Polyprotein processing in picornavirus replication

Biochimie 70 (1988) 119-130 ©Soci6t6 de Chimie biologique/Elsevier, Paris

119

Polyprotein processing in picornavirus replication Hans-Georg KILAUSSLICH, Martin J.H. NICKLIN, Chong-Kyo LEE and Eckard WIMMER

Department of Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, N. Y. 11794, U.S.A. (Received 9-7-198L accepted 13-10-1987)

Summary - The primary translation product of the picornavirus genome is a single large protein which is processed to the mature viral polypeptides by progressive, co- and post-translational cleavages. Replication of the picornaviruses is thus entirely dependent upon the proteolysis of viral precursor proteins. In poliovirus, two virus-encoded proteinases have been identified that catalyze all but the final cleavage of the viral polyprotein. The final processing event, maturation of the virion polypeptide VPO, appears to occur by an unusual autocatalytic serine proteinase-like mechanism. Proteolylic processing of viral precursor proteins is basically similar in all picornaviruses, but recently it has become clear that there are also important differences between these viruses. Understanding of the processing events in picornavirus replication may ultimately lead to the discovery of specific inhibitors of the viral enzymes that could prove clinically useful as anti-viral agents. picornavirus / proteolytic processing / ".,iral proteinase

Introduction

Picornaviruses cause a bewildering array of disease syndromes in man, spanning from fatal paralysis, encephalitis, meningitis, conjunctivitis, hepatitis, myocarditis and pancreatitis, to the common cold. The incidence of human infections with picornaviruses (namely with human entero- and rhinoviruses) is high, and although recovery appears to be complete in most cases, serious long-term effects may be more common than originally estimated (see, for example [1]). In the U.S., non-polio enteroviruses are the most common cause of meningitis in children, accounting for an estimated 75000 cases each summer. Coxsackieviruses (especially type B3) are the cause of serious heart disease, and enterovirus 72 (hepatitis A virus) is responsible for at least 25 000 cases of infectious hepatitis each year. Since vaccination against the more than 70 non-polio entero-

viruses and 110 rhinoviruses is impossible at the present time, the development of broadrange chemotherapeutic agents is desirable. It has been recognized that the detailed knowledge of the events leading to the proliferation of any family of viruses will greatly aid the search for anti-viral drugs. For reasons that will be outlined below, virus-encoded proteinases are prime targets for the development of specific inhibitors that may be tailored for use in vivo. Our studies of the molecular biology of poliovirus will hopefully contribute to the design of chemotherapeutica that can serve as an alternative means to control picornavirus infections. Picornaviridae comprise four genera (entero-, rhino-, cardio- and aphthoviruses, the latter two being strictly animal .viruses) whose genome structure, gene organization and strategy of replication are very similar. Poliovirus may currently be .the best known among all viruses studied. Its chemical structure (Fig. 1;

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Fig. 1. Gene organization and polypeptide processing of poliovirus. Virion RNA, terminated at the 5' end with the genomelinked protein VPg and at the 3' end with poly(A), is shown as a solid line, the translated region being more pronounced than the non-coding regions. Arrows indicate the sites at which imtiation (nucleotide (nt.) 743) and termination (nt. 7370) of translation occur. The numbers above the virion RNA line refer to the first nucleotide ofthe codon specifying the amino-terminal amino acid of each virus-specific protein. The coding region has been divided into three regions (Pl, P2, P3), corresponding to major cleavage products ofthe polyprotein. The nomenclature ofpicornavirus proteins is according to Rueckert and Wimmer [60]. Polypeptides are presented as wavy lines. Numbers between parentheses are molecular weights calculated from the amino acid sequences. Carboxy-terminai 'trimming' does not occur. Open circles indicate glycine in all cases except for VP2, where it is serine. The carboxy-terminai amino acid of 3 CD is phenylalanine. Solid circles indicate that the amino terminus is known to be blocked [10] with mvristic acid [8, 9]. Solid triangles: Q-(3; open triangles" Y-G; open diamond" N-S cleavage sites. Polypeptides 3C' and 3I)' are products of an alternative cleavage mode of 3CD. (Modified after [2].)

[2]) and crystal structure [3] have been solved; and the complete genetic map has been known for some time (Fig. 1; reviewed in [4]). The virus occurs in three different serotypes, whose genome sequences have all been determined (reviewed in [4]). Moreover, much energy has been expended to analyze the antigenic make-up of the virus [5] and the attenuation phenotype [6]. Finally, the molecular biology of poliovirus replication has been under investigation for nearly three decades. Although poliovirus is one of the smallest animal viruses m genome structure, a wealth of unsolved molecular problems awaits solution: the mechanism of virus neutralization and virus uptake, individual steps in proteolytic proceSsing, genome replication and vira morphogenesls.

