Proteolytic cleavage events in oncornavirus protein synthesis

Biochimica et Biophysica Acta, 458 (1976) 375-396 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 87031

PROTEOLYTIC CLEAVAGE EVENTS IN ONCORNAVIRUS SYNTHESIS

PROTEIN

STUART Z. SHAPIRO and J. THOMAS AUGUST Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, N.Y. 10461 (U.S.A.) (Received April 22nd, 1976)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II. Categories of virus protein proteolytic cleavages . . . . . . . . . . . . . . A. Proteolytic cleavage in virus particle morphogenesis . . . . . . . . . . . B. Proteolytic cleavage in virus protein activation . . . . . . . . . . . . . C. Cleavage of very large "polyproteins" during virus production . . . . . . . . . . 1. Picornaviruses . . . . . . . . . . . . . . . . . . . . . . . . . 2. a togaviruses . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . .

375

. . . . . . . .

376 376 377 378 378 380

11I. Type-C oncornavirus precursor proteins . . . . . . . . . . . . . . . . . . . . . A. Avian oncornavirus protein synthesis and cleavage . . . . . . . . . . . . . . . B. Mammalian oncornavirus protein synthesis and cleavage . . . . . . . . . . . . C. Studies of translation of oncornaviral RNA . . . . . . . . . . . . . . . . . .

381 382 385 388

IV. Virus protein precursor cleavage enzymes . . . . . . . . . . . . . . . . . . . . .

389

V.

391

Models of oncornavirus protein synthesis and virus genetic structure . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

393

I. INTRODUCTION Proteins of m a n y a n i m a l a n d bacterial viruses have been s h o w n to be the cleavage products of higher molecular weight precursor proteins. This widespread p h e n o m e n o n includes proteolytic cleavage events which appear to have evolved to fill different roles in various virus life cycles. F o r example, while for some viruses one or a few proteins are separately synthesized a n d subsequently cleaved d u r i n g virus f o r m a t i o n , for others all of their proteins are first synthesized together in one large polypeptide. Some o f the isolated p r o t e i n cleavages seem to be related to spatial p r o b l e m s in virus particle assembly while the more extensive cleavages seem to stem from restrictions imposed by the m e c h a n i s m of protein synthesis in a n i m a l cells. A n o t h e r possible role is observed in the cleavages of some m e m b r a n e proteins which

376 appear to be related to functional activation of those proteins; a phenomenon analogous to the cleavage activation of proenzymes. Examples of several of these types of virus protein cleavages are discussed in detail in Proteases and Biological Control (1975) [1,2,3]. Recent reports indicate that proteolytic cleavage plays an important role in the formation of oncornavirus proteins as well. This report will review the types of cleavage processes occurring in other viruses, the experiments that elucidated these processes, and the current data about oncornavirus protein cleavages.

1I. CATEGORIES OF VIRUS PROTEIN PROTEOLYTIC CLEAVAGES

IIA. Proteolytic cleavage in virus particle morphogenesis Jacobson and Baltimore [4] reported the earliest observed virus protein cleavage event, a cleavage linked to the process of poliovirus particle formation. During guanidine inhibition of poliovirus formation, lighter particles lacking virion RNA accumulated in the cell cytoplasm. While mature poliovirus particles contain four structural protein (VPI-VP4) the empty particles contained only VPI and VP3 plus an additional larger protein, VP0. If guanidine treated cells were pulse-labeled by incubation with radiolabeled amino acids for 15 min, and the guanidine block then released, radioactive VP0 was observed to disappear concomitant with the appearance of the radiolabel in VP2 and VP4. The results of this pulse-chase experiment led Jacobson and Baltimore to hypothesize that VP0 was a precursor protein cleaved to form the smaller proteins VP2 and VP4 in a process linked to the mechanism of virion RNA incorporation into the particle. In later studies biochemical evidence supporting this hypothesis was provided by peptide analysis [5]. Proteins VP0, VP1, VP2, VP3 and VP4 were digested by the protease, trypsin, which cleaves protein into peptides characteristic of each protein depending on its amino acid sequence. The peptides of each protein were resolved by column chromatography. The peptides of VP0 , VPI and VP3 were different indicating that the proteins were not related. However, most of the peptides produced by digestion of VP2 and VP4 were present in the digest product of VP0. These results are consistent with the inclusion of the primary sequences of VP2 and VP4 within VP0, the expected result if the cleavage of VP0 gives rise to VP2 and VP4. An analogous cleavage of an immature capsid protein to yield two mature virus capsid proteins has since been reported for the other picornaviruses, EMC (encephalomyocarditis) and human rhinovirus [6]. Also, similar protein cleavages have been reported to occur for core or head proteins during virion particle assembly for several other animal viruses and some bacterial viruses; these include vaccinia [7,8], adenovirus [9,10], bacteriophage 2 [11 ] and bacteriophage T4 [2,12-17]. The conclusions of these reports were based primarily on pulse-chase experiments. Evidence for protein cleavage was also provided by experiments in which virus proteins were synthesized in the presence of inhibitors of proteolysis [7,15]. Under such conditions

377 precursor proteins accumulated and cleavage products failed to appear. Some studies also included comparison of the amino acid composition of the precursor and products [8], and analysis of aberrant cleavage due to mutations [11,12,13,16,17]. Proteolytic processing was also observed for the glycoproteins of some enveloped viruses. The envelopes of the a togaviruses, semliki forest and sindbis-virus, were found to contain two glycoproteins, E~ and E2. Pulse label experiments did not reveal the presence of protein E2 intracellularly but did show a larger sized virus specific glycoprotein not seen in released virions [18-24]. However, upon incubation in medium lacking radiolabel, labeled E2 appeared suggesting a precursor-product relationship for the large glycoprotein with protein E 2. Studies with proteolysis inhibitors and analysis of peptides produced by tryptic digestion confirmed this relationship [18,23]. The precursor protein has not been detected in extracellular virus particles which suggests that its cleavage may be linked to a process in particle formation or release.

