Post-translational cleavage of polypeptide chains: role in assembly.

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POST-TRANSLATIONAL CLEAVAGE

x901

OF POLYPEPTIDE CHAINS: Annu. Rev. Biochem. 1975.44:775-795. Downloaded from www.annualreviews.org Access provided by University of Adelaide on 02/01/15. For personal use only.

ROLE IN ASSEMBLY Avram Hershko and Michael Fry Department of Clinical Biochemistry, Technion - Israel Institute of Technology, The Aba Khoushy School of Medicine, Haifa, Israel

CONTENTS

775 776 777 778 780 781 782

INTRODUCTION. PROCESSING OF PICORNAVIR US PROTEINS. Formation of Primary Translation Product(s)

.

.

Secondary Cleavages.

P roteolytic Cleavage in Picornavirus Assembly. Enzymic Mechanisms. POLYPEPTIDE CLEAVAGE IN VARIOUS ANIMAL VIRUSES. ROLE

OF

PROTEIN

CLEAVAGE

IN

THE

MORPHOGENESIS

OF

COMPLEX

BACTERIOPHAGES. Cleavage of Head Proteins During the Assembly of Bacteriophage T4..... .. .. .. .....

Maturation of the head. Post-translational cleavage events. .

Mechanisms of the cleavage reaction. Role of the host cell. Cleavage of Head and Tail Proteins in Some Other Large DNA-Containin" Bacteriophages. Bacteriophage T5 . Bacteriophage P2. Bacteriophage

). . . .

CONCLUDING REMARKS.

.

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

784 784 784 786 788 789 790 790 791 792 793

INTRODUCTION "You don't know how to manage looking-glass cakes," the Unicorn remarked, "Hand it round first, and cut it afterwards." Lewis Carrol, Through the Looking Glass

An increasing body of recent evidence indicates that postsynthetic processing of polypeptide and polynucleotide chains has widespread occurrence and considerable biological significance. Post-transcriptional cleavage mechanisms play

an

important

role in the formation of many RNA species in a variety of biological systems (1, 2). 775

Annu. Rev. Biochem. 1975.44:775-795. Downloaded from www.annualreviews.org Access provided by University of Adelaide on 02/01/15. For personal use only.

776

HERSHKO & FRY

Some well-known examples of specific scission of polypeptides are the activation of digestive enzyme precursors (3) and the cascade mechanisms involved in blood coagulation (4) and complement action (5). More recently, post-translational cleavage mechanisms have been shown to be involved in the form ation of proteins as different as insulin (6), collagen (7), and possibly albumin (8) and immunoglobulin light chains (9, 10). In addition, proteolytic cleaving enzymes of high specificity play a role in the inactivation (II, 12) and activation (13) of specific intracellular enzymes. Thus, post-translational cleavage mechanisms participate in a wide variety of biological processes, ranging from the formation of polypeptides from large bio synthetic precursors, to the modification of activity of both intracellular and extracellular enzyme systems. In this review, we discuss still another role of polypeptide cleavage, which has been discovered recently in several viral systems. The numerous cleavage phenomena of viru s-specific proteins can be classified, in general, into two distinct categories, which we shall call "formative" and "morphogenetic" cleavages. I n the first class, discovered in small RNA-containing animal viruses, the formation of all functional proteins proceeds through the cleavage of high molecular weight biosynthetic precursors. This process is probably a mechanism substituting for i nternal initiation in the synthesis of proteins on a polycistronic message in animal cells (14). The second class of viral protein cleavage, which is widespread in many animal viruses as well as in certain complex bacteriophages, is the specific scission of some structural proteins in the course of viral assembly. These morphogenetic cleavages seem therefore to play an integral part in the assembly of relatively complex viral structures in which simple self-assembly mechanisms may not be sufficient. We shall try to illustrate current progress in this field by describing in some detail two selected, relatively well-studied viral systems (i.e. pico rn aviruses and T4 bacteriophage). Post-translational cleavages occurring in other viruses will be discussed more briefly, with emphasis on biological principles which may be better illuminated in t hese systems. Part of the earlier literature concerning picornaviruses has been reviewed ( 15-20). Various other aspects of virus assembly are discussed in another review in this volume (21). PROCESSING OF PICORNAVIRUS PROTEINS

Since the discovery of the participation of post-translational cleavages in the formation of picornavirus proteins (14, 22, 23), this subject has been reviewed several times (15-20). We summarize here some of the more recent advances and outline the various stages in the processing of picornavirus proteins according to the current status of our k nowledge. Although most studies were performed with poliovirus, remarkably similar features of polypeptide processing were observed in other picornaviruses, such as encephalomyocarditis (EMC) virus (24-28) or rhinovirus (27, 28). Three different classes of cleavages seem to occur during the processing of picornavirus proteins, of which the first two may be regarded as different types of


777

formative cleavages. The first is involved i n the formation of the primary products arising directly from the translational process, and most probably represents cleavage of nascent polypeptide chains; this has been termed "nascent" cleavage (18). Another class, which we call "secondary" cleavages, is a series of transformation s that convert the primary precursors t o the final functional proteins. The third type is evidently a "morphogenetic" cleavage, since it is intimately associated with the final steps of picornavirus assembly. We shall discuss these various types of polypeptide cleavages separately.


Formation of Primary Translation Product(s) The translation of poliovirus-specific proteins takes! place on large polysomes, in which the whole viral RNA appears to function a � mRNA (29, 30). Baltimore and his associates have suggested that the total genonie of poliovirus is translated as a single unit to yield a large precursor polypeptide ("polyprotein"), from which all viral proteins are produced by subsequent cleavages (14, 16, 19). The size of poliovirus RNA is about 2.6 x 106 daltons (31, 32) and would thus code for a protein of �270,OOO daltons. Giant polypeptides of almost this size have been observed in poliovirus-infected cells in the presence of amino acid analogs (14, 33, 34), protease inhi bitors (35, 36), and in some temperature-sensitive mutants at the restrictive temperature (37, 38). Under normal conditions, however, such a gian t precursor cannot b e seen, and the earliest detectable products are polypeptides NCVPla, NCVPlb, and NCVPX, with molecular weights of 1 10,000, 93 000, and 34,000, respectively ! (see Figure I). It could be imagin ed that a polyprotein is formed but is cleaved thereafter at an extremely rapid rate. However, available evidence indicates that such a large precursor is not released from the polysome as such. Jacobson et al (33) have measured the size distribution of polysome bound nascent polypeptides of poliovirus-infected cells, and could not detect poly peptides of higher molecular weight than 130,000. Similar findings were obtained i n EMC virus (26). Furthermore, kinetic experiments have shown that the precursor of capsid proteins NCVP l a can be labeled and released even in a short pulse of 1-2 min (26, 42). Since it takes 10-12 min for the ribosome to traverse the entire length of the viral RNA molecule (25, 42), and since capsid proteins map near the 5' terminus (see below), the capsid protein precursor must be released independently soon after its synthesis. ,

1

The nomenclature of poliovirus proteins is somewhat discouraging. Summers et al (39),

in their initial work describing poliovirus-specific pro tein s, designated the four capsid

non capsid virus-specific p rotei ns were named in sodium· dodecylsulfate acrylamide gel the resolution te chnique s more poliovirus-specific

v iri on protei n s VPI-VP4, a nd the 10

NCVPI-NCVPIO, in order of their migration

electrophoresis. With the r efini ng

of

,

proteins were found, and these were marked by additional letters. For example, band NCVPl has been resolved to the two proteins NCVPla and NCVPlb (17), NCVP3 has been split to NCVP3a and NCVP3b (40), etc. To make matters worse, different groups of investigators are using different terminologies for some proteins. Thus, NCVPlb is called NCVPI! by Baltimore and his associates (33), NCVP6 is also VPO (41), and so on.