Virus

structure

Poliovirus is a naked (non-enveloped) icosahedron consisting of 60 copies each of the 4 capsid proteins VP4, VP2, VP3 and VPl. The capsid proteins, like all known poliovirus encoded proteins, are cleavage products of a precursor (see Fig. 1) [2, 3, 4, 7]. The ssRNA genome is of plus strand polarity; it is polyadenylated at the 3' end and covalently linked to a small protein (VPg) at the 5' end via a tyrosine-O4-uridylyl bond (Fig. 1). A novel observation was made recently [8, 9]: the chemical block that is present at the N-termini of VP0 and VP4 [l 0] is myristic acid. We had suspected such a large hydrophobic blocking group due to the properties of the N-terminal, tryptic peptide [10] of VP4.

Picornavirus polyprotein processing In

vitro

manipulations of the poliovirus

genome Two experimental achievements have greatly enhanced the opportunities for studying poliovirus molecular biology and genetics. The first achievement was the molecular cloning of the poliovirus genome [11, 12] and the successful construction of infectious eDNA clones by Racaniello and Baltimore [13] that was later repeated by Semler et al. [14] and Omata et al. [151. The second important achievement for in vitro manipulations of the poliovirus genome was the construction of a transcription system, using phage T7 RNA polymerase. For this purpose, the T7 RNA polymerase promoter was engineered directly in front of the cDNA sequence coding for the viral RNA. The resulting transcripts, that were found to carry only two additional G residues at the 5' end of the RNA (pppGpGpUpU etc., where the G residues are non-viral), have a specific infectivity of 105 pfu//zg of RNA which is within the range ofvirion RNA (106 pfu//zg of RNA) [16]. By this route, virtually unlimited amounts 6f highly infectious poliovirus genetic material, and derivatives thereof, can be produced in a simple test tube experiment. Four strategies have been followed to modify poliovirus genetic information in vitro. The first involved the expression of segments ofpoliovirus eDNA clones in suitable cells (usually E. coil), in order to study the function(s) of the expressed viral proteins. This approach was success(ully applied in investigations of the poliovirus-encoded proteinases [17, 18, 19]. Most recently, such gene segments have been transcribed with phage T7 or SP6 RNA polymerases and the transcripts have been used as mRNA in in vitro protein synthesis [20, 21]. Second, allele replacements between wild type and mutant strains of poliovirus have been performed to assess the effect of multiple or single known mutations on viral replication and pathogenicity. The third strategy involves the generation of mutations over the entire infectious eDNA clone by linker insertions or deletions (usually at restriction sites). The mutated clones are then screened for new phenotypes which can be correlated with specific genes or genetic elements of the viral RNA. In the fourth and final strategy, a specific segment of the viral genome can be selected and earmarked for 'saturation' mutagenesis in

121

vitro. This strategy has been followed with a segment coding for VPg: suitable restriction sites were genetically engineered into the genome located just outside the VPg coding sequence. Multiple mutations in VPg can be generated by chemical synthesis of derivatives of the entire gene segment [22] for further references of in vitro manipulations of poliovirus RNA, see [4].

The polyprotein of picornaviruses: general observations Post-translational modification of polypeptides through proteolytic cleavage is an important process by which functional proteins are formed in vivo. This conclusion is based upon ample evidence, some of which has been reviewed in other sections of.these proceedings. The replication of many RNA viruses is entirely dependent upon proteolysis of certain virus-specific proteins, and it is not unusual for the proteinase(s) involved in such processes to be encoded by the viral genome. Synthesis of their own proteinases confers an advantage to the virus, since this aspect of replication does not depend upon cellular enzymes and their intracellular localization. This, in turn, relieves the virus from potential host-range restrictions and, at the same time, allows it to independently evolve a system of highly specific proteinases and substrates [23, 24]. Virus-encoded proteinases generally act upon proteins in the cytoplasm. On the other hand, the precursors to viral-encoded membrane proteins (glycosylated envelope polypeptides) are cleaved by cellular proteinases that reside in specific compartments of the cell. This type of processing, for example, the removal of 'signal' sequences or the cleavage of glycosylated polypeptides to subunits, occurs during the transport of proteins to the membrane. In several virus families, a final cleavage of envelope proteins occurs during or after the assembly and release of the virion from the cell. Examples are the paramyxo- and orthomyxoviruses, and the enzymes involved in the extracellular maturation cleavages are proteinases of the host [25]. In contrast, picornaviruses do not use any cellular proteinases for the cleavage of their precursor polypeptides ('polyproteins'). Polyproteins are defined as large polypeptides containing numerous domains [26] that

122

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are proteolytically processed to separate proteins with different functions [18]. The domains of polyproteins may involve capsid proteins and non-structural proteins, such as RNA polymerases and proteinases. Most, eukaryotic plus-strand RNA viruses encode polyproteins. For example, polyproteins have been demonstrated in the replication of picornaviruses, togaviruses (Semliki forest virus, sindbis virus), flaviviruses (yellow fever virus), comoviruses (cowpea mosaic virus), tymoviruses (turnip yellow mosaic virus), potyviruses and nepoviruses. Retroviruses also synthesize polyproteins but their genomic RNA normally functions as mRNA only after its transcription from the pro-virus in the host nucleus. For the most part, the mechanism by which the viral polyproteins are proteolytically processed is poorly understood. Only in a few instances have virus-encoded proteinases been characterized; generally, their existence has only been implicated indirectly by either biochemical or genetic analyses. Notable exceptions are the picornaviruses and retroviruses. Our own research efforts in the past have focused on the two viral-encoded proteinases involved in poliovirus polyprotein processing. It should be stressed, however, that many research groups have contributed to the general understanding ofpicornavirus polyprotein processing. In particular, researchers studying encephalomyocarditis virus (EMCV)

tein synthesized at a supraoptimal temperature (43°C), for example, is not processed when the temperature is shifted down to 37°C [27]. Certain portions of the polyprotein must therefore fold properly so that it can serve as its own substrate. In addition, its individual components, once released from the polypeptide chain, must achieve proper conformations so that they can perform a variety of different structural and enzymatic functions.