liB. Proteolytic cleavage in virus protein activation Proteolytic events necessary for the functional activity of membrane proteins have been detected in studies of paramyxoviruses [3,25-28] and myxoviruses [29-38]. The paramyxovirus, sendai, when grown in mouse L cells, was much less infectious for L cells and had less hemolytic and cell fusion activity than the same virus grown in chicken eggs. Examination of the proteins of purified virions showed that sendai virus propagated in L cells lacked a membrane glycoprotein (F) found in the egg grown virus; however, it contained a larger sized protein (Fo) missing from the egg grown virus. Homma and Ohuchi observed that mild trypsin treatment resulted in the disappearance of the larger protein Fo and the appearance of the smaller protein F concomitant with the return of biological activity [25]. It was concluded that the cleavage of Fo to F is required for the normal biological activity of the virion membrane. The failure of pulse-chase experiments to reveal any mature F protein in extracts of Sendai virus infected cells is consistent with cleavage occurring during or after the process of virus release [28]. However, the presence of only the larger protein Fo in some virus particles indicates that cleavage is not necessary for particle formation or budding and therefore, this cleavage phenomenon is placed in a different category from those cleavages linked to particle assembly. Recent studies of paramyxovirus mutants by Scheid and Choppin [3] have provided elegant support for this cleavage activation model. Restoration of infectivity to non-infectious wild type sendai virus could be accomplished by in vitro digestion with trypsin but not with two other proteases, chymotrypsin or elastase. Mutants were isolated by selecting for virus whose infectivity was activated by chymotrypsin or elastase; such mutants demonstrated restored hemolysis and cleavage of the Fo protein only with the protease able to activate infectivity. Also mutants which could no longer be activated by trypsin no longer produced multiple cycles of replication in chicken eggs unless their specific activating protease was added to the allantoic fluid of the infected egg.

378 There have been several reports of a similar cleavage event involving a membrane protein of the related myxoviruses, influenza and fowl plague virus [29-38]. Two membrane glycoproteins, HA1 and HA2, associated with the hemagglutinin activity of the virus were observed in virions. These proteins were not detected after a short pulse labeling of infected cells, rather a larger virus specific glycoprotein, HA, was observed [29,31,32). Pulse chase experiments [29-33], proteolytic cleavage inhibition studies [29-33], comparison of relative amino acid composition [30], and analysis of tryptic digests [34], all indicated that the protein HA was a precursor to HA1 and HA2. Some cell lines were observed to produce particles containing only HA. While these particles had a normal morphology when examined by electron microscopy and retained normal hemagglutinating activity [35] studies by Lazarowitz and Choppin [37] and Klenk et al. [38] indicated that the cleavage of HA to HA1 and HA2 greatly enhanced the infectivity of virus particles.

HC. Cleavage of very large "polyproteins" during virus production HC.I. Picornaviruses. One cleavage event in the maturation of empty poliovirus capsids into complete virions was described by Jacobson and Baltimore in 1968 [4]. In the same year, reports by Summers and Maizel indicated that more extenfive proteolytic cleavage occurred during poliovirus production. It had been reported earlier that poliovirus infected cells contained ten noncapsid viral polypeptides (NCVPI-10) [39]. The molecular weights of these proteins plus the weights of the four capsid proteins (VP1-4) totaled 500 000 [40]. The genome of poliovirus, a single-stranded RNA of a molecular weight of about 2 • 1 0 6, is large enough to direct the synthesis of only about 200 000 daltons of protein. The discrepancy between the theoretical and actual amount of virus protein in addition to the instability of some of the intracellular viral proteins in pulse-chase experiments led Summers and Maizel to suggest that some of the viral proteins were cleavage products of other larger viral polypeptides [41]. Pulse-chase experiments [41,42] indicated that the intracellular virus protein NCVP1 was a precursor to all four viral capsid proteins (VP1-4) and tryptic digest analysis confirmed this relationship [5]. It was then discovered that labeling in the presence of protease inhibitors (diisopropyl fluorophosphate (iPrzP-F), l-Tosylamide2-phenylethyl-chloromethyl ketone (TosPheCHzCI), N-benzyloxycarbonyl-L-phenylalanine chloromethyl ketone (TosCbzPheCHzCl)) or amino acid analogs (p-fluorophenylalanine, canavanine, azetidine-2-carboxylic acid, and ethionine)* would reveal the synthesis of viral proteins larger than those previously detected and prevent the appearance of most of the normal intracellular virus proteins [5,42-44]. The molecular weight of the largest viral polypeptide observed by Jacobson et al. [5] was about 210 000, comparable to the coding capacity of the virus genome. It was suggested that the previous failure to detect these very large proteins was due to their * Some amino acid analogs may change the virus protein such that its cleavage site is not recognized by the cleavage enzyme.

379 cleavage on the polyribosome prior to completion of their synthesis [5,45,46]. To account for all the observed virus proteins, Jacobson and Baltimore proposed that all of the protein of poliovirus arose by cleavage of one large primary translation product, the observed 210000 dalton protein, which they termed a polyprotein [42].* The discovery that poliovirus messenger RNA was the same large size as the complete genome RNA [47] and that this large RNA appeared to contain only one ribosome binding site [48] supported this hypothesis. Because other data suggested that internal initiation, required for the separate synthesis of more than one polypeptide from a single messenger RNA, does not occur in eukaryotic cells [49] it was proposed that the extensive cleavage of one large polypeptide might exist as a mechanism to allow the encoding of several proteins on a single m R N A molecule [42,43]. Data from other laboratories support the polyprotein model of poliovirus protein synthesis. When Roumiantzeff, et al. [50] used polyribosomes isolated from poliovirus infected cells for in vitro protein synthesis, a large portion of the virus protein synthesized weighed about 200 000 daltons. In this system no viral proteins smaller than 65 000 daltons were observed. The purification of the protein synthesizing system away from cleavage enzymes could explain the restriction of the cleavage process observed by these authors. Garfinkle and Tershak described an inhibition of cleavage in a temperature sensitive virus strain [51]. At the restrictive temperature only 4 large virus polypeptides were observed. The largest protein weighed about 230 000 daltons. When the cells were shifted to a permissive temperature in the presence of cycloheximide (to prevent de novo protein synthesis), the high molecular weight proteins disappeared and smaller virus proteins appeared. Finally, the drug pactamycin has been used to map the order of all the observed virus specific intracellular proteins on the proposed polyprotein precursor molecule [6,45,52,53]. Since pactamycin inhibits the initiation ofpolypeptide synthesis without interfering with the elongation of polypeptide chains, amino acids are incorporated only into chains, whose synthesis began before the addition of the drug. Thus, if radioactive amino acids are given following treatment with pactamycin the specific activity of the amino acid sequences will be greater in peptides located nearer to the carboxyl terminus of the polyprotein. The pactamycin mapping technique presupposes the linkage of the proteins being mapped and that there is only one initiation site for protein synthesis. Its success in ordering the poliovirus proteins provides additional support for the polyprotein model. Precursor proteins of other related picornaviruses were also found. Early reports from Holland and Kiehn [54,55] confirmed the existence of precursor proteins for mengo and coxsackie viruses in addition to poliovirus. Other laboratories presented data showing virus protein synthesis via the mechanism of a precursor * In this review the term "polyprotein" will be reserved to designate a molecule which is the complete polypeptide product of a polycistronic messenger RNA and from which separate viral proteins are derived by the process of post-translational proteolytic cleavage.

