immune precipitates would provide evidence concerning the size of molecules carrying r:0 a‘ -J ‘ {oy‘i'al Lu '5'. p30 antigenic determinants in the two populations of intracellular proteins. The principal objective of this work was to determine if FeLV p30 was synthesized as part of a high molecular weight precursor polypeptide. Just prior to initiating this work, a single report appeared (80) which provided evidence for precursor proteins in avian oncorna- virus infected cells. Our experimental approach yielded presumptive precursors of 60,000 and 70,000 daltons. Tryptic peptide analysis was used to confirm the biochemical relationship between the precursor proteins and FeLV p30. Further experiments provided evidence that four of the five nonglycosylated proteins were synthesized as part of a 70,000 dalton precursor. A :"’]..a ‘IU . o . ‘ch‘r Vvoo 5‘. ‘I-v A knee: up a .. ,5 b . if”! I,“ C .g’ ‘ 515.6 'u: a LITERATURE REVIEW Since numerous reviews of oncorvaviruses have appeared during the past few years, this review will con- centrate on literature concerning the basic phenomenon investigated, that being post-translational cleavage and processing. Post—Translational Cleavage of Eukaryotic Proteins The earliest and best known examples of post- translational cleavage are found in the blood clotting and complement cascade systems. In the blood clotting system the conversion of prothrombin to thrombin proceeds via the action of Stuart factor (extrinsic pathway) or by the action of Hageman factor (intrinsic pathway). Thrombin itself acts enzymatically to convert fibrinogen to fibrin and thus complete clot formation (53). A second important plasma protein, plasma kinin, is generated by post- translational cleavage of inactive precursors. The pharmacologically active kinins are very small peptides (nine amino acid residues) generated by cleavage at basic amino acid residues. The inactive precursors are very .I'_~ 4 ‘ Iu.J 4p... .UAdU a RPPF1 vVn-‘HA . Cl-C9 9:2}: 5" 1'!- hu. '- A. 4 . AAA It A H'V‘vz “V 1‘. ME. 5 u ‘ ‘ . .V c‘_\ "t s~ga . A.. I F 8““ . Hp . - .~ 0“ H . (I, F) large polypeptides of greater than 50,000 molecular weight (21, 34). The complement system is a third example of this ubiquitous phenomenon of extracellular generation of bio- logically active peptides from inactive precursors. The complement system is composed of nine fragments termed Cl-C9 which are generated in a cascading fashion from enzymes within the system. There are at least five known enzymes in the complement system-~Clr, C13, C42, C423, and C3 activator. These all exist as inactive precursors which become activated to act enzymatically on other inactive precursors of the complement cascade (19, 41). In recent years it has become apparent that there are large precursor like polypeptide forms of many of the small biologically active peptide hormones. These large forms have been isolated in many cases from both the syn- thesizing cell of origin and from the circulation. Work by Steiner et al. (68) has clearly demonstrated that pro- insulin is a precursor of insulin. These workers have obtained the amino acid sequence of bovine proinsulin and found it to be a single polypeptide chain ordered: NHz-B chain—Arg-Arg-C peptide-Arg-A chain-COOH. Proinsulin is cleaved to yield insulin at the paired basic residues by trypsin like enzymes (68). The clearest biochemical evidence for biosynthetic precursor-product relationships for peptide hormones IiCl R’AJ ) . US.“ "-| a-. v-\ I); l I) »i ‘i “I D—d I). 7 exists in the case of parathyroid hormone (PTH) (18, 35) and glucagon (47, 74). PTH, long recognized as a prime factor in the regulation of calcium metabolism, is a simple polypeptide of 84 amino acid residues (18). Classical biosynthetic experiments employing pulse-chase labeling, immune precipitation, tryptic peptide mapping, and amino acid analysis have been used to demonstrate the synthesis of PTH. The PTH precursor is 109 amino acids long of which 84 are retained in the active hormone (18, 35). Cleavage of the precursor occurs by a trypsin like enzyme at the NHZ- terminus. Glucagon, important as a hyperglycemic agent as well as a stimulator of insulin secretion, also appears to be synthesized as a precursor (proglucagon) (47) whose size and properties are not entirely established. A strong candidate for a possible fragment of proglucagon is an isolated 37 residue peptide from bovine glucagon (74). This peptide contains 29 NH -terminal residues which 2 are identical to bovine glucagon with eight additional residues at the COOH- terminus. This molecule is believed to be a biosynthetic intermediate of glucagon (74). In addition to PTH and glucagon, large prohormone molecules have been identified for both vasopressin (55) and B-melanocyte stimulating hormone (B-MSH) (15). While direct biosynthetic evidence for precursors of these hor- mones is lacking, strong suggestive evidence exists for them. The evidence for precursors of vasopressin comes £3130 from labeled amino acid incorporation studies that demon- strate continual incorporation of amino acids into vaso- pressin long after protein synthesis is inhibited by puromycin (55). Additional evidence indicates that large immunoreactive forms of vasopressin are seen in gel fil- tration experiments (55). The best evidence for pre- cursors of B-MSH is the isolation of a 90 residue peptide containing the residues of the mature hormone (15). There is ample evidence for functional proteolytic cleavage of other eukaryotic proteins in addition to those of the small biologically active peptides. The synthesis and assembly of collagen is a well studied example of this phenomenon (7). Collagen is synthesized as a precursor molecule (procollagen) that readily assembles into the triple helical structure of tropocollagen. This molecular conformation can then readily undergo specific extra- cellular cleavage prior to assembly into collagen fibers (7). Evidence is accumulating which suggests that albumin may be synthesized as a proalbumin moiety containing five additional residues at the albumin NHz-terminus (76). Immunoglobulin light