Proteolytic processing of the polyprotein: specific events Proteolytic processing of the poliovirus polyprotein occurs in three stages. The primary cleavage takes place during polyprotein synthesis. It is carried out by proteinase 2A and leads to the rapid separation of the capsid precursor P1 from non-structural proteins (P2 and P3; see Fig. 1). The secondary cleavages are catalyzed by proteinase 3C that, probably after intramolecular liberation, acts at least partly in trans to cut at Q-G pairs. The tertiary cleavage occurs during morphogenesis of the virion (cleavage of VP0 to VP4 and VP2 at an N-S pair; see Fig. 1). As an exciting possibility, this cleavage may be an intramolecular event that involves elements of the viral RNA [28]. As suggested first by Rossmann et ai. [7], the serine ~° in VP2 may be the amino acid that, mter acuvauon by a proton-abstractor (a base of the nucleic acid), may catalyze cleavage between the asparagine and serine, the latter being the N-terminal amino acid of VP2 [29]. The three stages of proteolytic processing have been deduced on the basis of structural, genetic and enzymological data. The elucidation of the chemical structure of the poliovirus genome [2, 12] and the amino acid sequence analyses of all known poliovirus proteifis [10, 29-35] have led to the discovery of the cleavage sites in the polyprotein. The preponderance of Q-G cleavage sites immediately suggested that Q-G may be recognized by a virus-encoded proteinase. The identification of polypeptide 3C pr° as the enzyme that cleaves at Q-G pairs, but not at Y-G pairs, was possible with the use of monospecific anti-3C antibodies [36]. As shown in Fig. 2, these antibodies, when added in increasing amounts to an in vitro translation system, inhibited processing of precursors that arose by Q-G cleavages. Pre-immune serum (Fig. 2) or anti-2C serum (not shown here) had _ £'~a. . . . .

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ed proteinase activity and to map it to a specific genome segment. We have reviewed the subject recently [23, 24] and will only summarize the highlights here. The poliovirus polyprotein, the only known product of translation of poliovirus mRNA, harbors a number of very different functions, some of which are known to us. The biological significance of many of its features, however, remains obscure. Only recently has it been established that the macromolecule can cleave itself into different domains without the help of cellular components [18]. It does this so efficiently that, under normal circumstances, the polyprotein cannot be observed at all. Before its synthesis is completed, the P1 region (Fig. 1) is already severed from the growing polypeptide strand, possibly by an intramolecular cleavage event. It is not known, and even unlikely, that the completed, intact polyprotein could fold properly to induce its own cleavage (see discussions in [23~24]). Poliovirus polypro-

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no effect on proteolytic processing whatsoever [36]. Note that the intensity of band 3D', a product of Y-G cleavage .(Fig. 1) did not decrease upon addition of anti-3C antibodies, an observation suggesting that 3D' is generated by an. enzymatic activity distinct |?om 3C p'° [36]. This was later proven to be correct [18]. The question then arose as to how 3C pr° is generated in the initial stages of infection.

123

Cleavage in trans by a virion-associated proteinase appeared highly unlikely, since deproteinized virion RNA is infectious. A po!iovirusspecific precursor protein containing 3C pr° and adjacent polypeptide sequences was therefore expressed in E. coil with the objective of studying the occurrence of the Q-G" cleavages flanking 3C pr°. The mRNA and its translation products [17] are schematically shown in Fig. 3. Upon induction, we observed not only the expected precursor (polypeptide 1), but also its Q-G-specific cleavage products (polypeptides 2 and 3). Indeed, immunoprecipitation and terminal amino acid sequencing identified polypeptide 3 as being authentic poliovirus 3C p'° [17]. Linker mutation within the gene segment specifying 3C p'° abolished the appearance ofpolypeptides 2 and 3 in E. coil [17]. This result suggested that 3C p~''arose by intramolecular cleavage ofpolypeptide 1, a conclusion in agreement with earlier studies on polypeptide 3CD of EMCV [37]. Using a similar strategy of biochemical and genetic analyses of polypeptides expressed in E. co!i, we discovered that 2A is also a proteinase [18]. More recent experiments have dealt with an in vitro genetic study of poliovirus encoded proteinases [20, 38] (see below). Finally, a mechanism for the maturation cleavage (VP0 ~VP4 + VP2) was suggested on the basis of the crystallographic structures of human rhinovirus 14 [7] and ofpoliovirus [3]. A detailed di.~cll~.~ion o f the e o n ~ i d a r a t i n n ~ concerning this cleavage can be found in [28]. Other specific considerations regarding the processing events are as follows" 1) The nature of the active site in 3C p~° has been predicted by comparison of amino acid sequences of several picornaviruses [39] and contains L~ys 147 and His 161. This prediction received support by site-directed mutagenesis experiments [19]. Studies with inhibitors support the notion that 3C of EMCV and of poliovirus is a sulfhydryl proteinase ([37, 40, 41], Nicklin and Wimmer, unpublished results). Detailed enzymology with 3C p~°, however, has not been performed due to the lack of purified enzyme and substrate. From comparisons of amino acid sequences of picornavirus polypeptides, we suggest that 2A °~° is also a sulfhydryl proteinase [18, 42], but experiments to support this claim have not been carried out. 2) The proteinases cleave with high specificity" poliovirus 3C p~° does not cleave precursor