380 polyprotein for encephalomyocarditis [6,46,53,56-59], rhino [6,53,59,60] and mengo viruses [61,62]. A comparative study by Butterworth showed that not only was the general mechanism of polyprotein synthesis and cleavage similar among the picornaviruses, but the approximate size of the different proteins, the general map order, and the mechanism of capsid protein synthesis and maturational cleavage (i.e. NCVP1 -+ VP0, VPI, VP3, and then VP0 -~ VP2 ÷ VP4) were preserved for viruses that have diverged in evolution to the extent that their virion proteins are no longer antigenically related [6]. Support for the polyprotein model of virus protein synthesis has also come from studies of in vitro translation of purified picornavirus RNA. Interestingly, however, the first attempt to translate virus RNA failed to give any evidence supporting the polyprotein model. Rekosh et al. [63] reported that the protein synthesis product of an Escherichia coli cell-free system was a heterogeneous mixture of small proteins some of which appeared to be of the sizes of several virion capsid proteins, and tryptic digest analysis confirmed that this in vitro product was virus specific protein. Also, many of these small proteins could be labeled by N-formyl [35S]methionine indicating that they were each the product of an initiation of translation which was inconsistent with the virus RNA having only one initiation site. The authors suggested that these small proteins were the product of the bacterial system recognizing sequences in poliovirus RNA not ordinarily acting as initiating sequences in animal cells. Alternatively some of the heterogeneous protein observed in this system could have been the result of variable termination of protein synthesis. In later studies using eukaryotic cell-free systems the synthesis of larger polypeptides was demonstrated [64-66] and finally Esteban and Kerr [67] were able to demonstrate the in vitro synthesis of a very large virus protein which was comparable in size to the intracellular primary precursor. Also, the polyprotein model prediction of a single initiation site was supported by a report by Ob~rg and Shatkin [68] that the initiator amino acid formyl methionine could be incorporated into only one tryptic peptide in the in vitro eukaryotic cell system. When Jacobson and Baltimore first proposed the polyprotein cleavage model they noted that the virus would lose flexibility in controlling the time or rate of expression of different genes if all its proteins were translated as parts of one polypeptide [42]. One consequence of the model is that all virus protein should be made in equimolar amounts. Butterworth and Rueckert reported this was the case for encephalomyocarditis virus [57]. Recent studies of mengovirus, however, have indicated the possibility of nonequimolar virus protein synthesis. Two laboratories found that the capsid proteins of this virus were synthesized in twice the amount of the other viral proteins [61,62,69]. Two mechanisms for this phenomenon were offered: (1) there is either a position allowing variable termination of protein synthesis on the viral mRNA or (2) a viral protein which accumulates during infection can act as a termination factor. IIC.2. a togaviruses. If the polyprotein cleavage system of the picornaviruses has evolved to circumvent the lack of internal initiation on animal cell m R N A then

381 other viruses with large messenger molecules might also direct the synthesis of polyprotein precursor molecules. One known example is the a togaviruses. The major species of a togavirus specific RNA found in the cytoplasm of infected cells are 26 S RNA and 42 to 49 S (virion sized) RNA [70-74]. Minor amounts of virus specific RNA of 33 S and 15 to 18 S are also observed [71,73]. These RNAs are sufficiently large to encode several proteins. In early studies of the a togaviruses sindbis and Semliki Forest virus, several large intracellular virus-induced proteins including a polypeptide of 130 000 daltons were observed [75-77]. In addition, Scheele and Pfefferkorn [76] found that cells infected with a temperature-sensitive sindbis virus mutant accumulated a 90 000 dalton protein when incubated at the restrictive temperature while the concentrations of the smaller virion proteins were markedly reduced. These data suggested the possibility of precursor protein synthesis. Precursor protein synthesis was first actually shown to exist for one of the two membrane glycoproteins, E2 [18-24]. Next, evidence accumulated from pulse-chase, protease inhibitor and temperature sensitive mutant virus studies that indicated the presence of other larger precursor proteins. A 100 000 dalton polypeptide was observed to be a precursor to both glycoprotein E1 and the precursor of glycoprotein E2. Another polypeptide, one of 130 000 daltons was observed to be a precursor to the 100 000 dalton precursor plus the virion capsid protein [19,22,78-85]. Schlesinger and Schlesinger [80], Ranki et al. [79] and Lachmi et al. [86] confirmed these relationships by tryptic digest analysis. In addition, Lachmi and co-workers detected an 86 000 dalton precursor which contained amino acid sequences of the capsid protein and the envelope protein E2 but not protein E1 [86]. Finally, Morser and Burke [84] reported the detection of a virus protein weighing 165 000 daltons which is large enough to be the primary translation product of the viral 26 S RNA [84]. This 165 000 dalton product would be a polyprotein containing some nonstructural viral protein in addition to the 130 000 daltonprecursor which contains all the virion structural proteins. The translation of a togavirus RNA in cell-free protein synthesizing systems also was studied. The synthesis of protein products of virion (49 S) RNA having the size of the virion capsid protein and larger polypeptides was observed [87,88]. Tryptic digest analysis showed the capsid sized protein synthesized in vitro to be identical to the virion capsid protein and indicated that the larger polypeptides contained the sequence of the capsid protein [87]. Discrete polypeptides weighing 130000, 100000 and 30000 daltons were synthesized from sindbis virus-specific 26 S RNA purified from infected cells [88]. These polypeptides corresponded in size to the virion capsid protein and the precursors seen in cell extracts.