778

HERSlIKO

& FRY

Although ,the hypothetical polyprotein is not released normally in its entirety, several lines of experimental evidence indicate that initiation of translation occurs only at onc site on picornavirus RNA. Analysis of the products of cell-free protein synthesis directed by EMC virus o r poliovirus RNA indicates the existence of a single initiation site (43-45). As in some other eucaryotic systems (9, 10), translation starts with a short "lead-in" peptide which is subsequently cleaved off (43, 46, 47). The single initiation site hypothesis also requires that an equimolar production of the three primary proteins (together with their respective cleavage products) will be found in vivo. Such equimolar proportions were found in EMC virus (25, 28). Other in vivo experiments utilizing pactamycin, a specific inhibitor of initiation, are also consistent with a single site initiation mechanism (see below). The sum of these experiments is consistent with the notion that picornavirus translation is initiated at a single site, and that the three primary products are formed by the cleavage of nascent polypeptides.

Secondary Cleavages Figure 1 summarizes the presently known cleavage steps in the formation of poliovirus proteins, including the secondary cleavages. The methodology used for elucidating this scheme of proteolytic conversions requires some comment. m-RNA

"NASCENT CLEAVAGES'

{51--�/�1 NCVP1a (110)

NCVPX (34 )

1\

NCVP3a (68 )

SECONDARY CLEVAGES'

_ ", _3' ""

VPl

("34)

NCVP1b {93J

NCVP2 {81}

/\ �:.rJ1

VPO

VP3

7' \

I2Sl

VP4

VP2

!if

"""i3OJ

Post-translational cleavages in the formation of poliovirus proteins. Molecular (x 10- 3) are indicated in the brackets and are the average of the values reported in (28, 33, and 186). The locations NCVPlb and NCVP3a can be regarded as tentative; they are based mainly on pactamycin mapping (28, 40). Polypeptides NCVP3b and NCVP4 (not shown) map near the 3' end (28) and therefore are probably derived from NCVP2. The positions and relationship s of the other virus-specific proteins, not included in this scheme, are unknown. Further details are described in the text.

Figure 1

weights


POST-lRANSLATJONAL CLEAVAGE

779

The gene order of the primary, intermediate, and stable viral proteins has been determined mainly by the use of pactamycin, a specific inhibitor of protein synthesis initiation (25, 28, 40, 42, 48). When proteins are labeled several minutes after the addition of pactamycin, previously initiated nascent chains are completed, and the label is selectively incorporated into the part farthest from die NH2 terminus. Assuming a single initiation site in the translation of picornavirus proteins (see above) and that protein synthesis proceeds from the 5' to 3' end of mRNA, the labeling of polypeptides encoded near the 5' end of the genome will be most extensively inhibited. When initiation was selectively affected by treatment with hypertonic medium (49), essentially similar results were obta:ned. The validity of the assumptions involved in these methods (mainly the unique initiation site) is confirmed by the agreement of the results with genetic mapping data, where all capsid proteins are placed at one end of the genome (50). In determining the relationships between the various precursors and their cleavage products, several methods have been used. Kinetic pulse-chase experiments in conjunction with molecular weight determinations may give a strong clue for possible precursor-product relationships in many instances. A mathematical model has been developed for such multistep cleavage systems (26). The method of pactamycin mapping mentioned above is also valuable in defining precursor-product relation ships when used together with the other data. In some instances, the use of specific viral mutants may also be helpful. For example, in the defective interfering particles isolated by Cole et al (51 ), part of the genome coding for capsid proteins is deleted and the normal precursor of capsid proteins, NCVPla, is missing as well (52). The final and most conclusive proof of precursor-product relationships is the demonstration of similarity in amino acid sequences, as studied by the comparison of tryptic peptides or cyanogen bromide fragments of postulated precursors and products. The precursor-product relationships of poliovirus-specified proteins, as elucidated by the above methods, is depicted in Figure 1. Polypeptide NCVPla is clearly the precursor of the fou r capsid proteins VPI-VP4, as determined by kinetic studies ( 14, 17, 22), pactamycin technique (28), genetic methods (52), and tryptic peptide analysis ( 1 7, 33). The role of VPO as the immediate precursor of proteins VP2 and VP4 in the last steps of the assembly (see below) is also well established ( 1 7, 33). The gene order of the capsid proteins is 5' -> 3' VP4-VP2-VP3-VPl (28, 42). Much less is known about the formation, or even the identity, of nonstructural functional picornavirus-specific polypeptides. Such proteins, probably derived from NCVP2 and NCVPX, are those involved in viral RNA replication. Genetic (53) as well as recent biochemical (54) evidence indicates the existence of two distinct RNA replication complexes, one concerned with the formation of template ("minus") RNA strands and another that s ynthesizes progeny viral RNA. However, the polypeptide composition of the replication complexes is very heterogeneous, although they are enriched in non structural virus-specific proteins (50, 55). Some purification of EMC virus polymerase has been achieved (56), but its virus specific components have not yet been identified. The suppression of host macro molecular synthesis is another essential viral function (57). However, this function may not require a separate gene product. Cooper and his associates have recently

780

HERSHKO & FRY


proposed (58-60) that the inhibition of host protein synthesis is carried out by a precursor of viral structural unit. It should be pointed out that the presently known proteolytic conversions, as described in Figure 1, account for less than one half the multitude of virus specific polypeptides that can be detected in picornavirus-infected cells. Most of these are probably intermediates or waste fragments of the various cleavage processes. In addition, it is not known whether or not some proteolytic conversions may include the disposal of whole fragments by total proteolysis to acid-soluble products. For example, the cumulative mass of the three primary products is lower than the total coding capacity of picornavirus genome (2S). Proteolytic Cleavage in Picornavirus Assembly

The cleavage of VPO to the capsid proteins VP2 and VP4 is associated with the final stages of poliovirus assembly (33,41, 61). The icosahedral shell of picornaviruses is constructed of 60 identical structural units, or protomers; each proto mer is assumed to be composed of the four nonidentical capsid proteins (62-64). According to the postulates of Rueckert, Baltimore, Phillips, and their associates (16, 62, 6S), the morphogenesis of picoma viruses starts with a stepwise assembly of the "immature protomer" (64), which is a SS particle containing one molecule each of VPO, VP1, and VP3, into increasingly larger structures. Thus, the assembly of five 5S particles is assumed to form a 14S structure, and twelve 14S particles associate to produce 73S empty capsids ("procapsids") (16, 62). Viral RNA then combines with the empty shell to make a 1 25S "provirion" structure (61 ), and morphogenesis is culminated by the clcavage of VPO with the production of mature virions (41, 6 1 ). Evidence for this postulated sequence of events in picornavirus morphogenesis is based mainly on kinetic and pulse-chase experiments. During the course of poliovirus infection, 5S material can be detected about � hr before the appearance of empty capsids and virions (66). Upon chase with unlabeled amino acids, radioactivity in SS particles decreases rapidly (66, 67), whereas it continues to increase in 14S particles (67) and in the higher aggregates (66). In these types of experiments radioactivity in 14S particles begins to decline only after about 30 min of chase (68), while empty capsids are quite resistant to chase (66); this may be due to the presence of large pools of labeled precursor particles. The postulate that 73S empty capsids are the precursors of virions is based mainly on experiments with guanidine, an inhibitor of viral RNA formation. In the presence of guanidine, empty capsids containing uncleaved vpo accumulated (41 ). When guanidine was removed, radioactivity was lost from empty capsids, with a parallel increase in virions and a concomitant cleavage of VPO to VP2 and VP4 (41 ). The role of the recently discovered 1 25S particle (the provirion) (6 1 , 69) in virus morphogenesis is also based on pulse-chase experiments in the presence of guanidine and following its removal (61 ). Experimental evidence for some features of this proposed assembly mechanism is not conclusive. This model requires that the various proteins will be present in the intermediary structures in equimolar amounts, but the reported relative amounts of VPO, VP1, and VP3 in 5S, 14S, and 73S structures of poliovirus are not