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Fig. 3. Processing scheme for poliovirus-specific polypeptides synthesized in bacteria. The transcript of the plN-llI-C3-7c vector is shown as a bar. The open bars represent pIN-III-C3-~pecific regions present within the transcript. The poliovirus genomic RNA sequence is shown as a filled bar. The methionine residue present within protein P3-7c (3C) at nucleotide 5516 is indicated by M. Polypeptides are denoted by wavy lines. An arrow indicates the site ofcleavage of the signal peptide. Cleavages between Q-G pairs of amino acids are indicated by solid triangles. Polypeptide 3i is the product of a fortuitous internal initiation at the metbionine residue denoted as M. Cleavage between a Q-G amino acid pair within polypeptide 3i does not occur (shown as an open triangle). The numbers between parentheses are the molecular masses (in kDa)calculated from the amino acid sequences, assuming an average molecular mass of 1l0 for each amino acid. (Reproduced from [17].)

proteins ofencephalomyocarditis virus and vice versa (Nicklin and Palmenberg, unpublished results). This specificity of 3C pr° is remarkable in view of the fact that the cleavage signal recognized by 3C p~° of both poliovirus and EMCV is mainly Q - G . 3C °~° of poliovirus recognizes only 8-9 of the 13 Q - G pairs present in its own polyprotein. The Q - G pairs that are cleaved have surrounding amino acid sequences that differ from each other, although an additional determinant of recognition may be the amino acid in position - 4 relative to the Q - G cleavage site, in poliovirus this amino acid is most often an alanine residue [23, 24]. It is unlikely that the selection of the proper Q - G sites is based solely upon accessibility. We speculate that 3C p~° requires structurally flexible contexts surrounding the active Q - G sites, as has been proposedrecently by Arnold

et al. [281.

The high specificity of the viral proteinases may be an explanation for the observation that

no cellular proteins have been identified as yet that are cleaved by 3C pr° or 2A pr° [43-46]. 3) Synthetic peptides corresponding to poliovirus sequences (with Y - G or Q - G pairs in the center) that are cleaved specifically by the 2A or 3C proteinases have not been found. These studies will be easier when pure enzymes, free of contaminating peptidases, and a variety of synthetic peptides are available. 4) Poliovirus is the only virus whose 3C °r° cleavage sites are exclusively Q - G pairs. In other picornaviruses, the 3C pr° cleavage sites can vary considerably but are generally G l x Gly, where the glycine can be replaced by S, T, A, V or M residues (Q-G, -S, -T, -A, -V, -M; and E - G , -S) [23, 47]. 'Trimming' of the termini of polypeptides after cleavage at the amino acid pair has not been observed. Site directed mutagenesis of individual codons and the use of mutagenesis cartridges [22] is in progress to assess wh~it sequences can be recognized as cleavage signals by 3C prU.

125

Picornavirus pol)Trotein processing

5) Poliovirus rapidly and effectively turns off host cell protein synthesis [48], and this event is accompanied by the cleavage of a large polypeptide (termed p220 corresponding to its Mrof 220000) from the cell's cap binding complex [49, 50]. Neither 2A pr° nor 3C pr° cleaves p220 directly [44-46], but based upon genetic and biochemical experiments, polypeptide 2A pr° appears to 'activate' the degradation of p220 [38, 51] (see experiments described below). In agreement with this finding, infection with EMCV (a virus whose 2A is not homologous in function to polio's 2A) does not induce the cleavage of this cellular polypeptide [52]. 6) The Y - G specific cleavage in polypeptide 3CD that yields 3C' and 3D' (Fig. l) may be fortuitous and of no biological significance other than lowering the yield of3C pr° and 3D p°l. Sitedirected mutation of the tyrosine residue of

this Y - G site in 3CD to a phenylalanine residue (F-G) did not abolish the infectivity of the altered genome. Surprisingly, 3C' and 3D' were still produced from this altered cleavage site (C.-K. Lee and E. Wimmer, submitted). On the other hand, alteration of the cleavage site from T Y - G to A Y - G completely abolished the production of 3C' and 3C' without detectable effects on virus growth (C.-K. Lee and E. Wimmer, submitted). 7) Polypeptide 3AB, a membrane-associated protein [53-54] is relatively stable in pulsechase experiments of infected cells, although it contains an 'active' Q - G site. It has been speculated that 3AB is processed to 3A and VPg only after a tyrosine residue in the 3B portion of 3AB has been uridylylated [55-57]. 8) Substrates for 2A pr° and 3C or° have been synthesized in rabbit reticulocyte lysates