Ill. TYPE-C ONCORNAVIRUS PRECURSOR PROTEINS The widespread occurrence of proteolytic cleavage in virus protein formation m~kes it reasonable to speculate that cleavage events may occur with practically any

382 virus under study. Based on the data from studies ofpicornaviruses and a togaviruses proteolytic cleavage would appear to be particularly applicable to viruses which have large messenger RNA molecules that may be translated into large polyprotein precursors. Interestingly, the first report of oncornavirus precursor synthesis appeared at the same time that evidence was accumulating suggesting the synthesis of large messenger molecules by oncornaviruses. RNA tumor viruses appear to be polyploid with a 70 S virion nucleic acid separating upon denaturation into 2 identical or very similar segments of singlestranded RNA. Each RNA segment sediments at about 35 S. This haploid component is estimated to weigh 3.4" 106 daltons. It could encode approximately 350 000 daltons of protein. Several laboratories have purified messenger RNA from polyribosomes of oncornavirus infected cells and have examined its virus specific component: one reported that virus specific mRNA was primarily 35 S in size [89], while others observed 35 S and 20 S pieces [90] or 35 S, 20 S and 14 S m R N A [91]. All of these messenger RNAs are large enough to encode polyprotein polypeptides. In studying the RNA tumor viruses the usual experimental approaches for precursor synthesis study have had to be combined with an immunological step. Unlike infection with many other viruses, such as the picornaviruses and the a togaviruses, where synthesis of host cell protein is inhibited, cell growth continues after oncornavirus infection. Only a small percentage of total cellular protein synthesis is devoted to the production of viral proteins. In order to separate newly synthesized oncornavirus proteins from host cell proteins, Shanmugam et al. [92] and then other investigators, turned to precipitation of the oncornavirus specific polypeptides with antisera directed against virion proteins. This procedure has since been very helpful in the study of oncornavirus protein synthesis. However, thus far it has limited the investigator to the study of virion structural proteins and precursor proteins containing at [east one structural protein. Two groups of type-C RNA tumor viruses have been studied. They are the avian leukosis-sarcoma viruses and mammalian leukemia-sarcoma viruses.

IliA. Avian oncornavirus protein synthes& and cleavage Viruses of the avian group contain several structural proteins. The major components are five internal polypeptides (p27, p19, p15, p12 and pl0) and two glycoproteins found in the virus envelope (gp85 and gp37).* Precursor polypeptides containing virion component proteins were first detected by Vogt and Eisenman [94] utilizing an antiserum broadly reactive against the virion proteins p27, p19, p15 and p12. While immunopreeipitation of an extract from cells pulse labeled for ten minutes failed to reveal the mature virion proteins, several higher molecular weight proteins

* The nomenclature used in this paper to refer to RNA tumor virus structural proteins is that described in a paper by August et al. [93]. The relation of this terminology to the designations used by Vogt and Eisenman [94] is explained in another paper by these authors [96].

383 Pr 76 H2N-

p19 (p12) p27 (p12)

p15

/ Pr 66

Prl2 p15

Pr60 ~

;

-COOH

\

1I

Pr32

p27

"~, , pI9

Fig. 1. Pactamycinmap and cleavageschemeof avian oncornavirusprecursor protein Pr76 (adapted from Vogt et aL [96].

bearing virion protein antigenicity were observed. The two major components were a Polypeptide (Pr76) of 76 000 daltons and a polypeptide (Prl2) that was slightly larger than the p15 protein. During one hour of chase, label in the precursor protein peaks decreased while labeled virion sized proteins appeared in the extracted cells. Two dimensional analysis of tryptic digests of [aSS]methionine labeled proteins also supported the proposed precursor-product relationships. An attempt to elicit the synthesis of precursor proteins larger than Pr76 by using the amino acid analogs that enabled the detection of larger precursors in poliovirus infected cells was unsuccessful. The results of Vogt and Eisenman are supported by the work of Gupta et al. [95], who used monospecific antisera prepared against the purified AMV proteins, p27, p19 and p15 to immunoprecipitate newly synthesized viral proteins. They too, detected the synthesis of a 76 000 dalton precursor protein that contained antigenic determinants of each protein tested for. Their findings also were supported by tryptic digest analysis. Additional reports from Vogt, Eisenman and Diggelmann [96,97] have expanded the data on avian virus proteolytic cleavage events. Virion proteins p27, p19, p15 and p12 and detected precursors Pr76, Pr66, Pr60, Pr32 and Prl2 were analyzed by the tryptic digestion procedure. Pactamycin mapping of the order of the virion proteins on the 76 000 dalton precursor also was performed. These data were interpreted to give the map and cleavage scheme shown in Fig. 1. The map position of pl 2 is not yet certain. Tryptic digest analysis of this protein places it within the 76 000 dalton precursor protein [96] and the presence of its characteristic tryptic peptide in the digests of Pr66 and Pr60 and absence from the digest of Pr32 suggests that this protein maps to the C terminal side of pl 9. However, which side of protein p27 it maps on remains unclear. Vogt et al. [96] further investigated the mechanism of cleavage by studies of the in vitro cleavage of Pr76 during incubation of a cell lysate containing pulse labeled precursor in the presence of variety of agents. In the absence of any added inhibitors a significant fraction of Pr76 was cleaved to give p27 and pl 5. Agents that disrupt

384 cell membranes (chloroform and the detergents NP40 and deoxycholate) inhibited cleavage while several protease inhibitors (TosPheCH2CI, TosLysCHzC1, iPr2P-F and iodoacetamide) had no effect. Also, preliminary evidence suggested a significant copurification of Pr76 with the cell membrane fraction. These data led Vogt et al. [96] to postulate that Pr76 and its cleavage protease(s) may be closely associated during cleavage on a cellular membrane structure. Interestingly, an earlier finding by Vecchio et al. [98] that the predominant amount of virus specific messenger RNA was in membrane bound polyribosomes rather than free polyribosomes led to the similar speculation that some process(es) in virus maturation might require membrane associated enzymes. Kinetic data from pulse-chase experiments indicated that the cleavage of Pr76 to give Pr66 plus Prl2 was the first cleavage event [97]. Thus the placement of Prl2 at the C-terminal end of Pr76 indicates that the entire molecule is synthesized before its cleavage begins. The absence of cleavage before completion of Pr76 synthesis is in contrast to the early cleavages of the large picornavirus polyprotein. However, the situation indicated for Pr76 seems to be similar to that described for the intermediate sized picornaviral precursor, NCVPI, containing the virus capsid proteins. A detailed kinetic analysis of the capsid protein precursor of one picornavirus (EMC) by Butterworth and Rueckert [46], established that its synthesis also was completed before cleavage began. It was suggested that the apparent requisite release of the capsid protein precursor from polyribosomes before cleavage can occur might indicate that the cleavage of this polypeptide is not random but is involved in some specific step(s) in virion morphogenesis. The same conclusion is perhaps applicable to the cleavages of Pr76 of Rous Sarcoma Virus. Very recently, Hunter et al. [99] have described a Rous sarcoma virus mutant temperature sensitive for precursor protein cleavage. When this mutant, LA3342, is grown at the restrictive temperature Pr76 is cleaved at a significantly slower rate. In addition, the precursor appears to be cleaved aberrantly yielding novel viral polypeptides and virion particles with decreased infectivity. As yet, no evidence of proteolytic cleavage of a larger polypeptide has been found in the synthesis of gp85, the major envelope glycoprotein of the avian virus [100,101]. By pulse-chase immunoprecipitation experiments this protein was first detected intracellularly as a 70 000 dalton polypeptide. It was suggested that the addition of sugar residues to the 70 000 dalton protein produced the virion gp85. Treatment of virus infected cells with high concentrations of D-glucosamine inhibited the glycosylation of the p70 molecule. Under this condition virion gp85 was not produced and virus particles were not released from the cells. The glucosamine treatment in this study also appeared to depress the rate of synthesis of the protein component of the gp85 and of the nonglycosylated viral proteins as well. These data suggest that some aspect of regulation of the synthesis of viral structural proteins may be related to protein glycosylation. The virion enzyme reverse transcriptase also demonstrates a proteolytic cleavage event. The purified enzymes of avian myeloblastosis virus and of Rous