78 1

equimolar (70). Most importantly, precursor-product relationships between the various particles cannot be unambiguously defined by pulse-chase experiments in this system, due to the high number and apparently large pool sizes of the intermediary structures. In addition, it has to be taken into account that some particles may be the product of abortive assembly, rather than intermediary steps in virus morphogenesis. Thus, Ghendon et al (67) have shown that in poliovirus-infected MiO cells (as opposed to HeLa cells), 73S empty capsids are not formed, and even in the presence of guanidine, 14S particles rather than empty capsids accumulate. This casts some doubt on the obligatory role of empty capsids in poliovirus morphogenesis. Obviously, more definite conclusions can be obtained only by in vitro experiments. To date, attempts to reproduce in vitro poliovirus assembly have met only limited success. Phillips et al (68, 71) have discovered that 14S particles can be assembled in vitro into 73S structures in the presence of extracts from virus-infected, but not from uninfected cells. Subsequently, it was demonstrated that this activity is localized in the rough membranes (65), and that 1 4S can be self-assembled to 73S material even in the absence of cellular extracts, provided that the concentration of the 14S particles is high enough (72). It was suggested that virus-modified membranes may facilitate this assembly reaction by their ability to adsorb and concentrate 14S particles (72). It should be noted, however, that extracts from MiO cells can also carry out the 14S 73S in vitro conversion, whereas the 73S particle is not formed in vivo in these cells (67). Although the complete details of picornavirus assembly have not yet been elucidated, there seems to be no doubt about the close association of the cleavage of VPO to VP2 and VP4 with viral morphogenesis. This cleavage reaction occurs only in high ly assembled structures and, furthermore, there seems to be a definite requirement for the association of RNA with the assembled structure to allow this cleavage. Therefore, this type of morphogenetic cleavage reaction seems to fulfill a role biologically different from the formative cleavages described earlier. -+

Enzymic Mechanisms

Very little is known about the nature of proteolytic enzyme(s) participating in the cleavages of picornavirus proteins. Several protease inhibitors have been utilized to block these cleavages in vivo. Diisopropylfluorophosphate (DFP) at rather high concentrations partially inhibits the processing of viral proteins in cells infected with poliovirus (33) and EMC virus (73). When poliovirus proteins were labeled in the presence of DFP, radioactivity had accumulated in proteins larger than NCVP l a (33). Similarly, in poliovirus-infected cells treated with the inhibitor of chymotrypsin tolylsulfonyl-phenylalanyl-chloromethyl ketone (TPCK), the accumulation of high molccular weight polypeptides (> 200,(00) has been found (36). Korant (35) has reported that the action of the inhibit(}r varied with the cell line used: in monkey kidney cells, cleavage of poliovirus proteins was inhibited by TPCK, whereas in HeLa cells, only the trypsin inhibitor tolylsulfonyl-lysyl chloromethyl ketone (TLCK) was effective. The latter finding is at variance with the results of Summers et al in HeLa cells (36); the reason for this discrepancy is


782

HERSHKO & FRY

not clear. A different inhibitor, iodoacetamide, prevents the formation of capsid proteins and causes the accumulation of NCVPla, NCVP2, and NCVPX (74). It has been concluded that iodoacetamide blocks a later cleavage step than do serine protease inhibitors (74). In addition, zinc ions also inhibit the processing of picornavirus proteins by an as yet unknown mechanism (75). Although these experiments provide further evidence for the existence of cleavage mechanisms in the formation of poliovirus proteins, the action of these proteasc inhibitors on in vivo systems can hardly be regarded as specific. The chloromethyl ketones, TPCK and TLCK, may alkylate a variety of cellular proteins (36), and the actions of DFP and iodoacetamide are also rather nonspecific. Thus, the effect of serine protease inhibitors on intracellular protein breakdown (76) has been shown to be due to a nonspecific inhibition of cellular energy metabolism (77). It is well known that the breakdown of intracellular proteins is energy dependent (78-80); it remains to be seen whether similar energy-requiring proteolytic mechanisms participate also in the proccssing of virus-specific proteins. Available information concerning the characterization of these cleavage reactions by in vitro systems is very scanty. Holland et al have reported that purified virions contain some protease activities (81), b ut since this activity degrades mature capsid proteins, its relationship to the cleavage of precursor proteins is not clear. Korant (35) has shown that extracts from poliovirus-infected cells degraded high molecular weight precursor proteins to smaller polypeptides with molecular sizes similar to NCVP2, VPO, VPl, and VP3. In contrast, the cleaving activity contained in extracts of uninfected cells produced only proteins resembling NCVPla and NCVP2. Cleavage of NCVPla, accumulated by prior treatment with iodoacetamide, is carried out by infected but not uninfected extracts (74). It was suggested that the initial nascent cleavages are catalyzed by a host enzyme, while the subsequent splicing of NCVPla is carried o ut by a second protease which is either virus specified or activated by infection (74). Opposing these findings, high molecular weight precursors from EMC virus-infected Krebs carcinoma cells were degraded in vitro by extracts from both infected and uninfected cells, b ut the products of degradation had no resemblance to the proteins formed in vivo (73). In addition, while the processing of precursor proteins in vivo was inhibited by DFP, the in vitro degradation was not affected by this drug. The authors concluded that the cleavages observed in the in vitro system were probably due to nonspecific proteases liberated during homogenization of cells (73). It thus seems that much remains to be elucidated about the nature and mode of action of the enzyme systems participating in post-translational cleavages of viral proteins. POLYPEPTIDE CLEAVAGE IN VARIOUS ANIMAL VIRUSES

Apart from picornaviruses, cleavage of proteins has been described in other widely different types of animal viruses. In the arboviruses, Sindbis virus and Semliki forest virus, both formative and morphogenetic polypeptide cleavages apparently occur. Viruses of this group consist of an RNA-containing n ucleocapsid surrounded