chains also appear to be synthesized as precursor molecules (59). These precursors contain a leucine rich, hydrophobic, 20 residue peptide extending from the light chain NHZ-terminus. This brief survey of eukaryotic protein processing serves to illustrate the ubiquitous nature of b".: -1.» Cs :» .hU 52-34 . M. a.» D. l A, J Thu ‘3 ~51 post translational cleavage of linear precursor molecules. More examples will probably be found as other systems are investigated. One can conclude that eukaryotic cells utilize post-translational cleavage to generate and control the level of some biologically active proteins. As will be seen in the following sections, the availability of protein processing machinery is a valuable aid for virus replication. This host cell proteolytic machinery allows the infecting virion to maintain genetic simplicity by obviating the need for virion coded protein processing systems. Post-Translational Cleavage of Picornavirus Proteins The generation of mature virion proteins from high molecular weight precursor polypeptides is a well docu- mented phenomenon observed in many animal virus infected cells. Post-translational cleavage of virus proteins was first described for poliovirus infected cells (31, 70). Poliovirus is a small "plus" strand RNA virus of the picornavirus group. The virus contains four main struc- tural proteins termed: VP-l, VP-2, VP-3, and VP—4, with molecular weights of 35,000, 28,000, 23,000, and 6,000 respectively (30). The mRNA of poliovirus does not con- tain a blocked 5'-termina1 structure and is identical to the virion RNA in size and base composition and sediments as a 355 molecule in neutral sucrose gradients (27, 48). The mRNA of poliovirus has been estimated to code for 2 to 3 x 105 daltons of protein (30). If poliovirus mRNA is translated from a single initiation site, then one would expect the initial translation product to be a polyprotein of 2 to 3 x 105 daltons. When poliovirus infected cells are pulse labeled with radioactive amino acids, polypeptides of 34,000, loo-105,000, and 125-130,000 (NCVP-X, NCVP-lk and NCVP-l, respectively) daltons are detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis (31, 70). Mature virion structural proteins are unlabeled under these conditions (31, 70). Pulse chase labeling of these cells revealed a rapid loss of label from NCVP-l and the appear- ance of label in the viral structural proteins. These experiments strongly suggested a precursor product relation- ship between NCVP-l and the structural proteins of polio— virus (30, 31, 70). When cells are grown in the presence of the phenylalanine analog, p-fluorophenylalanine, the loss of label in NCVP-l can be prevented. The analog also inhibits the incorporation of label into the struc- tural proteins (30). A large poliovirus polyprotein could be detected in cells treated with chymotrypsin inhibitors (38, 72), amino acid analogs (30), or diisopropyl fluoro- phosphate (30). Under these conditions a polyprotein of greater than 200,000 daltons (NCVP-OO) could be isolated (30, 38, 72). Jacobson et al. (30) concluded that this ”he" Jyv a: burn.- Um h“. polyprotein may represent the entire genome of poliovirus. The initial translation product of poliovirus mRNA trans- lation is believed to be NCVP-OO (30, 38, 72). This polyprotein is believed to undergo proteolytic cleavage as a growing nascent polypeptide (4). This proteo- 1ytic activity has been termed "nascent" cleavage (4). These nascent cleavages result in the formation of NCVP-X, NCVP—l, and NCVP-lk. When monkey kidney cells or HeLa cells are infected with poliovirus, chymotrypsin inhibitors do not prevent nascent cleavage in the former but do in the latter (38). This evidence indicates that at least part of the proteolytic activity is supplied by the host cell. A combination of pulse chase labeling experiments and tryptic peptide analysis (30, 31, 70) as well as immune precipitation of in vivo and in vitro cleavage products (38) has provided a cleavage scheme for the generation of the structural proteins of poliovirus. The structural proteins (VP-1 - VP-4) are formed by sequential cleavage of NCVP-l (27, 30, 31, 70). The first cleavage results in the formation of VP-l and a precursor termed NCVP-3. NCVP-3 is further cleaved to form VP-3 and VP-O. VP-O, a procapsid protein, gives rise to VP-2 and VP-4 in an assembly related cleavage step. The precursors NCVP-X and NCVP-lk are believed to give rise to the nonstructural proteins responsible for virus replication. 25, 00C ;ri:1ar cursor 35 100 protei: and NC‘ Like p( “v . L 8‘3 'e;n :V; v, In.“ J - 10 Encephalomyocarditis virus (EMC), another picorna- virus, directs the synthesis of its structural proteins in a manner similar to that described for poliovirus (ll, 12). This virus contains five structural proteins termed a, B, y, o, and s with molecular weights of 34,000, 30,000, 25,000, 6,700, and 42,000, respectively (10, 12). The primary translation products of EMC mRNA are three pre- cursor proteins termed A, F, and C with molecular weights of 100,000, 38,000, and 84,000, respectively (12). These proteins are very similar to poliovirus NCVP-l, NCVP-X and NCVP-lk generated by nascent cleavage of NCVP-OO. Like poliovirus, all of the EMC structural proteins are generated by sequential cleavage of one precursor (EMC protein A) (ll, 12). The major difference between the two viruses is an additional cleavage step of EMC protein A to yield proteins B and H. Protein B gives rise by a single cleavage step to structural protein a and D1. This step is analogous to the cleavage of NCVP-l to form NCVP-3 and VP-l. EMC protein Dl is cleaved to form structural protein 6 and y in a manner similar to the formation VP-O and VP-l from poliovirus NCVP-3. Protein 6, a minor component of the virus, is cleaved to form 0 and 8. This step is analogous to the formation of VP-2 and VP-4 from VP-O in poliovirus infected cells. Both 6 and VP-O are ruinor components of their respective viruses and are incorporated into virions as uncleaved precursors (ll, 12, .3- 4 v... ’IlI'J KT nu rm "w .4 Jim (1).; (Y7 ( I) ' u 'Iflq ML- ‘I- sh a trim: g. ‘ L.