385 sarcoma virus consist of two subunits, a and ft. The a subunit is about 60 000 daltons and the fl subunit about 100 000 daltons. Analysis of the tryptic [102,103] and chymotryptic [103] digest peptides of both subunits suggests that the smaller a subunit is a proteolytic cleavage product of the large fl subunit. In the purified enzyme, a and fl subunits are present in equimolar amounts. In vitro, however, aging increases the amount of a subunit [103,104]. It was postulated that the large subunit is the molecule synthesized intracellularly and packaged into virions and that proteolytic cleavage then gives rise to the smaller a subunit during or after virion maturation [102,103]. The function of this cleavage event is, at present, unknown. Moelling [104] has suggested that cleavage modifies the efficiency of the enzyme with its natural substrate. Alternatively this cleavage event may be nonessential or artifactual.

IHB. Mammalian oncornavirus protein synthesis and cleavage Recently, studies from several laboratories have extended the analysis of oncornavirus protein synthesis to mammalian type-C viruses. Naso et al. [105] have studied protein synthesis in Rauscher murine leukemia virus infected JLSV-16 cells using antiserum prepared against disrupted whole Rauscher virus and an antiserum specific to the virus major core protein p30*. In a 10 min pulse none of the proteins labeled by 35S methionine and precipitated by anti-Rauscher virus serum were the size of the virion polypeptides. Most were larger than 30 000 daltons with major peaks of protein of the sizes 180 000, 140 000, 110 000, 65 000, 60 000 and 50 000 daltons. Following a chase of 1 h the level of radioactivity in all these high molecular weight polypeptides dropped while the smaller virion proteins p30 and pl 5 appeared. The use of anti-p30 serum suggested that the larger pulse-labeled proteins of 140 000, 65 000 and 50 000 daltons were precursors to the virion core protein p30. Experiments using [14C] glucosamine indicated that the 110 000 dalton component was a glycoprotein and therefore possibly a precursor to the major envelope glycoprotein. The precursor-product relationship of the intracellular glycoprotein and the virion envelope protein has since been confirmed by tryptic digest analysis [106]. Also, tryptic digest analysis of the detected pl 5 protein indicated that it too was a cleavage product of the intracellular glycoprotein precursor. It should be noted that the protein analyzed by Naso and co-workers appears to be the pl 5e protein described by Ikeda et al. [107] and related to the envelope glycoprotein. Two Rauscher virus proteins of about 15 000 daltons have been described. The other pl 5, purified and characterized by Strand et al. [108] is slightly smaller, is immunochemically unrelated to the pl 5e and may be analogous to one of the small molecular weight internal proteins of the avian oncornaviruses. Studies of Rauscher virus precursor protein synthesis were also conducted by van Zaane et al. [109]. Using an antiserum reactive with disrupted whole virus, they detected possible precursors of 82 000 and 65 000 daltons after a pulse label. Pulse* Rauscher murine leukemia virus contains five major structural proteins: four nonglycosylated internal polypeptides, p30, p15, p12 and pl0, and an envelope glycoprotein, gp69/71 [93].

386 chase experiments demonstrated the appearance of virion proteins of 30 000, 15 000 and 12 000 daltons, concomitant with the disappearance of the two large polypeptides. Monospecific anti-p30 serum precipitated the 65 000 protein but not the 82 000 dalton protein. It was also found that the 82 000 protein was glycosylated. It is probable that the unstable 82 000 dalton protein is a precursor to gp69/71 while the 65 000 dalton protein is a precursor to the smaller proteins. This model would be consistent with the data of Naso et al. [105], Stephenson et al. [110] and the data of this laboratory [111]. Stephenson, Tronick and Aaronson [110] have studied the synthesis of virus proteins in cells infected with two Rauscher virus mutants temperature-sensitive for the release of virus particles. They examined the size, as measured by agarose gel filtration of proteins carrying the antigenicity of p30 or p12 which were present in extracts of cells grown at different temperatures. Proteins of the size of p30 and p12 predominated in extracts of cultures grown at a permissive temperature. However, cultures grown at a non-permissive temperature yielded chiefly a protein of 60 000 to 70 000 daltons. When cultures were shifted from the nonpermissive to the permissive temperature, in the presence of cycloheximide to prevent new protein synthesis, the concentration of larger protein decreased concomitant with an increase in immunologically specific protein the size of p30 and p12. P15 antigenicity also was associated with protein of 60 000 to 70 000 daltons synthesized in cultures grown at a nonpermissive temperature. These data suggest that these three nonglycosylated structural proteins are synthesized as part of a 60 000 to 70 000 dalton precursor polypeptide. Studies in this laboratory have employed monospecific antisera to three purified Rauscher virus proteins, the envelope glycoprotein gp69/71, the core protein p30, and protein pl5 [112]. Anti-p30 serum and anti-pl5 serum precipitated a pulse labeled protein of 65 000 daltons (Pr65) as the major virus-specific polypeptide [111]. In addition, polypeptides of 350 000, 260 000, 76 000 and 60 000 daltons were precipitated by both antisera. With chase, a decrease in the concentration of the large and intermediate sized labeled polypeptedes was observed and p30 protein appeared. The mature p15 protein does not contain methionine so this protein was not detected in these pulsechase studies which used [asS] methionine as label. Tryptic digest analysis showed that the 65 000 dalton precursors precipitated by anti-p30 and anti-pl 5 serum were indeed the same molecule and confirmed the precursor-product relationship of Pr65 with the virion core protein p30. Tryptic digest analysis to confirm the identities of the other possible precursor proteins has not yet been performed. Anti-gp69/71 serum precipitated a pulse labeled protein of 90 000 daltons (Pr90) as the major envelope glycoprotein specific polypeptide. In addition, this serum precipitated the same 350 000 dalton and 260 000 dalton polypeptides as did the antip30 and anti-p 15 sera. Label appeared in virion sized glycoprotein concomitant with the disappearance of the very large polypeptides and a decrease in concentration of labeled Pr90 following a chase period. Labeling experiments using tritiated sugars