POST-TRANSLATIONAL eLEA VAGE

783

by . a lipoprotein envelope (82). There are only three proteins in the virion, one nucleocapsid and two envelope proteins (83-85). On the other hand, there are as many as 9-13 additional virus-specific proteins in Sindbis virus-infected cells, some of them considerably larger than virion proteins (83). In pulse-chase experi ments, the label is shifted from high molecular weight proteins to the smaller structural polypeptides (86-89). As in the case of poliovirus, extremely large Sindbis virus-specific proteins accumulate in the presence of protease inhibitors (90, 91) or in temperature-sensitive mutants at the nonpermissive temperature (92-94). It is believed, therefore, that Sindbis virus proteins originate from a largc common precursor (91, 93). In fact, tryptic peptide similarity between the large protein accumulated in a temperature-sensitive mutant of Sindbis virus and all three virion proteins has been demonstrated (93). In addition, a large molecular weight peptide which contains tryptic peptides of the capsid protein is synthesized in a cell-free system with Sindbis virus mRNA (95). There is some evidence for a single initiation site in the translation of Sindbis virus proteins, since pactamycin treatment inhibits the labeling of capsid proteins much more than that of envelope proteins (87). Another type of cleavage appears to be the conversion of a precursor of intermediary size to one of the envelope proteins in both Sindbis virus (87) and Semliki forest virus (85). This conversion occurs at the plasma membranes and may therefore be associated with virus morphogenesis (87, 96). Although evidence is still incomplete, these findings indicate that the formation of group A arbovirus proteins is quite analogous to the processing of picornavirus polypeptides. In the large DNA-containing poxviruses, protein cleavages are closely linked to morphogenetic events. At least two major proteins of the vaccinia virus core are derived from larger precursors by post-translational processing (97-100). These cleavages are rather slow and occur at a time coincident with the maturation of virus particles (98). In the morphogenesis of this complex virus, assembly begins with the formation of envelope units (101). Rifampicin, which inhibits vaccinia virus morphogenesis at the stage of envelope formation (102, 103), also prevents cleavagc of these precursor proteins (97). Upon removal of the drug, the formation of normal envelope units precedes restoration of the cleavage of core protein precursors (97, 103). Thus, the influence of the drug on polypeptide cleavage is probably secondary to the block in envelope asscmbly (103). In a rifampicin resistant strain of vaccinia virus, the drug had no influence on either envelope formation or protein cleavage (104). The action of rifampicin is rather specific to this stage of morphogenesis, since the processing of a third protein, which is located outside the virus core, is not influenced by the drug (100). In the morphogenesis of another group of relatively large DNA viruses, the adenoviruses, proteolytic cleavage mechanisms play a somewhat similar role. Coincidentally with the conversion of empty capsids to mature adenovirus-2 virions, five different polypeptides are converted to smaller virion proteins (105-107). The precursors are only slightly larger than the products, and there is no evidence for the existence of high molecular weight precursors (107). It appears that the processing mechanisms of adenovirus, as those of vaccinia virus and T4 bacterio phage, are restricted to morphogenetic events. Post-translational proteolytic con-

784

HERSHKO

&

FRY

versions have been reported in viral systems as different as influenza virus (108-110), paramyxovirus (11 1), avian RNA tumor virus ( 1 12), and possibly polyoma virus ( 1 13). However, these conversions do not play a significant role in viral development in all cases. For example, in influenza virus, two glycoproteins located on the surface of the viral envelope are derived by the cleavage of a large precursor (108-110, 114). It has been shown more recently, however, that this cleavage is due to the action of serum plasmin (115) and is not required for virus assembly or for any other biological properties of the virion (116).


ROLE OF PROTEIN CLEAVAGE IN THE MORPHOGENESIS OF COMPLEX BACTERIOPHAGES

Cleavage of proteins during the assembly of large DNA-containing bacteriophages is a widespread phenomenon, although it may not be a general mechanism common to the assembly of all phages of this type. Since cleavage of coliphage T4 head components was first described in 1970 (117-120), proteolytic cleavages were found to occur during the morphogenesis of the head of bacteriophz.ge P2 (121) and in the maturation of the head and tail of coliphage A (122-125) and T5 (126). On the other hand, no proteolytic cleavage phenomena could be detected in the morphopoiesis of other DNA containing bacterial viruses, such as Salmonella phage P22 (127, 128) and coliphage T3 ( 1 29). Cleavage of Head Proteins During the Assembly of Bacteriophage

T4

The assembly of the head of T4 has been recently reviewed (130) and is described in full elsewhere in this volume (21). We limit our discussion to the outline of the assembly process as related to the proteolytic cleavage events associated with it. The head of the phage appears to be based on an icosahedral design with a triangulation number of 21 (131, 132). The mature T4 DNA, which is a linear uninterrupted duplex molecule with a cyclically permuted, terminally repetitious n ucleotide sequence (133, 134), occupies most of the internal volume of the head (135, 136). About 11 different protein species compose the capsid of this phage (119). Three additional internal proteins designated IP I, IP II, and IP III* (137) and three acid-soluble internal peptides (138) are located inside the head and are found in association with the DNA. The assembly of T4 head is controlled by some 18 genes (139, 140). Genes 20, 21, 22, 23, 24, and 40 are required for determination of the size and shape of the head (139, 141). Genes 2, 4, 13, 14, 16, 17, 49, 50, 64, and 65 control the subsequent steps of the head maturation (140). Gene 3 1 is thought to control the solubility of the major head protein (119, 142). The events leading to the packaging of the DNA molecule within its protein shell are still largely unknown. Three possible models for the assembly of T4 head have been offered. One model proposes that an empty capsid is formed first and is then converted into mature phage head with the gradual penetration and packaging of the DNA (143, 144). An alternative model suggests the formation of a DNA condensate around which the protein shell is later assembled (145). A third model MATURATION OF THE HEAD

785

POST-TRANSLATJONAL CLEAVAGE

hypothesizes that capsid formation and DNA packaging are concomitant and that a small piece of DNA serves as a core for head formation (130). Available experi mental evidence is most consistent with a pathway of head maturation that involves formation of a precursor protein shell largely devoid of DNA, into which nucleic acid is packaged at a later stage. Identification of intermediates in the pathway of T4 head maturation was attempted through experiments of three types : (a) ultrastructural studies of head-related structures formed at various stages IPf____


IPJl:---.,.

�:::::::...

D

P20-_-.P20" [20,23,31,40J

T�EMBRANE

0

P23_P23' [21, 247J

=r

P 23 P 22 IP ]I P 20 r ?] (a)

Figure 2

O

�

" I�� [16 , 17J

"

.--�

[.9]

•

P 23· P 22 ][ REDUCED IP IP ][' P 2q'7]

P 23 P 22 IP ]I P 2c{'?]

(b)

1�-=:iP�'

}

(d)

(e)

(e)

Protein cleavages associated with a tentative pathway of the assembly of the

head of bacteriophage T4. The numbers in brackets underneath the arrows designate genes that are probably involved in particular steps of the assembly of the head. The cleavage events are indicated above the main arrows. The prefix P designates a gene product. Asterisk marks a cleaved form of the gene product. The various "prohead" designations are according to Laemmli & Favre (143).

(a) Assembly core

Biochemical (146) and ultrastructural ( 144,

157)

studies suggest the

in vivo existence of such early structures. It was suggested (146) that the internal ' proteins, shortly followed by P22, become localized in a complex with a bacterial membrane and serve as a nucleation site around which the capsid proteins are later assembled. Since the internal proteins seem to be dispensable (146, 187), the assembly core probably serves as an initiation complex rather than a shape-determining structure ( 1 87).

(b) Prohead I

(400S particles)

These structures were identified and partially isolated

by Laemmli & Favre (143) and observed electron microscopically by Simon (144). It is a short lived particle composed mainly of uncleaved P23, P20, P22, IP III, and perhaps other minor proteins (143). It is possible that P20 is modified prior to the prohead I stage (164). Prohead I is membrane bound and contains less than 1 % of the phage DNA complement (143). This structure is analogous to gene 24 defective

T

(147-149, 188). (c)

Prohead II (350S particles)

particle

The major event leading to the conversion of prohead I

into prohead II is the cleavage of P23 to form P23* (143). It is possible that P24 joins the capsid at this stage (143, 147). This pa rti cle does not seem to contain DNA (143). (d) Prohead III These intermediates, containing 10-30% (152) to 40-50% (143) of the phage DNA complement and sedimenting at

320-550S,

are bound to the replicative

DNA (143, 152). During the conversion of prohead II into prohead III, about

50%

of

P22 and IP III are cleaved into an unknown product and IP III*, respectively (143). A structure somewhat analogous to prohead III is the gene 49 defective head-related particle, which is also partially filled with DNA (143, 1 52). It should be emphasized, however, that genes 16, 17, and 49 defective particles contain the capsid polypeptides in their cleaved form (143).