‘ 11 30). A similar sequential cleavage scheme has also been demonstrated for human rhinovirus-lA (HRV-lA) and for Mengo virus infected cells (11, 51). One distinguishing feature of poliovirus and EMC, HRV—lA, and Mengo virus is the absence in the latter of a giant polyprotein analogous to NCVP-OO (4, 12, 72). Although only poliovirus infected cells contain a detectable giant polyprotein, several lines of evidence indicate that the other picornavirus mRNAs are translated in a similar fashion. If picornavirus mRNA is translated into giant polyproteins which undergo nascent cleavages, several features of the translation products should be apparent. These translation products should arise from a single initiation site on the mRNA. If translation proceeds via a single initiation site, equimolar production of the three primary proteins (EMC precursors A, F, and C and poliovirus precursors NCVP-l, NCVP-x, and NCVP-lk) should be found in ziyg. Analysis of the products of EMC and inengo virus RNA stimulated cell free translation products indicates that these RNAs are translated from a single initiation site (49). Oberg and Shatkin (49) labeled t cell-free translation products with Met-tRNA$e and with fMET-tRNAget. Analysis of tryptic digests of the labeled translation products yielded a single labeled peptide when labeling with fMet-tRNAI;et and 30 tryptic peptides labeled ‘with Met-tRNA.$et precursor (49). Analysis of the in vivo . D \nieg .50. . SIO‘ l ”ha in: . g‘ ; S‘Tz" uvui’y pro: FEM mm t 12 translation products from EMC, poliovirus, and HRV—lA infected cells demonstrated that the three primary proteins of these viruses were present in equimolar amounts (11, 12) - However, examination of mengo virus infected cells failed to yield equimolar amounts of the three primary proteins (51) . The reason for this discrepancy is unclear. The isolation of poliovirus NCVP-OO combined with the demonstration of a single initiation site of cell-free Protein synthesis and the presence of equimolar amounts of the three primary proteins of these viruses provides strong eVidence that picornavirus mRNA is translated from a single initiation site into giant polyproteins. Assuming a single initiation site for picornavirus mRNA translation, the gene order of the primary and mature Vi rion proteins can be determined by the use of pactamycin, a specific inhibitor of proteins synthesis initiation (71, 73). Radioactive labeling of proteins synthesized in the presence of pactamycin will result in selective incor- Poration of the label into the COOH terminal end of the molecule. This experimental approach results in extensive inhibition of labeling of residues near the NHZ-terminus and thus reflects the order of corresponding nucleotide secIuences of the 5'+3' mRNA. This technique has been used by Several workers as a means of genetic mapping of poliovirus genes (13, 42, 54, 71, 73). Pactamycin genetic maPping of poliovirus provides the gene order: \"J - .‘n 1 "F" no” 5v»; h .~V‘ .Cmt . 5"- the PY‘B s .ivk CEII m“ u; _ 13 NHZ—NCVP-l - NCVP-X - NCVP-lk - COOH for the three primary proteins (73) . The corresponding proteins of EMC and HRV- 1A have the same order (13, 42) . The four major structural proteins of poliovirus, formed by cleavage of NCVP-l, are ordered: N112 - VP-4 - VP—2 - VP—3 - VP-l - COOH (54) . The corresponding EMC structural proteins are ordered NH2 - o - B - y — a - COOH (13) as are the analogous proteins of HRV-lA (42) . The picornaviruses are very similar in polypeptide Composition, translation, post-translational processing, and gene arrangement. The most likely explanation for the role of post-translational cleavage seems to be that proposed by Jacobson and Baltimore (31) , namely that eukaryotic protein synthesizing machinery is unable to carry out internal initiation in the translation of poly- cistronic mRNA. Since these post-translational cleavages are highly specific they can effectively eliminate the requirement for multiple initiation sites on polycistronic messages. P\<3§_§-Translational Cleavage of A\J‘EDL'lavirus Proteins Although the picornaviruses were the first group of viruses rigorously examined for post-translational cleavage, alphavirus mRNA is translated in a similar manner. The alphaviruses are "plus" strand viruses with a 428 gel'lomic RNA and a 263 mRNA (65) . These viruses contain 0'. (“I va1 ‘.n :‘i: cps;- ‘- Dion ‘1; pp O in .ll‘ ‘oa‘ 'UQ in ‘ nafi‘ ‘¥ ‘- a p 1 11" V H 14 three structural proteins, the core protein (CP) and two envelope proteins (EPl and EP2) with molecular weights of 30,000, 45,000, and 53,000, respectively (52). The two alphaviruses most frequently studied have been Sindbis virus and Semliki Forest virus (SFV) . Using a temperature sensitive mutant of Sindbis virus, Scheele and Pfefferkorn (60) were able to demonstrate the accumulation of a 130,000 dalton putative precursor at the expense of the viral structural proteins in pulse labeled cells. Tryptic peptide analysis was used to con- firm a precursor product relationship between the 130,000 dalton protein and Sindbis virus CP, EPl, and EPZ (61, 62) . This precursor has been isolated more recently without the aid of a temperature sensitive mutant in Sindbis virus infected HeLa cells (66) . Both direct and indirect evi- dence exists for a single initiation site on Sindbis Virus 268 mRNA (14, 62). Pulse labeling in the presence of pactamycin selectively inhibits incorporation of radio- aCtive amino acids into the CP, thus inferring a single Site of protein synthesis initiation (62) . Recent work by Cancedda et al. (14) , employing incorporation of labeled methionine from fMIE:T-—tRNAr§et into cell free translation products, demonstrates a single labeled peptide upon tKYPtic peptide analysis of 26S mRNA stimulated translation products (14). These same workers, however, could detect two initiation sites by the same technique when Sindbis "1!“ In. .5 V! .v u “1' a .Vo9\ hn+l UV ba ."