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126

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(RRLs) and used to define aspects of the cleavage activity by and to develop assays for 2A pr° and 3C pr° [20, 21, 38] (see below). It has become apparent that the relationship between substrate and proteinase is complex in that some truncated, virus-specific polypeptides, although containing bona fide cleavage sites, are no longer processed b.y the enzyme. In the past, we have studied the properties of 3C r'~° and 2A P", by expressing the enzyme with suitable vectors in E. coil [17, 18]. Whereas these experiments served to define the enzymatic functions of the poliovirus gene products, the context of the prokaryotic cell extract did not allow a detailed study of the proteins without purification. Isolation of the enzyme, on the other hand, was difficult because we did not have a simple assay to monitor progress of purification. More recently, therefore, we have used expression vectors for in vitro transcription of viral genetic information by phage T7 RNA polymerase. The synthetic RNAs were then translated in vitro in reticulocyte extracts and thus biologically active, virus-specific macromolecules (substrates and enzymes) were generated [20, 38]. The plasmid constructs used for these studies are shown in Fig. 4, the translation products of these constructs are shown in Fig. 5A [381. Plasmid pMN22 yielded polypeptide P1, the plli~l~Ul:SUl UI tllli~ I.;PIIJ:~IU p l U t ~ l l l 3 .

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Fig. 5. Products of in vitro translation of synthetic RNAs and effect on p220 processing. Synthetic RNAs were runoff transcripts of plasmids as shown in Fig. 4. A. Aliquots of translation reactions performed is rabbit reticulocyte lysate were analyzed on a 12.5% NaDodSO4-polyacrylamide gel. Note that a protein is present in the no RNA lane, a phenomenon often seen in this system. 2ABC' and 2AB' denote truncated polypeptides 2C and 2B, respectively. Because of an insertion in 2A in piasmid pMN28, the 2ABC~I cleavage product migrates slightly differently from the 2ABC' obtained from pMN27. Lane M represents a lysate of[35S]methionine-labeled poliovirus-infected HeLa cells. B. Samples of each translation mixture were incubated with an S10 extract from mock-infected HeLa cells for 2 h at 30"C. Aliquots of each sample were analyzed on an 8% NaDod, SOa-polyacrylamide gel and transferred onto nitrocellulose sheets. The blots were probed with a monoclonal antibody directed against p220. U-S10 represents S10 extract from mock-infected HeLa celis~ I-SI0 represents S10 extract from poliovirus-infected HeLa cells. C.P. refers to cleavage products of p220. Note that p220 was cleaved only if unmodified 2A was produced, regardless o f w h e t h e r 2A is bound to downstream proteins as in 2AB" or 2ABC". Purified 2A (kindly provided by H. K/Snig and B. Rosenwirth, data not shown) and 2A obtained by in vitro translation (lane pS32A) also induced cleavage of p220. (Data from Kdiusslich et al., [381.)

127

Picornavirus polyprotein processing

supplied either by an infected HeLa cell cytoplasmic extract or produced by translation in vitro (data not shown). In the latter case, a plasmid vector containing a highly efficient 5' non-translated region of EMCV and the coding sequence of2A pr° ofpoliovirus was transcribed and translated in vitro [38]. Apart from cleaving the pMN29 product, the in vitro synthesized 2A pr° also induced the cleavage of the cellular protein p220 in vitro (Fig. 5B; [38]). Studies with anti-2A antibodies established that 2A pr° itself does not directly cleave p220 [38, 45]. The cellular enzyme activated by 2A pr°, and responsible for the degradation of p220, is unknown. These data clearly demonstrate that virusencoded proteinases can be studied using in vitro transcription of suitable plasmids to generate synthetic RNAs for translation in cellfree extracts. The synthesis of proteins in vitro produced the substrates needed for assays of the enzymes. This, in turn, has allowed us recently to develop highly efficient expression systems for 3C pr° from both poliovirus and encephalomyocarditis virus that have been used to produce large amounts of active 3C pr° in E. coli. Purification of the poliovirus enzyme from E. coli lysates has been achieved, a result that will finally allow us to carry out much needed characterization of this interesting proteinase (Nicklin and Wimmer, unpublished results). Using a similar approach, we can now ~l . . . . .I.~.A. l.,.).l ~ ' 3 )

dl~lJ

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Comparison of picornavirus proteinases All picornaviruses studied produce 3C pr°, but an active 2A pr° appears only to be a component of the polyproteinsofentero- and rhinoviruses (Fig. 6). Cardioviruses and aphthoviruses have a leader polypeptide ('L') preceding the P1 region. In foot-and-mouth disease virus (FMDV) (an aphthovirus), the L polypeptide is a proteinase that appears to be capable of selfcleavage from PI [581. A polypeptide corresponding to 2A in FMDV is absent altogether (Fig. 6). In EMCV (a cardiovirus), the L polypeptide has no known proteolytic activity, and the function of 2A is as yet uncertain [59]. It has thus become clear that the mechanism of polyprotein processing of entero- and rhinoviruses vs. cardio- and aphthoviruses is quite different in regard to the separation of P1 from P2. These differences are summarized in Fig. 7 where 3 strategies ofpolyprotein processing are