(e) Mature Head

The mature T4 head, sedimenting at 1 1 00S, contains a full DNA

complement and all the capsid proteins in their final cleaved forms.

786

HERSHKO & FRY

of t he phage development (144); (b) biochemical (119, 146--156) and ultrastructural 1 57, 158) studies of head-related p article s accumulated in ce lls infected

(14 1, 147,

by phages defective in genes controlling various steps of head assembly; and

(c)

pulse-chase experiments in cells infected with either wild-type or mutant

phages establish the percursor-product relationships between successive inter

(143, 147, 148). However, all these argued that empty capsids could aris e from previously filled intermediates which, by virtue of th eir fragility, tend to lose their DNA (121, 145, 147, i57, 1 59; see also 146 for a possible association of the "assembly" core with T4 DNA). In addition, a recent paper by Chao et al (160) . indicated the involvement of host DNA rep ai r enzymes in the assembly of T4 heads. Such connection between DNA metabolism and the morphogenesis of the head suggests that DNA does play a morphopoietic role in the assembly of the p hage T4 he ad. A postulated pathway of T4 head maturation based on the model of the formation of empty heads and their sequential filling with DNA is shown diagrammatically in Figure 2. This scheme. is based. mainly on the tentative path way ofT4 head maturation presented by Laemmli & Favre (143). More details on t he various steps of t he a ss embly process are given elsewhere in this volume (21). The role of the host membrane in T4 morphogenesis was reviewed (161). Additional data are supplied in several recent papers (143, 146, 147). The functions a ss ume d mediates in the pathway of head formation


types

of

experiments are

not conclusive; it

was

�

for the various phage genes that control head development were discussed by several authors

(14 1 , 143, 146, 147, 149, 150, 152).

Fo u r head-associated ph age -specified proteins are cleaved during t he morphogenesis of coliphage T4. Three of the head proteins cle�ved are structural components of the capsid coded for by gcnes 22, 23, and 24 and synthesized la te in infection (1 19). The fourth is internal protein IP III, which is an "early" product of an unidentified T4 gene (1 1 9, 1 37, 162). Cleavage of head-related precursor proteins was first described in 1 970 by four separate groups of investigators whose experimental approaches were similar (1 17120). Essentially, protein s of the purified capsid and total lysates of wild-type and head defective T4-infected cells were compar ed by m ean s of sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis. Comparative studies of this type allowed identification of the products of genes 18, 1 9, 20, 22, 23, and 24, which POST-TRANSLATIONAL CLEAVAGE EVENTS

Were all present in wild-type lysates and missing from the respective defective gene

(119). In the process of assigning polypeptides to their genes of origin, have noted significant di ffere nc es between the molecular weights of certain gene products present in wild-type and head defective lysates and capsids ( 1 17-120). Specifically, it was reported that during wild-type infection, P23 is converted from a protein of 55,000-56,000 daltons to the major head protein P23* with a molecular weight of 46,000-48, 000 (1 1 7-120). Similarly, Laemmli (1 1 9) and Hosoda & Cone (117) have shown that P22 of 31,000 mol wt is cleaved during wild-type infection to form an unidentified product. Laemmli (1 19) has also demonstrated that P24 (molecular weight 45,000) is converted during n(lTm8J T4 maturation into a minor head component designated P24* with a lysates

several authors



787

molecular weight of 43,000. Likewise, IP III (23 ,500 daltons) is cleaved to form the internal protein IP 111* (21,000 daltons) (119, 137). All the precursor protein s described are accumulated in their uncleaved form during infection with T4 mutants defective in any of the head genes 20, 2 1, 22, 23, 24, and 3 1 (1 1 7-120). In addition to the above well-defined proteolytic cleavage events, several additional cleavages of T4 polypeptides were proposed: Coppo et al (163) and Laemmli & Favre (143) have reported that a T4 protein designated B l (163) appears to be a cleavage product of an unknown precursor. Recently, Bolin & Cummings (164) have shown that the arginine analog L-canavanine inhibits the cleavage reaction s of P22, P23, P24, and IP III. This drug also prevents the appearance of P20 (head protein) and PIO, P 1 2, and P18 (tail proteins), indicating that these poly peptides may also arise by proteolytic splicing during normal T4 assembly. However, c1ea �age of P20 and any tail protein was not demonstrated in infected cells unexpo sed to amino acid analogs (1 19, 143, 165-167). Also, the identities of t he precursors to these proteins are unknown. The kinetics of the proteolytic reactions was established by pulse-chase experi ments. This technique, in conjunction with the use of specific mutants, allowed the establi shment of precursor-product relationships between P23 and P23*, P24 and P24*, and IP III and IP III*. In the case of P22, although it was found to disappear rapidly during the chase, no band that could be derived from this protein has been traced in the gel pattern (1 1 7, 11 9). The cleavage reactions for P22 and P23 were found to be very rapid; about 50% of the precursor proteins disappeared within 2-3 min following chase of the label. In contrast, the cleavage o flP III is much slower (11 9). Cleavages bring about the loss of small fragments from P23, IP III, and P24 of about 10,000, 2,500, and 1,500 daltons, respectively (1 1 7-120). Attempts (0 find t hese fragments have failed, possibly because peptides of such small size were not sieved on the acrylamide gels at the concentrations used (119). It is also possible t hat the fragments are further broken down to undetectable sizes. A possibility was raised that t he internal peptides II and VII having molecular weights of 3900 and 2500, respectively, could be the products of the extensive cleavage and degradation of P22 (146, 168). This contention is supported by the finding that P22 is an internal component associated with the assembly core (146) and also by a study inferring the sharing of common antigenic determinants by P22 and the internal peptides (169). Several lines of evidence suggest that proteolytic cleavages are intimately linked to the assembly process. As mentioned above, the cleavage of P22, P23, and IP III is blocked in mutants defective in any of t he head genes 20, 2 1, 22, 23, 24, and 3 1. As a result, mature heads are not formed and aberrant head-related structures accumulate instead (120, 141, 147-149). Also, IP III, which is an early protein synthesized 4-5 min after infection, starts to be cleaved only at late times (about 14-17 min), indicating the coupling of its proteolytic cleavage to the assembly process (119, 170). Recently, LaemmIi & Favre (143) were able to differentiate between the cleavages of P23 and those of other t hree hcad proteins and to correlate these events with defined stages of the head assembly process.