~ . l ”1‘ a. 15 virus 42$ genomic RNA was used to stimulate cell free pro- tein synthesis. The translation of SFV mRNA is very similar to that seen with Sindbis virus mRNA. SFV infected cells have proven to be more efficient for the study of post- translational cleavage, hence a more complete picture is available. The structural proteins CP, EPl, EP2, and a small glycoprotein EP3 (10,000 daltons) are synthesized as part of a 130,000 dalton precursor (36, 39, 40, 43). This precursor undergoes several cleavage steps to generate both the CP and EPl. A 62,000 dalton cleavage product of the 130,000 dalton polypeptide is relatively stable and ultimately gives rise to EP2 and EP3 (43, 67) . Pactamycin gene ordering experiments provide the gene order: NHz—CP-EPB-EPZ-EPl-COOH for the structural proteins of SFV (40). This order is analogous to that of S indbis virus with the exception of the additional component ED 3 of SFV. Analysis of tryptic digests of cell free translation products labeled with 358 fMET-tRNAIEet indi- Q a-";es a single initiation site for translation of SFV 26S mRNA (16) . While no giant polyprotein analogous to poliovirus 00 has been isolated in alphavirus infected cells, the NCVP- QPglleration of viral structural from precursors of 130,000 Q a ltons is similar to that in poliovirus infected cells. '1'}: Q suggestive evidence from pactamycin gene ordering Q "(26’ (l u an R ‘. (.5 ‘vv I’v- ‘ I G l . stru: Q i '9‘ ~ ulSl 16 experiments and selective labeling of cell free translation products with fMET---tRNAI;Elet hint that alphavirus mRNA may indeed be translated into an as yet undetectable giant po 1 yprote in . Post-Translational Cleavage of DNA Virus Proteins Post-translational cleavage of DNA virus proteins appears to be linked to virus assembly rather than as a me ans of translating a polycistronic mRNA. Vaccinia virus infected cells contain precursor proteins P4a and P4b of Structural proteins 4a and 4b, respectively (32, 33, 44). Pulse chase labeling (32) and tryptic peptide analysis (44) We re used to establish a precursor-product relationship. Ri fampicin inhibited the formation of 4a from P4a (32, 33). C(Dzr'itinued rifampicin treatment resulted in the incorporation of P4a into assembled virions as a core component (33) . I1f T4 protein maturation (20). This is in Contrast to the primary role of post-translational cleavage in the picorna- viruses and the alphaviruses. The brief review of endo- ge nous eukaryotic proteolytic mechanisms presented initially gains perspective when taken in concern with "plus" strand RNA virus mRNA translation. The pre-existing proteolytic machinery of eukaryotic cells has not only eliminated the requirement of these viruses to code for all of the enzymes necessary for protein maturation, but has allowed the translation of viral polycistronic mRNA without evolutionary Se lection, by eukaryotes, for the ability to translate mRNA with internal initiation sites. This molecular arrangement thus allows the translation of a cumbersome viral mRNA m0 lecule by a more evolutionarily advanced host cell. Q St-Translational Cleavage of IE) ‘*~—~—i£E£gTumor Virus Proteins During the past three years there have been several onrts of the eXistence of high molecular weight precursors Before reviewing this C) IE , oncornaVirus structural proteins. a - Vldence, a general summary of oncornaVirus structure and 13% plication will be presented. 18 Oncornavirus Structure These enveloped viruses contain a 60-70 S RNA genome, which under denaturation conditions sediments as 28—358 subunits (75) . A unique feature of the oncorna- viruses is the presence of an RNA directed DNA polymerase, which makes possible the synthesis of a full length DNA copy of the virus genome (75) . The avian and mammalian oncornaviruses have a rather complex protein structure which is, however, similar among both groups of viruses. The avian oncorna- Viruses contain five nonglycosylated structural proteins termed: p10, p12, p15, p19, and p27 (9). Oncornavirus Proteins are designated p or gp for protein or glycoprotein with molecular weight values times 10.3 (e.g., p30 = P30 ,000) (3) . The mammalian viruses contain four nongly- cosylated proteins: p10, p12, p15, and p30 (9). Recently a fifth nonglycosylated protein, termed p15E, has been identified for the murine viruses (28) . These viruses generally contain a major glycoprotein, gp85 in the avian Vj-I‘uses and gp69/7l in the mammalian viruses as well as a II'linor glyc0protein, gp37 and gp45 in the avian and Ina~1'Hl'nalian viruses, respectively (9) . Recently two groups of workers (6, 8, 17, 84) have determined the genetic complexity of avian oncornavirus genOmic RNA as well as physical and genetic maps of the gellome. Evidence from chemical analysis (8) and genetic 19 recombination (6) demonstrates that the 60-708 genome is 6 polyploid with a genetic complexity of 3.5 x 10 daltons. RNAase Tl oligonucleotide mapping reveals a unique arrange- ment of the oligonucleotides demonstrating‘that all 358 subunits are similar in sequence (8) . Tl oligonucleotide mapping of recombinant avian leukosis and sarcoma viruses with specific markers for the viral glycoprotein, the sarcoma phenotype, and the RNA directed DNA polymerase produced the gene order: 5'- nonglycosylated structural proteins - RNA directed DNA polymerase - envelope glycoproteins - sarcoma gene product - 3' (84) . Oncornavirus Replication The existence of a virion RNA directed DNA poly- merase suggests that these viruses synthesize a DNA copy of their genetic information. Viral specific DNA sequences haVe been detected in infected cells in a stable integrated association with host cell DNA (5, 78) . The obvious link in the conversion of ssRNA to ds integrated DNA is a ds II'EDIlintegrated DNA intermediate. This unintegrated DNA, teZIZ‘I'ned proviral DNA, has recently been isolated as a closed c:i—Zt‘cular form and is a precursor of the integrated form of the viral specific DNA (23, 26). The evidence to date indicates that shortly after infection viral DNA is made in the cytoplasm of infected cells. This DNA is a linear or open circular form similar 11‘ length to genomic RNA subunits. Viral DNA migrates to 20 the nucleus and assumes a closed circular form just prior to its integration