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Fig. 6. Structure of the picornaviral genome for some picornaviruses. Peptide nomenclature is according to standard convention, as shown in Fig. 1, except that the capsid proteins are shown as 1A, 1B, IC and 1D. 'L' denotes a leader peptide found only in cardio- and aphthoviruses. "CCCC' denotes a poly(C) stretch in the 5' non-coding region of EMCV and FMDV; the dot at the left (5') end is VPg. Proteolytic cleavages of the polyproteins occur be~W~i'i ami~o :A •~,,.,, pairs ;.n.r.l ;.~.Q.t.~. . . . .i.n . .c.t.a.n.r.l a. r. d. . . .c l n o~,l.~...l .a.t .t o. .r code. Cleavages produced by proteinase 3C are shown above each genome. Proteinase 2A (hatched arrowhead) and VPO (lAB) (stippled arrowhead) cleavage sites are shown below the genome. Sites for which the proteolytic agent has not been specifically identified are between parentheses. Question marks denote sites where the sequence has not been established precisely. (Reproduced

from [281.)

shown for three representative members of the Picornaviridae. Only in entel3- and rhinovirus protein synthesis is P1 severed from P2 by 2A pr°. EMCV and FMDV lack an active 2A pr°. Instead, the cleavage between P1 and 2A is thought to be catalyzed by 3C pr° after the latter has excised itself from the polyprotein. In the case of EMCV, the leader polypeptide L also is cut from the polyprotein by 3C pr°, whereas in FMDV protein synthesis polypeptide L severs itself from the growing polypeptide cnain. The VP4 capsid proteins of all picornaviruses are

128

H.-G. Krdusslich et al.

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Fig. 7. Polyprotein cleavage strategies of picornaviruses. Polyproteins are drawn as lines, several cleavage activities being represented as boxes. Only those t r a n s cleavages of 3C are shown that distinguish processing of the three polyproteins. Polioviruses, and most likely all human entero- and rhinoviruses, process their polyprotein in a similar fashion. 2A of EMCV does not appear to have proteolytic activity; the activity cleaving at the Q-G site located C-terminal to 2A is unknown (see question mark). The same problem exists for the G-P cleavage in the polyprotein of FMDV where '2A' is truncated to a peptide of 16 amino acids. The 'L' polypeptide of FMDV can sever itself from P! of the polyprotein; the 'L' polypeptide of EMCV, however, does not have this ability. Cleavage between VP2 and VP4 occurs only during maturation of the virion (after association of all capsid proteins with the viral RNA). For further details, see text.

myristylated [8, 9]. In poliovirus protein synthesis, blocking of the N-terminus of VP4 occurs after the N-terminal initiator methionine has been .,~,,,u,~u . . . . . . "~'" " ' For EMCV and ..... t:', ',uj. FMDV, myristylation of VP4 must be preceded by the cleavage between polypeptide L and polypeptide P1. As yet unsolved is the question of how 2A of EMCV is cleaved from 2B at its C-terminus; similarly mysterious is the homologous cleavage at the G - P pair in the FMDV polyprotein (see question marks in Fig. 7). Available evidence suggests that these cleavages are intramolecular (A. Palmenberg, personal communication). Finally, the maturation cleavage between VP4 and VP2 may follow a ~,lechanism common to all picornaviruses [7, 28]: an intramolecular event catalyzed by a specific serine residue in VP2 that is thought to have been activated by a base of the viral ribonucleic acid. No direct evidence, however, for this intriguing proposal exists as yet, and a serine I° of VP2 which is conserved in the other three genera of picornaviruses (entero-, rhino- and cardioviruses) is absent from the aphthoviruses.

Conclusion Poiyprotein processing of picornaviruses follows a complex pattern and evolved to be independent from cellular proteolytJc activities. Although the virus-encoded activities have been recognized, their properties remain largely unknown due to previous difficulties in producing the enzymes in sufficient quantities and in developing feasible assays for enzyme activity. Both these problems have been solved recently. Nevertheless, very recently it has become apparent that specific cleavages by 3C or° may involve additional virus-encoded factors: 3C p'°, in fact, may not efficiently process the 2 Q - G pairs in P1 unless complexed to polypeptide components of P3 ([21], Nicklin, Kr~iusslich and Wimmer, unpublished results; B. Enger-Valk, personal communication). Thus, cleavage at the 8 essential Q - G sites in the poliovirus polypro~ein may be delicately modulated to serve the ~pecific requirements of polypeptide production during virus replication.