788

HERSHKO & FRY

. These investigators reported that prohead I, which contains the uncleaved P22, P23, and IP III, is converted into prohead II with the concomitant cleavage of P23 to P23*, whereas the cleavage of other proteins occurs at later stages of the morphogenesis (see Figure 2). Bijlenga et al (155) have also shown that under permissive conditions, P23 of the gene 24 defective head-related T particle i s transformed in situ into P23* of the capsid. This transformation is presumably analogous to the cleavage of P23 during the conversion of prohead I to prohead II, and thus genes 21 and 24 defective r particles are analogs of prohead I (147149). However, whereas the gene 24 defective particles are maturable, gene 21 defective structures are abortive nonmaturable particles (147). Since both proheads I and II are thought to be devoid of DNA (143, 147-149), it was concluded that the cleavage of P23 precedes the DNA packaging step. The cleavage of P22 and IP III, on the other h and, occurs concomitantly with the packaging of phage DNA: Prohead III, into which only part of the DNA complement is packed, contains P22 at a reduced amount and both IP III and IP 111* (Figure 2) (143). Cleavage of P22 and IP III is completed with the conversion of prohead III into mature T4 head, which contains a full complement of DNA, P23*, P24*, and IP III* and no uncleaved precursor polypeptides (119, 143). These results strongly suggest a close link between the cleavage of P22, IP 1lI, and possibly P24 and the process of packaging the DNA molecul e within its protein shell. In addition, the above set of experiments confirms that all the cleavage reactions occur i n large multimeric structures as originally proposed by Laemmli (119). Very little is k nown about the m echanism of the proteolytic cleavage reactions associated with T4 head assembly. The origins, number, and i dentity of the cleavage enzymes, their mechanisms of action, and specific requirements for activity are completely unknown. Some insight into the mode of the proteolytic digestion that converts P23 into P23* was gained in the work of Celis et al (171), which demonstrated that fragments synthesized by several amber mutants in gene 23 contained tryptic peptides present in the uncleaved P23 but not in the modified P23*. It was concluded th erefore that it is the N-terminal portion of P23 that is cleaved during T4 maturation. Some further aspects of the cleavage reactions were recently described in cell-free proteolytic syst ems (170, 172, 173). It was reported (172) that T4 precursor proteins could be cleaved in vitro with the formation of a peptide that was indistinguishable from internal peptide II-a presumed in vivo cleavage product with a molecular weight of 3900 (172, 1 74). The i n vitro formation of this short peptide was dependent on the presence of a factor present in extracts of T4i nfected cells but absent from extracts of uninfected cells. It was also shown that both native and acid-denatured precursor s could serve as substrates for cleavage in vitro. This result is somewhat surprising, since it would be expected that it is the native conformation o f precursor proteins that determines their recognition by the cleaving system. Bachrach & Benchetrit (170) have recently reported that lysates of T4-infected cells possess proteolytic activity which converts an early protein-presumably IP III into a polypeptide with an electrophoretic mobility MECHANISMS OF THE CLEAVAGE REACTION

-



789

similar to that of IP 111*. This putative cleavage enzyme appears in T4-infected Iysates at 1 4--16 min postinfection, in accordance with earlier observations on the cleavage of I P III at a late stage of the infection (1 1 9). Proteins labeled late in infection were not cleaved under the same conditions, indicating that the activity described is not involved in the cleavage of P22, P23, and P24. These authors have also reported that whereas the in vitro cleaving activity is present in mutants defective in genes 20, 22, 23, and 24, it is missing in amber mutants of gene 21. It was proposed, therefore, that the product of gene 21 is involved in the cleavage of IP III. It should be noted, however, that this differs from the in vivo findings, in which mutation in any of these genes prevents the cleavage of IP III (1 1 7-120). In a more recent communication (173), the above authors have demonstrated that only the cleaved form of the internal proteins, spliced either in vivo or in vitro, binds to T4 DNA and not with heterologous DNA. Poglazov & Levshenko (1 75) have found that a T4-induced trypsin-like proteolytic activity reaches a maximum 18 min postinfection. The relevance of this activity to phage assembly is not clear as yet. Amino acid analogs which have been widely used for the characterization of cleavage reactions occurring in animal viruses (1 4, 33, 3 4) were used to a limited extent in the study of T4 protein cleavage. Couse et al (176) have shown that the arginine analog L-canavanine prevents the intracellular development of T4 and induces the formation of aberrant tubular heads and polyheads. This analog did not markedly inhibit total protein and RNA synthesis but DNA synthesis and cleavage of P23 were blocked (177, 1 78). It is not clear as yet whether the primary effect of the analog is to block synthesis of DNA or to inhibit the scission of capsid proteins. Interestingly, exposure of cells infected by T4 to L canavanine, followed by addition of arginine, causes the production of normal phages together with enormous infectious particles named "lollipops," which contain P23*, P24*, and IP 111* and no precursor proteins (179). A recent study on the effects of L-canavanine on the maturation and utilization of specific T4 gene products (164) revealed that this analog prevents the cleavage reactions of P22, P23, P24, and IP III but, surprisingly, it also inhibits the appearance of P20 (head protein) and PlO, P12, and P18 (tail proteins). As a consequence of these effects on specific phage proteins, tail assembly as well as head assembly are inhibited. It was suggested that in addition to the already known cleavage events, P20, PlO, P 1 2, and P18 are also processed. However, it remains to be seen whether the proposed protein modification events can be demonstrated in T4-infected cells not exposed to L-canavanine. Proteolytic cleavage of phage-specified proteins could be either a phage function, a host function, or both. As mentioned, the in vitro cleavage of some T4 precursor proteins requires the presence of infected cell extracts (170, 172). However, the isolation of mutant strains of Escherichia coli, in which cleavage and assembly are specifically blocked, may allude to a possible host participation in the cleavage of phage-specified proteins. Such host mutants, isolated in several laboratories, were designated tab B (163), mop (180), and

ROLE OF THE HOST CELL


790

HERSHKO

&

FRY

gro E (181) and seem to be closcly related (163, 181). While phage adsorption, DNA injection, and tail production are unaffected by the bacterial mutation (180, 181), the cleavage of the phage precursor proteins an'd capsid formation are blocked ( 1 63 , 1 80, 1 8 1 ). The development of other phages, such as all T-even phages (163, 180), 1>80 (180, 181), and 434 (181), is also often inhibited in these mutant strains. The uncleaved precursor polypeptides P23 (163, 18 1), P22, P24, and IP III (163) accumulate in membrane-associated lump-like aggregates similar to those found in wild-type bacteria infected with gene 3 1 defective phage ( 1 63, 180, 1 8 1). Indeed, phage mutations ComB (163), "Roo (180), and T4e (181), mapped in the region of gene 3 1, enable the phage to overcome the block exerted by the host mutation and allow the precursor proteins to be cleaved and head assembly to proceed normalIy (163, 180). It was suggested (163, 180, 18 1) that the bacterial mutation impairs the fun ction of T4 gene 3 1 and that a modified P3 1 produced by the mutant phage overcomes the bacterial lesion. Thus, solubilization of precursor proteins of the head, their modification, and ordered assembly require interaction between a wild-type bacterial gene product and P31. It is not known, however, whether the bacterial mutation primarily affects the cleavage reaction or some other critical event of the head morphogenesis. There are two possible mechanisms by which phage and host products may interact : 1. P31 together with the bacterial product, or under its influence, could be part of the proteolytic system responsible for the cleavage of T4 precursor proteins (180) ; or 2. P 3 1 together with the host factor are involved in some early step of T4 morphogenesis, such as the interaction between the head components and the bacterial membrane on which they are assembled ( 1 63, 1 80). There is some evidence that the membranes of the mop mutant are indeed biochemically and fun ctionally modified (180) . Thus, the possibility of a lesion in the bacterial membrane as a primary cause for inhibition of the assembly of T4 head was favored in that case.