into host cell DNA (26) . The integrated DNA can serve as template for the production of "plus" strand RNA which could be translated into Viral proteins and assembled into mature viral proteins (75) . Translation of Oncornavirus mRNA Evidence from murine leukemia virus infected cells indicates that oncornavirus mRNA may consist of three distinct molecules which sediment as 358, 218, and 168 entities (22, 25, 79) . These molecules are all poly-A Containing, polyribosome associated RNAs and presumably Should function as mRNAs. The mechanism for generating these size classes of mRNA is unknown, however, recent work (Nicholaus Mueller-Lantzsch personal communication) (63) Suggest that a post-translational cleavage of oncornavirus mRNA may occur. If the 358 mRNA molecule is functional, and were translated 5'+3' from a single initiation site, one would expect a product of 250-3oo,ooo daltons. In examining oncornavirus translation products one is severely hampered by the immortalizing nature of this viral infection. Viral protein synthesis accounts for approximately 2% of the total host macromolecular syn- thesis. This biological relationship necessitates a SpeCific immune probe for viral translation products. This probe has invariably been antibody generated against 21 viral structural proteins. The inadequacy of this probe lies in its inability to monitor non-virion associated proteins coded for by the virus. During the past three years, evidence has accumu- lated that oncornaviruses direct the synthesis of their structural proteins as precursors which are cleaved to form mature viral proteins. Vogt and Eisenman first reported tzrlea isolation of an oncornavirus precursor polypeptide ( 8 O) . These workers used a combination of pulse-chase labeling and immune precipitation with antisera to avian myeloblastosis virus (AMV) to isolate a 76,000 dalton Precursor (Pr76) of AMV structural proteins. Tryptic pep- tide analysis of Pr76 and AMV confirmed this precursor prOduct relationship (80). More recent work employing tryPtic peptide analysis of Pr76 and cleavage intermediates with tryptic peptide maps of purified AMV structural pro- te ins is evidence that‘Pr76 is a precursor of AMV p10, p14, 91-7 , and p28 (81) . Two groups of workers have been able to S3’1’lthesize Pr76 in cell-free translation systems stimulated with AMV genomic RNA (56, 57, 82). Work with mammalian oncornaviruses has been limited, a‘ll't'lost exclusively, to Rauscher leukemia virus infected QQJels. One group of workers have been able to isolate 2 Q 0 ,000, 90,000, 80,000 and 65,000 dalton precursors of the S.‘tz'i‘uctural proteins of this murine leukemia virus (2, 45). '1' lllese workers have been able to translate RLV genomic RNA 22 in.a cell-free system into 180,000 dalton products (46). Recently the genomic RNA of Moloney murine leukemia virus (MLV) has been translated in a cell-free system (37). Polypeptides of 60,000, 70,000, and 160,000 daltons were detected in response to added MLV RNA (37). This work (2, 37, 45, 46) employing both i2_vivo and in_vitro methods is the only support for a giant polyprotein as the initial translation product of oncornavirus mRNA. A second group of workers using similar techniques both in yiyg_and in zitrg have not been able to detect translation products larger than 82,000 daltons (24, 58,77). These results are in direct conflict with the isolation of a 200,000 dalton oncornavirus polyprotein (2, 45). The reasons for these conflicting results are not clear. Three recent reports have appeared which support 60-70,000 dalton precursors of RLV nonglycosylated structural proteins. Stephenson et a1. (69) have isolated a 60-70,000 dalton protein in temperature sensitive mutants of RLV infected cells, which appears to be an uncleaved precursor of RLV p30, p15, and p12. Mouse L929 cells also contain a 70,000 dalton uncleaved precursor of murine p30 (50). Shapiro et a1. (64) were able to identify a 65,000 dalton precursor of RLV p30 and p15 in RLV infected rat kidney cells as well as a 90,000 dalton precursor of the main glycoprotein (gp69/7l). 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Detection of early and late virus-induced polypeptides and their distribution in subcellular fractions. Virology 57:402-408. Wang, L. H.; P. Duesberg; P. Mellon; and P. K. Vogt. 1976. Distribution of envelope-specific and sarcoma-specific nucleotide sequences from different parents in the RNAs of avian tumor virus recom- binants. Proc. Nat. Acad. Sci. U.S.A. 73:1073-1077. MANUSCRIPT I Analysis of Intracellular Feline Leukemia Virus Proteins. V. Identification of a 60,000 Dalton Precursor of FeLV p30 ‘ BY Gregory F. Okasinski and Leland F. Velicer J. Virol. (in press) 1976 32 ANALYSIS OF INTRACELLULAR FELINE LEUKEMIA VIRUS PROTEINS. I. IDENTIFICATION OF A 60,000 DALTON PRECURSOR OF FeLV p30 Gregory F. Okasinski and Leland F. Velicer Department of Microbiology and Public Health Michigan State University East Lansing, MI 48824 Running Title: FeLV p30 precursor polypeptide 1Article No. 7557 from the Michigan Agricultural Experiment Station. Most of this work was submitted by G. F. Okasinski in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Presented in part at the 75th annual meeting of the American Society for Nficrobiology 27 April-2 May 1975. New York, NY and at the Cbld Spring Harbor meeting on RNA Tumor Viruses, 28 May- 1 June, 1975. Cold Spring Harbor, NY. 33 ABSTRACT The synthesis and release of feline leukemia virus (FeLV) p30 was studied using a permanently infected feline thymus tumor cell line. Disrupted cells were divided into two subcellular fractions; a cytoplasmic extract (CE) representing cellular material soluble in 0.5% NP-40 and a particulate fraction (PF) insoluble in 0.5% NP-40 but soluble in 0.2% deoxycholate and 0.5% NP-40. Intracellular FeLV p30 was isolated from infected cells by immune precipi- tation with antiserum to p30 and subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the precipitated proteins. Cells labeled for 3 h with 358 methionine contained equal amounts of p30 in both the CE and the PF. P30 synthesis was