Picornavirus polyprotein processing

Acknowledgments We thank our colleagues, particularly Haruka Toyoda, Ann Palmenberg, Bert Semler and Richard J. Kuhn for stimulating discussions and Lynn Zawacki for the preparation of the manuscript. Work reported here was supported in part by Public Health Service grants AI15122 and CA28146 to E.W. from the National Institutes of Health. M.N. is fellow DRG-848 of the D a m o n R u n y o n - W a l t e r Wincheil Cancer Fund. H.G.K. is supported by fellowship Kr906/1-2 from the Deutsche Forschungsgemeinschaft.

References 1. Wilfert C.M., Thompson R.J., Sunder T.R., O'Quinn A., Zeller J. & Blacharsh J. (1981) Pediatrics 67, 811815 2. Kitamura N., Semler B.L., Rothberg P.G., Larsen G.R., Adler C.J., Dorner A.J., Emini E.A., Hanecak R., Lee J.J., van der Werf S., Anderson C.W. & Wimmer E. (1981) Nature 291,547-553 3. Hogle J.M., Chow M. & Filman D.J. (1985) Science 229, 1358-1363 4. Wimmer E., Kuhn R.J., Pincus S., Yang C.-F., Toyoda H., Nicklin M. & Takeda N. (1987) in: Virus Replication and Genome intereactions (Woolhouse H.V., Ellis T.H.N., Chater K.F., Davies J.W. & Hull R., eds.), 7th John Innes Symposium, Co. of Biologists Ltd., Cambridge, pp. 251-276 5. Wimmer E., Emini E.A. & Diamond D.C. (1986) in: Concepts in Clinical Pathogenesis H (Notkins A.L. & Oldstone M.B.A., eds.), Springer-Verlag, New York, pp. i59-i73 6. Nomoto A. & Wimmer E. (1987) in : Molecular Basis of Virus Disease (Russell W.C. & Almond J.W., eds.), Society for General Microbiology Symposium, Vol. 40, Cambridge University Press, Cambridge, pp. 107134 7. Rossmann M.G., Arnold E., Erickson J.W., Frankenberger E.A., Griffth J.P., Hecht H.-J., Johnson J., Kamer G., Luo M~, Mosser A.G., Rueckert R.R., Sherry B. & Vriend G. (1985) Nature 317, 145-153 8. Chow M., Newman J.F.E., Filman D., Hogle J.M., Rowlands D.J. & Brown F. (1987) Nature327, 482-486 9. Paul A.V., Schultz A., Pincus S.E., Oroszlan S. & Wimmer E., (1987) Proc. Nail Acad. Sci. USA 84, 78277831 10. Dorner A.J., Dorner L.F., Larsen G.R., Wimmer E. & Anderson G.W. (1982) J. ViroL 42, 1017-1028 11. van der Werf S., Bregegere F., Kopecka H., Kitamura N., Rothberg P.G., Kourilsky P., Wimmer E. & Girard M. (1981) Proc. Natl. Acad. Sci. USA 78, 5983-5987 12 Racaniello V.R. & Baltimore D. (1981) Proc. Natl. t~,,'ad. Sci. USA 78, 4887-4891 13. Rac~,niello V.R. & Baltimore D. (1981) Science 214, 916-91~ 14. Semler B.L.~ Dorner A.J. & Wimmer E. (1984) Nucleic Acids Res. 12, 5123-5141 15. Omata T., Kohara M., Sakai Y., Kameda A., Imura N. & Nomoto A. (1984) Gene 32, 1-10