387 showed that Pr90 is a glycoprotein and tryptic digest analysis of Pr90 confirmed its role as precursor to gp69/71. This finding of a glycoprotein precursor to the gp69/71 is similar to those o f N a s o et al. [105] and van Zaane et al. [109]. The discrepancies in the reported weights of these molecules may be attributed to differences in electrophoresis procedures used in the three laboratories. Other studies in this laboratory have investigated the glycosylation of the virus envelope glycoprotein. In the presence of 2-deoxy-D-glucose, an inhibitor of glycosylation, a carbohydrate deficient 35S-labeled polypeptide of about 70 000 daltons was precipitated by anti-gp69/71 serum and no Pr90 was detectable. With chase the concentration of the 70 000 dalton polypeptide in the cell extract decreased but smaller proteins which might be specific cleavage products were not observed. These data suggest that the carbohydrate side chains of the glycoprotein may play a role in specifying the precursor cleavage site and in protecting this virus protein from nonspecific proteolytic degradation. Other laboratories studying the glycoproteins of both the a togaviruses and of influenza virus have previously observed similar phenomena [22,30,33,85]. Okasinski and Velicer [113] have studied another mammalian oncornavirus, feline leukemia virus. Their data from pulse-chase experiments indicate that the major core protein of that virus, p30, is also synthesized from a 60 000 to 70 000 dalton precursor polypeptide. Recently, tryptic digest analysis has confirmed this proposed precursor-product relationship [114]. A cleavage event in the formation of the mammalian oncornavirus reverse transcriptase may also take place. Gerwin et al. [115] have detected an enzyme with reverse transcriptase activity weighing 95 000 daltons in cells infected with RD-114 virus. The antigenic and catalytic properties of this intracellular enzyme were indistinguishable from the 67 000 dalton virion enzyme. Although further studies with either pulse-chase experiments or tryptic digest analysis are necessary to confirm a precursor-product relationship for these proteins, the situation does seem very similar to the cleavage event observed with the avian oncornavirus reverse transcriptase. Very high molecular weight polypeptides carrying virus protein antigenic determinants have been detected by Naso et al. [105] and in this laboratory. Naso and co-workers reported observing polypeptides of 180000 and 140000 daltons in Rauscher virus infected N I H Swiss mouse embryo cells (JLSV-16) and we observed polypeptides of 350 000 and 260 000 daltons in Rauscher virus infected rat kidney cells (NRK). When Rauscher virus infected mouse cells (NI H/3T3)were examined in our laboratory, the 350 000 and 260 000 dalton polypeptides were not detected, however, a polypeptide of 145 000 daltons was present and precipitable by anti-gp69/71, anti-p30 and anti- 15 sera [116]. In an attempt to enhance the concentration of the very large possible precursors or reveal the synthesis of other large precursor proteins, several protease inhibitors useful in picornavirus and a togavirus studies were tested in the murine leukemia virus system. We observed that a combination of three amino acid analogs (FPA, canava-

388 nine and azetidine-2-carboxylic acid) prevented the cleavage of Pr65. Precursor made in the presence of these analogs migrated as a diffuse band of protein close to 76 000 daltons rather than a sharp peak of Pr65. The enhancement of 76 000 dalton protein at the expense of Pr65 suggests that Pr65 is a product of the larger polypeptide. Van Zaane et al [109] also observed a polypeptide of about 72 000 daltons synthesized instead of Pr65 when labeling was performed in the presence ofcanavanine. We observed that TPCK, also, was somewhat effective in blocking the cleavage of Pr65, and ZnC12 was effective in blocking cleavage of Pr90. However, neither these inhibitors nor other protease inhibitors or amino acid analogs enabled the detection of any previously undetected precursor polypeptides or enhanced the level of the very large possible precursor molecules already detected. Oncornavirus precursor proteins have also been detected in extracellular virus particles. A 60 000 dalton precursor to virion p30 was found by Oskarsson et al. [117] in virions released by a cat cell line coinfected with a replication defective murine sarcoma virus and a feline leukemia helper virus. Immunologic analysis and tryptic digest analysis showed the observed 60 000 dalton protein to be related to the murine virus p30 protein. These workers suggested that the presence of this uncleaved precursor protein in virions might indicate some type of defect in protease activity in cat cells. The presence of a 70 000 dalton possible precursor polypeptide in extracellular Rauscher virus was also reported by Jamjoon et al. [118]. Tryptic digest analysis suggested that this polypeptide was also a precursor to the p30 protein. In this laboratory, immunoprecipitation with both anti-p30 serum and anti-pl 5 serum revealed the presence of the precursor protein, Pr65, in newly harvested virions released by infected N R K cells. These three reports indicate that complete cleavage of Pr65 is not required for virus to be released from cells. However, it is not known whether the presence of precursor reflects part of the viral post-budding maturation process or has any effect on the infectivity of virus particles.

IIIC. Studies of translation of oncornaviral RNA Support for the precursor pathway of protein synthesis with oncornaviruses has also come from studies of in vitro translation of purified virus RNA. However, in an interesting parallel with the initial picornavirus in vitro translation work, the earliest such studies with oncornaviruses failed to indicate precursor synthesis. Siegert et al. [119] reported in 1972, that the product of in vitro translation of avian myeloblastosis virus RNA was a heterogeneous mixture of small proteins some of which resembled mature virion proteins in size. Gielkens et al. [120] using Rauscher virus RNA also observed only the production of small to medium sized polypeptides. The failure of these early studies to detect the synthesis of large precursor proteins may possibly be attributed to the use of cell-free protein synthesizing systems prepared from bacterial cells. As described above, in vitro synthesis studies with poliovirus RNA [63] utilizing a bacterial system had also failed to reveal any precursor polyproteins although ample evidence now exists that such polyproteins are made. More recently, Von der Helm and Duesberg [121], using a cell-free system prepared from ascites Krebs

389 (mouse) cells observed a 75 000 to 80 000 dalton polypeptide in studies of the & vitro translation of Rous sarcoma virus 3 ~ 4 0 S RNA. Relatedness of the large polypeptide to smaller molecular weight virion proteins was demonstrated by tryptic digest analysis. In a study of the translation of Rauscher murine leukemia virus 35S RNA, Naso et al. [122] employing a cell-free system prepared from JLSV-5 (mouse) cells observed the synthesis of two very large polypeptides weighing 180 000 daltons and 125 000 daltons; very little protein of the size of mature virion polypeptides was observed. Most recently, Kerr et al. [123] studying the translation of Moloney murine leukemia virus 35 S RNA observed the synthesis of 60 000, 70 000 and 180000 dalton polypeptides which were shown to be virus specific by tryptic digest analysis.