estimated to be 0.8% of the total host cell protein synthesis. Immune precipitates from cells pulse labeled for 2.5 min contained a labeled 60,000 dalton polypeptide (Pp60) in the PF and a polypep- tide in the CE which co-migrated with FeLV p30 in SDS-PAGE. When cells were chased after a pulse label there was a rapid loss of Pp60 in the PF and an accumulation of p30 in the CE within 30 min followed by distribution of p30 in .bOth the PF and the CE. Estimation of intracellular and 34 35 extracellular p30 levels during a 0.5-24 h chase period suggested that most of the newly synthesized p30 was incorporated into extracellular virus. Tryptic peptide analysis of labeled Pp60 and p30 demonstrated the presence of 13 of 15 p30 peptides within the Pp60 molecule. The tryptic peptide analysis in concert with the pulse chase labeling data provides strong evidence that Pp60 is a precursor of p30. :3 $31 the Pa viruse weight polyp rigorc and pr appear INTRODUCT ION The polypeptide composition of both the avian and mammalian oncornaviruses has been thoroughly studied in the past several years (13, 14, 17, 18, 25). The oncorna- viruses contain 5-7 major structural proteins with molecular weights ranging from 10,000-85,000 daltons (5). While the polypeptide composition of the oncornaviruses has been rigorously studied, information concerning the synthesis and processing of these polypeptides has only recently appeared (l, 10, 23, 24, 31, 33, 34). Evidence obtained from picornavirus, paramxovirus, and reovirus infected cells indicates that nononcogenic RNA virus mRNA is translated from a single initiation site (4). The mRNA of these viruses is well characterized (3, 36, 6). All three types of nononcogenic RNA virus mRNA are translated into polypeptides which correspond in size 'with the viral mRNA (6, 21, 22). In poliovirus infected cells, the initial translation product is a large precursor polypeptide, which is subsequently cleaved to yield mature virion polypeptides (21). Oncornaviruses contain a high molecular weight genome composed of 28-358 subunits with a molecular weight 36 ofapprox subunits rparent En vitrc corbined specific subunits very simi istransl m, the 05 about 37 of approximately 3 x 106 daltons (2, 7, 9). These RNA subunits contain 3' poly (A) sequences (7, 20, 27). The apparent ability of these subunits to serve as mRNA in "in vitro" protein synthesizing systems (24, 30, 35), combined with the presence of polyribosomes of viral specific RNA with a mol. wt. similar to that of genomic subunits (12, 16, 32), suggests that oncornavirus mRNA is very similar to genomic subunits. If oncornavirus mRNA is translated in a manner similar to nononcogenic RNA virus mRNA, then one would expect an initial translation product of about 300,000 daltons. Attempts to isolate the initial translation product of oncornavirus protein synthesis have been directed to "in vitro" protein synthesizing systems and immunoprecipi— tation of viral polypeptides from infected cells. Various "in vitro" protein synthesizing systems have been used with limited success (8, 30, 35). Recently, however, polypep- tides (mol. wts. of 140,000-185,000 and 50,000-75,000) have been synthesized using Rauscher leukemia virus (RLV) genomic RNA in a cell free protein synthesizing system (24), and 75,000-80,000 dalton polypeptides in response to added 30-408 RNA of Rous Sarcoma virus (35). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of immune precipitates from avian myeloblastosis virus (AMV) (10, 33, 34) and RLV (l, 31) infected cells provides eVidence for a 76,000 dalton precursor in the former and 38 200,000, 80,000 and 65,000 dalton precursor polypeptides in the latter. While precursor polypeptides have been isolated, evidence for a 300,000 dalton precursor polypep- tide is lacking. The work reported here was undertaken (l) to deter- mine if a precursor polypeptide of FeLV p30 existed, and (2) to monitor the incorporation of intracellular p30 into extracellular virus. Data is presented which demonstrates a 60,000 dalton precursor polypeptide (Pp60) of FeLV p30 and suggests that most of the newly synthesized intracellular p30 is incorporated into extracellular FeLV. MATERIALS AND METHODS Source of cells and virus. The permanently infected feline thymus tumor cell suspension (F-422) was used throughout these experiments. This cell line produces the Rickard strain of FeLV and was propagated as previously described (7). Radioactive labeling of cells and FeLV. Labeled intracellular protein and extracellular FeLV was obtained from cells incubated for 3 h or 20 h respectively with 35 either S methionine, 3H amino acid mixture, or 14C amino acid mixture (New England Nuclear Corp.). All labeling was 6 cell/m1 with 14 done at a starting cell density of 2 x 10 luCi of isotope/106 cells. Labeling with 3H or C amino acid mixtures was done in medium containing 10% of the 358 methionine labeling normal supplement of amino acids. was done in growth medium containing 5% of the normal supplement of methionine. Pulse chase labeling was done with cells previously incubated in methionine deficient or amino acid deficient growth media for 45 min to deplete the amino acid pools. 35 The cells were then labeled for 2.5 min with S methionine of 14C amino acid mixture (luCi/lO6 cells) at a cell 39 tines fshas colle grow at I fart min 4O density of 50 x 106 cells/ml. The pulse was terminated by placing the labeled cells on frozen media containing 10 times the normal concentration of methionine or amino acids (chase medium), adding 20 volumes of cold chase medium, and collecting the cells by centrifugation. Cells to be chased were then incubated in warm chase medium for various times at a cell density of 106 cells/ml. Purification of virus. Cells were removed from the growth medium by centrifugation at 1000 rpm for 5 min in an International PR-6 centrifuge. The growth medium was further clarified by centrifugation at 10,000 rpm for 10 min in a Sorvall GSA rotor. Clarified medium was then overlayed onto a discontinuous gradient, consisting of 5 ml of 40% sucrose (wt/wt) in TNE buffer (0.01M Tris, 0.1M NaCl, 0.001M EDTA, pH 7.5) and 5 m1 of 20% sucrose (wt/wt) in TNE. The virus was banded on the 40% sucrose layer by centrifugation at 25,000 rpm for 1.5 h in a SW 27 rotor (Beckman). The banded virus was collected, diluted with an equal volume of TNE buffer, and pelletized by centri- fugation at 25,000 rpm for 1.5 h in a SW 27 rotor. The viral pellet was resuspended in sample buffer or lysis buffer (see below and Figure l) for SDS-PAGE and detergent disruption respectively. 