129

16. van der WeftS., Bradley J., Wimmer E., Studier F.W, & Dunn J.J. (1986) Proc. Natl. Acad. Sci. USA 83, 23302334 17. Hanecak R., Semler B.L., Ariga H., Anderson C.W. & Wimmer E. (1984) Cell 37, 1063-1073 18. Toyoda M., Nicklin M.J.H., Murray M.G., Anderson C.W., Dunr, J.J., Studier F.W. & Wimmer E. (1986) Cell 45, 76~_-770 i9. IvanoffL.A., Towatari T., Ray J., Korant B.D. & Petteway S.R. Jr. (1986) Proc. Natl. Acad. Sci. USA 83, 53925396 20. Nicklin M.J.H., Kr/iusslich H.-G., Toyoda H., Dunn J.J. & Wimmer E. (1987) Proc. Natl. Acad. Sci. USA 84, 4002-4006 21. Ypma-Wong M.F. & Semler B.L. (1987) Nucleic Acids Res. 15, 2069-2088 22. Kuhn R.J., Tada H., Ypma-Wong M.F., Dunn J.J., Semler B.L. & Wimmer E. (1988) Proc. Natl. Acad. Sci. USA 85, On press) 23. Nicklin M.J.H., Toyoda H., Murray M.G. & Wimmer E. (1986) Biotechnology 4, 33-42 24. Toyoda H., Nicklin M.J.H., Murray M.G. & Wimmer E. (1986a) in: Protein Engineering: Applications in Science, Medicine and Industry (Inouye M. & Sarma R., eds.), Academic Press, New York, pp. 319-337 25. Scheid A. & Choppin P.W. (1984) in: Concepts in Viral Pathogenesis (Notkins A.L. & Oldstone M.B.A., eds.), Springer-Verlag, New York, pp. 26-31 26. Jacobson M.F. & Baltimore D. (1968) Proc. Natl. Acad. Sci. USA 61, 77-84 27, Baltimore D. (1971) in" Perspectives in Virology (Pollard M., ed.), Vol. 7, Academic Press, New York, pp. 1-14 28. Arnold E., Luo M., Vriend G., Rossmann M.G., Palmenberg A.C., Parks G.D., Nicklin M.J.H. & Wimmer E. (1987) Proc. Natl. Acad. Sci. USA 84, 21-25 29. Larsen G.R., Anderson C.W., Dorner A.J., Semler B.L. & Wimmer E., (1982) J. Viroi. 41,340-344 30. Kitamura N., Adler C.J., Rothberg P.G., Martinko J., Nathenson S.G. & Wimmer E. (1980) Cell21, 295-302 31. Semler B.L., Anderson C.W., Kitamura N., Rothberg P.G., Wishart W.L. & Wimmer E. (1981) Proc. Natl, Acad. Sci. USA 78, 3464-3468 32. Semler B.L., Hanecak R., Anderson C.W. & Wimmer E. (1981) Virology 114, 589-594 33. Emini E.A., Elzinga M. & Wimmer E. (1982) J. Virol. 42, 194-199 34. Adler C.J., Elzinga M. & Wimmer E. (1983) J. Gen. Virol. 64, 349-355 35. Pallansch M., Kew O.M., Semler B.L., Omilianowski D.R., Anderson C.W., Wimmer E. & Rueckert R.R. (1984) J. Virol. 49, 873-880 36. Hanecak R., Semler B.L., Anderson C.W. & Wimmer E. (1982) Proc. Natl. Acad. Sci. USA 79, 3973-3977 37. Palmenberg A.C. & Rueckert R.R. (1982) J. Virol. 41, 244-249 38. Kriiusslich H.-G., Nicklin M.J.H., Toyoda H., Etchison D. & Wimmer E. (1987) Z Virol. 61, 2711-2718 39. Argos P., Kamer G., Nicklin M.J.H. & Wimmer E. (1984) Nucleic Acids Res. 12, 7251-7276 40. Pelham H.R.B. (1978) Eur. J. Biochem. 85, 457-462 41. Gorbalenya A.E. & Svitkin Yu. V. (1983) Biochemistry (U.S.S.R.) 48, 442-453 42. Blinov V.M., Donchenke A.P. & Gorbalenya A.E. (1985) Proc. Acad. Sci. U.S.S.R. 281,984-987 43. Korant B.D., Chow N.L., Lively M.O. & Powers J.C. (1980) Ann. N.Y. Acad. Sci. 343, 304-318 44. Lloyd R.E., Etchison D. & Ehrenfeld E. (1985) Proc. Natl. Acad. Sci. USA 82, 2723-2727

130

H.-G. K r i i u s s l i c h et al.

45. Lloyd R.E., Toyoda H., Etchison D., Wimmer E. & Ehrenfeld E. (1986) Virology 150, 299-303 46. Lee K.A.W., Edery I., Hanecak R., Wimmer E & Sonenberg N. (1985) J. Virol. 55, 489-493 47. Paimenberg A.C. (1987) in: Positive Strand RNA Viruses (Brinton M.A. & Rueckert R.R., eds.), UCLA Symposia Cellular Biology, Vol. 54, Alan R. Liss Inc., New York, pp.25-34 48. Ehrenfeid E. (1982) Cell 28, 435-436 49. Etchison D., Milburn S.G., Edery I., Sonenberg N. & Hershey J.W.B. (1982) J. Biol. Chem. 257, 14806-14810 50. Etchison D., Hansen J., Ehrenfeld E., Edery I., Sonenberg N., Milburn S., & Hershey J.W.B. (1984) J. ViroL 51,832-837 51. Bernstein H.D., Sonenberg N. & Baltimore D. (!985) Mol. Cell. Biol. 5, 2913-2923 52. Mosenkis J., Danieis-McQueen S., Janovec S., Duncan R., Hershey J.W.B., Grifo J.A., Merrick W.C. &

Thach R.E. (1985) J. Virol. 54, 643-645 53. Semler B.L., Anderson C.W., Hanecak R., Dorner L.F. & Wimmer E. (1982) Cell 28, 405-412 54. Takegami T., Kuhn R.J., Anderson C.W. & Wimmer E. (1983) Proc. NatL Acad. Sci. USA 80, 7447-7451 55. Takegami T., Semler B.L., Anderson C.W. & Wimmer E. (1983) Virology 128, 33-47 56. Takeda N., Kuhn R.J., Yang C.-F., Takegami T. & Wimmer E. (1986) J. Virol. 60, 43-53 57. Kuhn R.J. & Wimmer E. (1987) in: The Molecular BioIogy of Positive Strand RNA Viruses (Rowlands D.J., Mahy B.W.J. & Mayo M.), Academic Press, London, pp. 17-51 58. Strebel K. & Beck E. (1986) J. Virol. 58, 893-899 59. Parks G.D., Duke G.M. & Palmenberg A,C. (1986) J. ViroL 60, 376-384 60. Rueckert R.R. & Wimmer E. (1984) J. Virol. 50, 957959