IV. VIRUS PROTEIN PRECURSOR CLEAVAGE ENZYMES The proteolytic enzymes that process virus precursor proteins may be either normal host cell constituents or induced by virus infection (i.e. virus induced or modified host cell enzymes or viral encoded enzymes). Examples of both constitutive and induced types of protease activity have been found associated with virus protein cleavage events. The cleavages of the envelope proteins of the myxo and paramyxoviruses, a 2 phage head protein, and the large polyprotein of the picornaviruses appear to be catalyzed by host cell enzymes. In the case of the myxo and paramyxoviruses, influenza and Sendai, the extent of cleavage of the envelope glycoproteins was shown to vary depending on the host cell lines of the viruses [25,29,35]. For 2 phage there is genetic evidence that during the process of head formation a host cell enzyme is required for conversion of the phage head protein, C, to a smaller molecular weight protein, h3. GroE mutants of the host cell, E. coli, are lacking in a function required for this proteolytic conversion of the phage C protein [11]. Several types of data suggest that some cleavages of the picornavirus polyprotein also are performed by host cell enzymes. Korant observed that in HeLa cells poliovirus polyprotein cleavage is inhibited by TosLys CH2C1, an inhibitor specific for trypsin-like proteases but not by TosPheCH2CI, an inhibitor of chymotrypsin-like proteases [124]. However, chymotrypsin-like protease was necessary for cleavage in poliovirus infected L L C M K 2 (monkey) cells which suggested that the precursor was cleaved by a host cell enzyme. Korant also observed that extracted poliovirus polyprotein could be cleaved in vitro to form virus polypeptides of the intermediate size, NCVP1 and NCVP2, by extracts from uninfected HeLa cells. Lastly, the sizes of the intermediate cleavage products NCVP1 and NCVPX of polio grown in HeLa and L L C M K 2 cells differed by about 1000 daltons; this was attributed to different cleavage enzymes recognizing different cleavage sites on the precursor molecule in the different cell lines [124]. Cleavage enzymes have also been shown to be virus coded or induced proteins. A possible case is the late cleavages of picornavirus intermediate sized precursors to

390 form the smaller virus proteins. Kiehn and Holland [55] observed that in cells infected for only two hours most pulse-labeled virus-specific proteins weighed about 90 000 to 100 000 daltons whereas a pulse label at 4 h following infection demonstrated smaller molecular weight virus polypeptides. Next, Korant found that the intermediate sized precursor, N C V P l a , containing the poliovirus structural proteins was cleaved by extracts of infected cells but not by extracts of uninfected cells [45,125] and also Esteban and Kerr, showed that a 130 000 dalton EMC precursor protein (NCVP1) was cleaved to form a 105 000 dalton protein during incubation with an EMC infected cell extract but not with an uninfected cell extract [67]. Weber [126] reported that a temperature sensitive adenovirus mutant (tsl)when grown at nonpermissive temperature failed to produce six viral proteins which are all cleavage products of higher molecular weight precursor proteins. Complementation, reversion and recombination data indicated that the tsl defect was at a single gene locus. Weber suggested that the tsl defect was in either a virally encoded protease or a virus inducer or co-factor for a cellular protease. Finally, at least one cleavage event in the life cycle of a bacteriophage, T4, appears to be performed by a viral enzyme. Pulse labeled precursor protein was broken down by incubation with cell extracts of infected cells but not by extracts of uninfected cells [14,16,17]. Bachrach and Benchetrit [16] showed that this cleavage activity was absent from cells infected with virus mutants in gene 21, suggesting that this virus gene may encode the cleavage enzyme. For oncornaviruses, the available evidence suggests that the 76 000 dalton precursor of the Rous sarcoma virus structural proteins is processed by a host cell enzyme(s) [97]. When Rous virus is grown in hamster cells the cells are transformed but no virus particles are produced. Examination of the virus specific proteins of these cells reveals only Pr76; no cleavage products appear with chase. However, when these infected hamster cells are fused with uninfected chick embryo fibroblasts the precursor is cleaved to form virion proteins. This suggests that a specific protease(s) capable of cleaving the avian virus precursor protein is present or inducible in avian cells but not in hamster cells. Although at present there is no evidence for an oncornavirus encoded or induced protease, the existence of such an enzyme would be of interest because it might provide the mechanism by which the virus could affect the regulation of cell division. Proteolytic enzymes have been observed to stimulate cell growth [127] and some protease inhibitors can cause a phenotypic reversion of transformed cells to a nontransformed state [128]. Also, a strong correlation has been observed between increased cell surface protease activity and some of the morphological and growth characteristics of transformed cells [129]. This increased level of protease activity occurs however, whether the cell was transformed by an R N A tumor virus, a D N A tumor virus or a chemical carcinogen [130]. Therefore, if this protease is an oncornavirus encoded enzyme then other oncogenic stimuli seem to be able to induce the protease of an endogenous oncornavirus. Alternatively if the oncornavirus induces a host cell enzyme then the other ongenic stimuli also function in a similar manner. Selective pressures in evolution would predict that the viral encoded cell trans-

391 formation factor(s) may play a functional role in the viral life cycle. A protease model for oncornavirus transformation while currently speculative is attractive because it would be consistent with such selective pressures.