Cleavage of Head and Tail Proteins in Some Other Large DNA-Containing Bacteriophages

Several authors have recently described proteolytic cleavage events occurring during the assembly of bacteriophages T5 (126), A (122-125), and P2 ( 121). Although these reactions are not as well characterized as those associated with the morphogenesis of coliphage T4, their study adds new insight into the role and scope of protein cleavage in the assembly of bacteriophages. In two cases, T5 and A. precursor proteins of the tail undergo cleavage during maturation of the phage (124, 126). Since assembly of the tail does not involve any interaction between DNA and proteins, these cases indicate that post-translational cleavage may also play a role in the protein-protein interactions responsible for ordered assembly of the tail. Coliphage T5 contains at least 15 different structural poly peptides, among them a major head component with a molecular weight of 32,000 and a major tail component that has a molecular weight of 55,000 (182). Zweig & Cummings ( 126) have recently shown that at least three of the phage specified BACTERIOPHAGE TS

-



791

proteins undergo proteolytic cleavage during the assembly of T5. Two of the polypeptides cleaved are related to the morphogenesis of the head, whereas the third is a precursor to a minor component of the tail. Specifically, the following cleavage events have been reported : A tail-related precursor protein with a molecular weight of 1 35,000 is cleaved to form a minor tail component having a molecular weight of 1 28,000. A second precursor protein with a molecular weight of 50,000 undergoes cleavage to form the major protein component of the head, which has a molecular weight of 32,000. A third T5-specified minor component of the head (molecular weight 43,0(0) also seems to be a cleavage product of a slightly larger precursor protein. The close linkage between the scission of the tail-specific precursor protein band a and the morphogenesis of the tail is indicated by the finding that all studied tail-defective phages fail to induce cleavage of the band a protein under restrictive conditions. A block in the synthesis of the major head protein, on the other hand, does not interfere with the proteolytic modilication of the tail-related precursor protein or with the assembly of the tail. Interestingly, cleavage of the tail-related polypeptide is blocked in E. coli mutants groEA639 and groEA36 infected with wild-type T5. This block is specific to tail proteins since cleavage of the head-related proteins proceeds normally auc normal heads are produced in these mutant hosts. A T5 mutant designated T5e6 is able to overcome the bacterial lesion and to propagate normally in the groEA hosts. The above finding raises the possibility of a requirement for interaction between host and phage products for cleavage and assembly of the tail-related proteins. Cleavage of the head-related proteins and assembly of the head are specifically inhibited by the arginine analog L-canavanine. Although the drug interferes to some extent with the assembly of the tail, a significant amount of competent tails is produced in its presence. It seems, therefore, that the head- and tail-specific cleavage events are independent and under different controls. BACTERIOPHAGE P2 In a recent paper, Lengyel et al ( 1 2 1 ) have described the cleavage of two head-related precursor proteins during the morphogenesis of coliphage P2. The product of gene N, having a molecular weight of 44,000, is cleaved to form mainly the major capsid protein with a molecular weight of 36,000. In addition, four other minor head components with molecular weights ranging between 37,400 and 42,200 seem also to be cleavage products of the same precursor protein. The product of gene 0 with a molecular weight of 30,000 also undergoes cleavage with approximately the same kinetics as that of the N protein. The product of the cleavage of the 0 protein is unidentified but it is certainly smaller than 17,000 daltons. The cleavage reactions of the N and 0 proteins are coupled : All amber mutants in the 0 gene produce measurable amounts of the N gene product but this precursor protein does not undergo cleavage. Conversely, an amber mutation in the N gene blocks cleavage of the 0 protein. In contrast to the cleavage of the T4 precursor proteins, which is closely linked to DNA synthesis, both DNA and protein synthesis are not required for the cleavage of the two P2-specified precursor head proteins.

792

HERSHKO & FRY

Several instances of post-translational scission of head and tail proteins of bacteriophage A have been described. Two proteins of molecular weights 3 5,000 and 1 2,000, specified by A genes E and D respectively, make up about 94% of the protein mass of the A capsid (183). Of the several remaining minor protein components of the head, a polypeptide designated h3 of molecular weight 56,000 accounts for 2�3% of the shell protein mass (1 22). The kinetics of the formation of h3 suggests that it is a conversion derivative rather than a primary product of any }, gene ( 1 22). Murialdo & Siminovitch ( 123) and Georgopoulos et al (122) have proposed that the precursor to h3 is either the product of head gene B (pB) or the product of head gene C (pC). More recently, Hendrix & Casjens (184) have shown by tryptic peptide analysis that pB is the precursor of h3. It was also demonstrated that in addition to p8 and pC, the product of gene E (PE) is needed for this conversion to occur (1 23). Georgopoulos et al (122) have found that a host function is also required for either the production or the functioning of h3. During normal }, head morphogenesis, h3 is prominent in fast sedimenting head-related structures. In contrast, this protein is missing from the fast sedimenting particle in extracts of graE mutant strains that specifically block the assembly of A heads. It was proposed therefore, that the graE mutation either inhibits the cleavage reaction forming h3 or blocks the association of h3 with the fast sedimenting


BACTERIOPHAGE }"

structure. Since some graE mutants called graEB can be compensated by mutations

head genes E or B, it was suggested that the proteins specified by A genes E, B, and possibly C interact with a bacterial component defined by the groE mutation. Thus, the bacterial gro function could be directly or indirectly involved in the formation of h3. A novel cleavage and possibly a fusion reaction involved with A head assembly has been described recently by Hendrix & Casjens ( 125). Bacteriophage }, heads contain two additional minor proteins, Xl and X2, having molecular weights of 3 1 ,000 and 29,000, respectively. On the basis of the tryptic fingerprints of 35S labeled X l and X2, it was concluded that X2 is a proteolytic cleavage product of Xl' In addition, both X l and X2 contain tryptic peptides also present in either pE or pc. Only part of the different sequences of pE and pC appears in X l and X2 and these sequences are found in the products in equimoJar amounts. This evidence indicates, therefore, that Xl and hence X2 may be the products of a fusion reaction between pE and pc. The covalent bond joining pE and pC does not seem to be a disulfide bridge. The recent study of Hendrix & Casjens ( 1 84) indicates that the processing of pC, as well as the cleavage of the head protein pB, occur on nascent multimeric structures rather than prior to the assembly of the protein subunits. Hendrix & Casjens ( 1 24) have reported recently that a }. tail protein is also cleaved prior to the attachment of tails to heads. The tryptic digest of a minor tail protein designated t2 with a molecular weight of 78,000 was found to share many peptides in common with a digest of another protein pH, which is found in ),-infected lysates but not in phage particles and has a molecular weight of 90,000. It was proposed, therefore, that t2 is derived from pH by proteolytic cleavage. It was also found that tails that accumulate in head-defective ), mutants contain polypeptide t2 rather than pH. The authors concluded, therefore, that the cleavage of pH to t2 occurs independently of the attachment of tails to heads ( 1 24). in the A

POST-TRANSLATIONAL eLEAVAGE

793


CONCLUDING REMARKS

In considering the possible functions of post-translational cleavage of polypeptides in various viral systems, a clear distinction should be made between formative and morphogenetic proteolytic scission. Formative cleavage of proteins is an integral part of the translation mechanism of certain animal viruses. The most plausible assumption concerning its role seems to be that originally proposed by Jacobson & Baltimore (14), namely that the protein synthesizing machinery of animal cells, in contrast to that of bacteria, is incapable of carrying out internal initiation in the translation of polycistronic viral messages. Since post-translational scissions are characterized by a high degree of specificity and precision, they can effectively replace translational punctuation signals. There may be some biological advantages in this mechanism, such as the equimolar production of polypeptides required in identical amounts (for example, picornavirus capsid proteins). On the other hand, it is evident that this mechanism does not enable the regulation of synthesis of individual polypeptides, in contrast to other viral systems in which coat proteins are synthesized in much larger amounts than polypeptides involved in RNA replication. Since the yield of viral RNA production in picornaviruses appears to be well balanced with the rate of formation of capsid proteins, additional and as yet unidentified regulatory mechanisms have to exist which influence either the activity or the stability of picornaviral replication complexes (20, 59, 1 85). In contrast to formative cleavages, the much more widespread morphogenetic proteolytic scissions are characterized by their occurrence in multimeric structures that are intermediary stages in viral assembly. Furthermore, these proteolytic cleavages occur at rather specific steps of viral morphogenesis, and they are invariably blocked when the normal course of morphogenesis is prevented by several specific agents and viral or host cell mutations. However, the exact roles of the proteolytic scissions in viral assembly are still largely unknown. It appears that protein cleavagcs associated with viral morphogenesis are not limited to a single type of molecular interaction and that the cleaved polypeptides may interact with either nucleic acids or with other proteins. Thus, in the maturation of the head of bacteriophage T4, cleavage of the core proteins P22 and IP III seems to be intimately linked to the packaging of DNA, and it has been suggested that in this case protein cleavage could either provide DNA binding sites or bring about condensation of the DNA through the production of acidic internal peptides ( 143). In contrast to the cleavage of the core proteins, the scission of P23 is completed before packaging of the DNA begins (see Figure 2). That protein cleavage is not limited to DNA-protein interactions is also illustrated by the finding that such reactions occur in the assembly of the tails of bacteriophages T5 and l In the latter case, it has been proposed that protein cleavage may produce a higher energy configuration of tail proteins, providing the energy required for DNA injection ( 1 24). On the other hand, in poliovirus morphogenesis, the cleavage of VPO, which converts the provirion to mature virion, may have a role in the stabilization of the RNA-containing particle, since the former but not the latter structure can be disrupted by denaturing agents (61).