41 Preparation of subcellular fractions. Cells were collected by centrifugation, washed in Hank's balanced salt solution (HBSS), resuspended in lysis buffer (0.5% NP-40, 0.15 M NaCl, 0.01 M Tris, pH 7.4), vortexed for 20 sec, and then incubated for 5 min at 4°C. The disrupted cells were then centrifuged at 2400 rpm for 5 min in an Inter- national PR-6 centrifuge. The supernate was removed and centrifuged at 100,000 x g for l h in a SW 50.1 rotor (Beckman). The 100,000 g supernate (cytoplasmic extract) was carefully removed and the pellet resuspended in lysis buffer containing 0.2% deoxycholate. The cytoplasmic extract (CE) was also made 0.2% deoxycholate in one experi- ment (Figure 6). Both the resuspended pellet and CE were rapidly freeze-thawed 8 times. The solubilized pellet and the CE were then centrifuged at 100,000 x g for l h in a SW 50.1 rotor. The supernate from the solubilized pellet was termed the particulate fraction (PF) or NP-40 insoluble fraction, while the CE was also termed the NP-40 soluble fraction. Preparation of antisera. Antiserum to p30 was pre- pared as previously described (17). Antisera to bovine serum albumin (BSA) was obtained from Dr. E. Sanders (Michigan State University). 42 Immunodiffusion analysis. Double diffusion was performed, using 2% Noble agar (Difco), as previously described (17). Immune precipitation. Antiserum used for immune precipitation was clarified by centrifugation at 100,000 x g for 0.5 h in a SW 50.1 rotor. Clarified antisera was added to subcellular fractions or disrupted virus and incubated for 30 min at 37°C and then overnight at 4°C. Immune precipitates were collected by layering the incu- bation mixture over 1 ml of 5% sucrose (wt/wt) in lysis buffer, followed by centrifugation at 2,000 rpm for 20 min. The immune precipitates were resuspended in 0.5 ml lysis buffer, layered over 5% sucrose and centrifuged. This was repeated one additional time. The final precipitate was solubilized for SDS-PAGE as described below or TCA precipi- table radioactivity was assayed as previously described (17). SDS-PAGE. Electrophoresis in the presence of 1% SDS was done using a 9% polyacrylamide gel similar to that described by Fairbanks et al. (11). Samples were solub- ilized in sample buffer (0.01 M Tris HCl, SmM EDTA, 1% SDS, and 2% mercaptoethanol) and heated for 3 min at 100°C. Electrophoresis was performed at 70 V for 3 h. The gels were fractionated and assayed for radioactivity as previously described using 3a 70B scintillation cocktail (l7). 43 Tryptic peptide analysis. Immune precipitates from cells pulse-labeled with 3H amino acids were electrophoresed in the presence of 1% SDS. The gels were fractionated into 2 mm slices as described above and the polypeptides eluted with 0.4 ml of 0.1% SDS at 37°C for 24 h. Small aliquots of each fraction were assayed for radioactivity to locate labeled polypeptides. 3H amino acid labeled FeLV was prepared, electrophoresed, and p30 eluted in a similar manner. The eluted polypeptides plus 1 mg of BSA as carrier, were precipitated with 15% TCA and 1 volume of ethanol. The precipitated protein was centrifuged and the pellet washed 4 times with ethanol and once with ether. The final pellet was dried under a stream of nitrogen. The precipitated protein was oxidized as described by Hirs (19) with 1 ml of performic acid (4.5 m1 formic acid + 0.5 ml 30% hydrogen peroxide kept at 25°C for 1.5 h) for l h at 4°C. Fifteen m1 of distilled H20 was added followed by 1yophilization. The lyophilized proteins were resuspended in 15 ml of distilled H O and lyophilized again. 2 The oxidized proteins were resuspended in 3 ml of 0.15 M NH4HCO3 containing 300 ug of TPCK treated trypsin (Worthington Biochemical Corp.) and 10 pl toluene then incubated for 4 h at 37°C. An additional 300 ug of TPCK treated trypsin was added and digestion continued for 15 h at 37°C. The digested polypeptides were lyophilized, 44 resuspended in 3 ml of distilled H O and lyophilized again. 2 The digested peptides were stored at -76°C. Cation exchange chromatography of the tryptic peptides was done by a modification of the technique of Schroeder (29), using a high pressure column of type P chromobeads (Technicon) maintained at 52.5°C. The tryptic peptides were resuspended in 1.5 m1 of pH 3.1 buffer (16 ml pyridine, 278 ml acetic acid/liter) and then centrifuged at 1000 rpm for 5 min to remove insoluble cores. The pep- tides were loaded onto the column under pressure developed from a 30 ml disposable syringe and tight fitting tygon tubing. The peptides were eluted with a linear gradient of 300 ml pH 3.1 buffer and 300 ml of pH 5.0 buffer (161 ml pyridine, 143 ml acetic acid/liter) at a flow rate of 30 ml/h. Fractions (3 ml) were collected, evaporated at 60°C, and radioactively assayed with 10 m1 of 3a70B scintillation cocktail (RPI Corp., Elk Grove Village, 111.). RESULTS Immune precipitation of FeLV p30 from disrupted girgg. 358 methionine labeled FeLV was prepared and electrophoresed in the presence of 1% SDS. The polypeptide profile (Figure la) obtained was similar to that seen with 3H amino acid labeled FeLV (17, and Figure 4). There was, however, little methionine label in the p10 and p11 posi- tion of the profile. A shoulder on the high molecular weight side of the p15 peak was routinely seen and may correspond to the previously reported p21 of FeLV (17). The majority of label was distributed among p15, p30 and p70. To demonstrate the specificity of anti-p30, 358 methionine labeled FeLV was disrupted with 0.5% NP-40 and 15 rapid freeze-thaw cycles and then immune precipitated. This disruption procedure solubilizes all of the major structural proteins except p70 (Manuscript in Preparation). SDS-PAGE of the immune precipitate (Figure lb) demonstrated a single polypeptide, which migrated at the position of FeLV p30. Immune precipitation of 14C amino acid labeled FeLV yielded similar results (data not shown). The data indicated that antiserum to p30 was monospecific with 45 Fig. 46 l.