V. MODELS OF ONCORNAVIRUS PROTEIN SYNTHESIS AND VIRUS GENETIC STRUCTURE While the analysis of oncornavirus precursor protein synthesis is not yet complete, enough is known to speculate about possible models of virus protein synthesis. The synthesis of the major nonglycosylated structural proteins on one large precursor of 70 000 to 80 000 daltons is a consistent event of type-C oncornaviruses. The virus envelope glycoprotein appears to be synthesized separately from the major nonglycosylated proteins. The synthesis of a 70000 dalton polypeptide to which sugar residues are added to form a glycoprotein of 85 000 to 90 000 daltons has been observed with both the avian Rous sarcoma virus and the mammalian Rauscher leukemia virus. The mammalian virus protein is further processed by cleavage and additional glycosylation while the avian virus protein is found in its uncleared state in released virus particles. These cleavages giving rise to the major non-glycosylated virion proteins and to the Rauscher virus envelope protein resemble examples of protein processing during virion morphogenesis. Also, in production of the virus reverse transcriptase the synthesis of a catalytically active higher molecular weight precursor protein appears to occur for both avian and mammalian oncornaviruses. The cleavage of this precursor may be of the zymogen activation variety or it may be a nonessential event. It is unclear whether cleavage of a large primary polyprotein containing the total viral genome equivalent of protein, as found in picornavirus replication, is a step in the production of oncornavirus proteins. As an alternative the intermediate sized precursors and other virus proteins may be synthesized separately. Some evidence for large polyprotein synthesis does exist. Naso et al. [105, 122] observed large polypeptides weighing 180 000 and 140 000 daltons in studies of murine leukemia virus protein synthesis. In our laboratory, small amounts of possible precursor polypeptides of 350 000, 260 000 and 145 000 daltons have been detected. The 350 000 dalton protein is large enough to be the primary translation product of the entire 35 S RNA genome of the virus. Oncornavirus protein synthesis could occur by way of such a large primary polyprotein. If this is the case, very rapid cleavage of this polypetide to form intermediate sized precursors could explain why the very large proteins are only observed in such small quantities. Nonequimolar amounts of virus protein could be the result either of selective degradation or of variable sites of termination of protein synthesis along the 35 S virus messenger RNA. The variation in size of the very large" viral protein precursors observed in different cell lines could be due to varying levels of activity of host cell proteases involved in their cleavage processing. Such a model of oncornavirus protein synthesis, however, cannot explain the inability of protease in-

392 hibitors or amino acid analogs, to enhance the concentration of the large oncornavirus polypeptides. Also, this model does not explain the presence of different size classes of oncornavirus mRNA whose existence indicates that oncornavirus protein need not be synthesized as one long polyprotein precursor molecule. As an alternative, oncornavirus protein could be synthesized not as a very high molecular weight polyprotein but as intermediate sized precursors. These precursors would be the primary translation products of different oncornavirus mRNA molecules. While the 35 S virus mRNA could encode a 350 000 dalton polypeptide its complete translation could be prevented by an early termination site. The synthesis of only a distinct 75 000 to 80 000 dalton polypeptide from purified Rous virus 35 S RNA in the in vitro translation study of Von der Helm and Duesberg [121] supports this hypothesis. The observed very large polypeptides, in this model could possibly be host cell proteins precipitated as background in immunoprecipitation or they might represent occasional readthrough past termination sites on the virus messenger. If the amount of these very large polypeptides observed depended on an imperfect response to a termination signal rather than on any proteolytic cleavage processes then this could explain the failure of amino acid analogs or protease inhibitors to enhance the concentrations of these proteins. In addition to a general model of virus protein synthesis the present knowledge of virus precursor protein synthesis has contributed to understanding the genetic structure of oncornaviruses. Data obtained from the precursor protein studies of Vogt et al [96] in conjunction with data from RNAase T~ resistant oligonucleotide studies [131-133] allow the construction of a preliminary map of most of the known genes of an avian sarcoma virus. The oligonucleotide mapping data, indicates an order of 3' terminal-poly(A)-transformation factor (sarc)-envelope glycoprotein (env)-5' terminal. The genes for the transformation factor(s) and for the envelope protein map in the regions 6.5 20')(, and 28-50')(, of the wild type RNA from the poly (A) terminus respectively [131,132]. The unmapped regions between the poly (A) and the transformation gene and between the transformation gene and the envelope protein gene appear to be insufficient in size to encode either the virus internal protein precursor Pr76 or the reverse transcriptase enzyme. Therefore these genes probably map on the 5' terminus side of the envelope protein gene. Data from study of the structure of oncornavirus mRNA and its in vitro translation makes it possible to speculate on the order of the reverse transcriptase (pol) and Pr76 genes. It was suggested by Furuichi et al. [134] that ribosome binding was associated with the methylated oligonucleotide sequences observed at the 5' termini of many mRNAs (called "caps"). Such a "cap" was detected at the 5' terminal of an avian sarcoma virus 35 S RNA [135]. The study of Naso et al. [122] indicated that there was only one ribosome binding site per 35 S oncornavirus RNA molecule. The major product of in vitro translation of purified viral 35 S RNA should be protein synthesized from this ribosome binding site which should be at the 5' terminal. Von der Helm and Duesberg reported this product to be the internal protein precursor Pr76 [121]. Finally, the pactamycin mapping data from the precursor studies of Vogt et al [96], gives the gene order of the internal virus

393

,| Pr76 pol 5 ,,I p19,p27,p151

env I

sorc I

poly A l

~OH 5'

Fig. 2. Gene order of avian sarcoma virus.

proteins included in Pr76 as 5' t e r m i n a l - p l 9 , p27, p15 - 3' terminal. A l l o f this inf o r m a t i o n suggests a gene o r d e r for the a v i a n s a r c o m a virus as shown in Fig. 2. This sequence w o u l d a c c o u n t for all o f the k n o w n p r o t e i n s o f a v i a n s a r c o m a viruses with the exception o f the small internal proteins p l 0 a n d p12. P12 is included s o m e w h e r e in the Pr76 region. It is n o t yet clear whether this gene o r d e r is the same for the m a m m a l i a n oncornaviruses. The n o n g l y c o s y l a t e d internal virus proteins a p p e a r to be linked in one p r e c u r s o r as is the case for the a v i a n viruses, however, the o r d e r o f these p r o t e i n s within the p r e c u r s o r is n o t yet known. I f the 145 000 d a l t o n possible p r e c u r s o r observed in this l a b o r a t o r y actually c o n t a i n s the virion internal proteins a n d the envelope p r o t e i n p r e c u r s o r o f R a u s c h e r virus then the genes for these proteins m u s t be linked w i t h o u t the reverse t r a n s c r i p t a s e gene between t h e m as i n d i c a t e d for the a v i a n virus genome. Also, the o r g a n i z a t i o n o f the t r a n s f o r m a t i o n f a c t o r gene(s) within the g e n o m e o f m a m m a l i a n s a r c o m a viruses m a y differ significantly from the avian virus gene order. Indeed, since the m a m m a l i a n s a r c o m a viruses are believed to each have resulted f r o m a r e c o m b i n a t i o n a l event between a l e u k e m i a virus a n d the h o s t cell genome, the t r a n s f o r m a t i o n gene(s) m a y be in a different p o s i t i o n in different m a m m a l i a n s a r c o m a virus isolates.

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