794

HERSHKO

&

FRY

A common feature of these various types of molecular interactions might be that all proteolytic modifications convert the assembly reactions in which they participate to irreversible processes ( 130). This characteristic and the dependence of protein cleavage on correct particle conformation may provide a mechanism for the sequential assembly of the components of relatively complex viral structures. Possibly, structural proteins remain in their uncleaved form until a specific intermediary structure is assembled and only then does cleavage occur, making that specific assembly step irreversible and enabling the association of the particle with additional components. Obviously, further advance in elucidating the pathways of morphogenesis of complex viruses is needed in order to gain a better understanding of the role of proteolytic cleavages in viral assembly. ACK NOWLEDGME N TS

The authors wish to thank Drs. B. E. Butterworth, S. R. Casjens, P. D. Cooper, R. W. Hendrix, E. Kellenberger, G. Koch, and B. A. Phillips for making their results available to us prior to publication, and Drs. A. Cohen, H. Engelberg, B. D. Korant, and B. Moss for helpful comments. Special thanks are due to Mrs. Rachel Neiger for devoted secretarial assistance. Literature Cited 1. Burdon, R. H. 1971. Progr. Nuc/. Acid Res. Mol. Bioi. 1 1 : 3 3-79 2. Bautz, E. K. F. 1972. Progr. Nuc/. Acid Res. Mol. Bioi. 1 2 : 129-60 3. Neurath, H., Walsh, K. A., Winter, W. P.

4. 5. 6.

1967. Science 158 : 1638-44

Davie, E. W., Kirby, E. P.

Top. Cell. Regul. 7 : 5 1-86

Miiller-Eberhard, H. J.

1973. Curro

1971. Harvey

Lect. 66 : 75-104

Steiner, D. F. et al 1969. Recent Progr. Horm. Res. 25 : 207-82 7. Bellamy, G., Bornstein, P. 197 1 . Proc. Nat. Acad. Sci. USA 68 : 1 138-42 8. Judah,J. D., Gamble, M., Steadman, J. H. 1973. Biochem. J. 1 34 : 1083-91 9. Milstein, C, Brownlee, G. G., Harrison, T. M., Mathews, M. B. 1972. Nature New BioI. 239 : 1 1 7-20 10. Mach, B., Faust, C, Vassali, P. 1973. Proc. Nat. Acad. Sci. USA 70 : 451-55 1 1. Katunuma, N. 1973. Curro Top. Cell. Regul. 7 : 175-203 1 2. Holzer, H. et al 1 973. Advan. Enzyme Regul. 1 1 : 53-60 13. Cabib, E., Ulane, R. 1973. Biochem. Biophys. Res. Commun. 50 : 186-91 14. Jacobson, M. F., Baltimore, D. 1968. Proc. Nat. Acad. Sci. USA 6 1 : 77-84 15. Baltimore, D. 1969. The Biochemistry of Viruses, ed. H. B. Levy, 101-76. New

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York : Dekker Baltimore, D. 1971.

From Molecules to

17.

Man, Perspectives in Biology, ed. M. Pollard, 1-14. New York : Academic Summers, D. F., Roumiantzeff, M., Maizel, J. V. 1971. Strategy Viral Genome,

Ciba Found. Symp., 1 971, 1 1 1-24 Baltimore, D. 1971. Bacteriol. Rev. 35 : 235-41 19. Baltimore, D. 1971. Trans. NY A cad. Sci. 33 : 327-32 20. Sugiyama, T., Korant, B. D., Lonberg Holm, K. K. 1972. Ann. Rev. Microbiol. 26 : 467-502 2 1 . Casj ens S., King, J. 1975. Ann. Rev. Biochem. 44 : 555-6 1 1 22. Summers, D . F., Maizel, J . V . 1968. Proc. Nat. Acad. Sci. USA 59 : 966-71 23. Holland, 1. 1., K iehn, E. D. 1 968. Proc. Nat. Acad. Sci. USA 60 : 101 5-22 24. Butterworth, B. E., Hall, L., Stoltzfus, e M., Rueckert, R. R. 1971. Proc. Nat. Acad. Sci. USA 68 : 3083-87 18.

,

25. Butterworth, B. E., Rueckert, R. R. 1972.

26. 27. 28. 29. 30.

J. Virol. 9 : 823-28 Butterworth, B. E., Rueckert, R. R. 1 972.

Virology 5 0 : 535-49 McLean, C, Rueckert,

J. Virol. 1 1 : 341-44 Butterworth, B. E.

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Penman, S., Becker, Y., Darnell, J. E. 1964. J. Mol. Bioi. 8 : 541-55 Summers, D. F., Levintow, L. 1965.

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Protein cleavage during virus assembly: a novel specificity of assembly dependent cleavage in bacteriophage T4.

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Moments and distribution functions for polypeptide chains. Poly-L-alanine.

O-GlcNAc occurs cotranslationally to stabilize nascent polypeptide chains.

Thermodynamics of polypeptide supramolecular assembly in the short chain limit.

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Self-assembly of magnetic balls: from chains to tubes.

Directed self-assembly of gold nanoparticles into plasmonic chains.

Light-assisted, templated self-assembly of gold nanoparticle chains.

DDIT4 through cleavage by caspase 3 modifies its cellular function.

Role of positions e and g in the fibrous assembly formation of an amphipathic α-helix-forming polypeptide.

Acetylation of nascent polypeptide chains on rat liver polyribosomes in vivo and in vitro.

Assembly of dimeric myeloperoxidase during posttranslational maturation in human leukemic HL-60 cells.

Differences in alpha and beta polypeptide chains of tubulin resolved by electron microscopy with image reconstruction.

Extracellular labeling of growing secreted polypeptide chains in Bacillus subtilis with diazoiodosulfanilic acid.

Identification of beta,beta-turns and unordered conformations in polypeptide chains by vacuum ultraviolet circular dichroism.

Cooperative interactions in hybrids of aspartate transcarbamylase containing succinylated regulatory polypeptide chains.

The two polypeptide chains in fibronectin are joined in antiparallel fashion: NMR structural characterization.

Defective cleavage of a precursor polypeptide in a temperature-sensitive mutant of avian sarcoma virus.

Antibody response to the streptococcal group A-variant polysaccharide in BASILEA rabbits lacking kappa-polypeptide chains.

Cell-mediated immunity to insulin and its polypeptide chains in insulin-treated diabectics.