--SDS-PAGE of immungsprecipitated p30 from NP-40 disrupted FeLg. 8 labeled FeLV was prepared 35 from 100 x 10 cells incubated with 100 uCi of S methionine for 24 h in 50 m1 of growth medium. The virus was purified as described in materials and methods. (A) FeLV (20,000 cpm) was resuspended in sample buffer and electrophoresed. (B) FeLV (30,000 cpm) was resuspended in lysis buffer, incubated for 0.5 h at 37°C then rapidly freeze- thawed 15 times. The disrupted virus was incubated with 200 pl of anti-p30 and an immune precipitate collected, resuspended in sample buffer, and elec- trophoresed, as described in materials and methods. 35$ cpm/fraction x IO"2 .__. 47 IO A. 355 labeled FeLV l ., a. W ) D ‘4r ‘ ' D 1* IO 2" D Opl'Id 4 pH L 0 g Immune precipitated p30 3 “~de 6. . 4. a 2. o—‘miL—r O 20 4O 60 80 FRACTION NUMBER Figure l 48 respect to FeLV structural proteins. Control experiments using 5 ug of BSA and 200 ul of anti-BSA showed virtually no precipitation of labeled viral proteins, indicating little or no nonspecific trapping. Immunodiffusion of intracellular proteins. A cytoplasmic extract and particulate fraction were prepared and examined for the presence of p30 by immunodiffusion with anti-p30. Both the CE and the PF were positive for p30 as judged by the presence of a line of identity with disrupted FeLV (Figure 2a). This antiserum had previously been shown to be monospecific with respect to FeLV proteins in both immune precipitation (Figure lb) and immunodif- fusion (17). Antiserum to BSA was used in similar immuno- diffusion experiments with no precipitin lines evident (data now shown). Estimation of the level of intracellular p30 synthesis. To estimate the percent of host cell protein synthesis directed toward synthesis of p30 a PF and CE from long term labeled cells (200 x 106) were each divided into equal aliquots (cpm/aliquot) and incubated with increasing amounts of anti-p30. The immune precipitable cpm in each aliquot of the CE and PF are expressed as the percent of total cpm (cpm in the CE aliquot and cpm in the PF aliquot). The CE contained 90% of the total cpm in this experiment (data not shown). Maximal immune precipitation Fig. 49 2.--Immunodiffusion of the cytoplasmic extract (CE), particulate fraction (PF), and NP-40 disrupted FeLV with anti-p30. Wells A, B, and C, contained CE, NP-40 disrupted FeLV, and PF respectively. Well D, contained antiép30. The CE and PF were prepared from 100 x 10 cells disrupted with 0.6 ml of lysis buffer as described in materials and methods. NP-40 disrupted FeLV was prepared from unlabeled virus as described in Figure l. 50 Figure 2 . ‘- k nu. '11:}? U.‘ . g.- t v; , ‘3" 51 occurred with 50 pl of antiserum (Figure 3), which was equivalent to 400 pl of anti-p30 to maximally immune pre- cipitate intracellular p30 from the CE or PF of 100 x 106 cells. The data in Figure 3 indicated that approxi- mately 0.8% of the total host cell protein synthesis was directed toward production of FeLV p30. The data further suggested that intracellular p30 was equally distributed between the CE (an NP-40 soluble form) and the PF (an NP-40 insoluble form) and indicated a 10 fold enrichment of intracellular p30 in the PF relative to the total cpm present in this fraction. Nonspecific precipitation was determined from a parallel experiment employing 5 ug of BSA per aliquot and increasing anti-BSA. Total nonspecific precipitation was less than 5% of the anti-p30 immune precipitable cpm. Tht total cpm (cpm in CE + cpm in PF) in this experiment represented greater than 95% of the total TCA precipitable cpm incorporated during a 3 h labeling period (data not shown) and indicated that the cell fractionation procedure allowed examination of greater than 95% of the total protein content of these cells. SDS-PAGE of intracellular p30 immuneyprecipitated from long term labeled cells. SDS-PAGE of immune precipi- tates from the CE and PF of long term labeled cells routinely yielded a labeled polypeptide which co-migrated with FeLV p30 (Figure 4a and 4c). In addition two very small peaks (a and b) are consistently seen in both Fig. 52 3.--Maximal immune precipitation of intracellular p30. 250 x 105 cells were labeled for 3 h with 250 pCi of H amino acid mixture in 125 ml of growth medium, and a CE and PF prepared as described in materials and methods. Each subcellular fractions was divided into six equal aliquots (cpm/aliquot) and 5 ug of unlabeled NP-40 disrupted virus (pre- pared as in Figure 1) added. The aliquots were incubated with either 5, 10, 25, 50, 100, or 200 ul of anti-p30 and immune precipitates collected as described in materials and methods. The immune precipitates were resuspended in 1% SDS and the radioactivity was assayed. The cpm immune pre- cipitated from each aliquot are expressed as the percent of total cpm (cpm of a CE aliquot + cpm of PF aliquot). OI 53 m musmflm :3 02125 So oo. oo. ‘ uuuuuuuuuu n..:: to \\ .ootxw o_Emo_oo;o Paloudgoald wdo 0/0 md Fig. 54 4.--SDS-PAGE of immune precipitates from the cyto- plasmic extract and the particulate fraction of long term labeled c8115. A CE and PF were pre- pared from 180 x 10 cells labeled for 3 h with 100 uCi of S methionine in 50 ml of growth medium as described in materials and methods. The CE and the PF were each divided into two equal aliquots (cpm/aliquot) and incubated with either 200 ul of anti-p30 or with 5 ug of BSA and 200 ul of anti-BSA. The immune precipitat s were col- lected and co-electrophoresed with H amino acid labeled FeLV as described in materials and methods. (A) CE and anti-p30. (B) PF and anti-p30. (C) CE with 5 ug BSA and anti-BSA. (D) PF with 5 ug BSA and anti-BSA. 55 o-u-o z-O' x uouomywdo Hg .0 ~‘Z' I O... . Q ..... O ..... O’Doov §#-‘-..- A m-..------9--........°. V? 04 (O