Proteolytic processing of picornaviral polyprotein.

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Further

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PROTEOLYTIC PROCESSING OF Annu. Rev. Microbiol. 1990.44:603-623. Downloaded from www.annualreviews.org Access provided by Mahidol University on 02/07/15. For personal use only.

PICO]�NAVIRAL POLYPROTEIN Ann C. Palmenberg Institute for Molecular Virology and Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706

KEY WORDS:

protease, poliovirus, autocatalysis, maturation processing

CONTENTS THE PICORNAVIRUS FAMILy............ .. ....................... . ................ ............ Family Organization . .......... . . ....... ...... ... . . ............ ....... ...... ........ ....... ......

603

604

The Genomic RNA................................................................................ The Viral Proteins................................................................................

605 607

PRIMARY CLEAVAGE EVENTS.... . . . ..... . . . ..... . . ..... . . . ..... . ... . . . . .... . . ... ... . .... . .. . Primary Cleavage in Entero- and R hinoviruses . .. ......... ..... ... . .. ....... ...... . .. ... .. Primary Cleavage in Cardio- and Aphthoviruses .. ......... ...... .. . . . .. ........ ....... ...

609 610 6ll

SECONDARY CLEAVAGE EVENTS ...... . . . ........ ... ................ ... . ... . . . ............. The 3C Protease.. ..... ......... ........... .. .... . ....... ........ .. .... . . . ... .. . . . ... .. . . .... . . . . .

613 614

MATURATION CLEAVAGE . .... . . . . ...... . . . ...... . ..... . . . ..... . .. .... . . ..... . . . ... . . . . . .. . . . . .

616

REPASS AND SUMMARy...... . . ..... . . . . ..... . . .. . .... . . ...... . . . ..... . . . ... . . . . ..... . . ....... .

618

Leader-Catalyzed Cl eavage in FMDV........................................................

616

THE PICORNAVIRUS FAMILY The family of picomaviruses lists among its members a diverse variety of pathogenic agents. The modest protein capsid structures and small RNA genomes that characterize this

family (pieD

=

small,

rna

=

RNA), belay the

medical and agricultural importance of picornavirus-caused diseases.

Infection with rhinovirus for example, better known as the common cold, is among the most prevalent and acute of human upper-respiratory illnesses. Each year this virus exacts high tolls from every economic and social institu603

0066-4227/90/1001-0603$02. 00

604

PALMENBERG

Annu. Rev. Microbiol. 1990.44:603-623. Downloaded from www.annualreviews.org Access provided by Mahidol University on 02/07/15. For personal use only.

tion, as cold-related absenteeism is the single largest cause of lost work days for schools and businesses. Poliovirus, another well known member of the picornavirus family, has likewise afflicted human populations for as long as recorded history. The tombs of ancient pharaohs faithfully depict the withered limbs and crippling paralysis effected by this virus. Even today, in the presence of modem vaccines and sanitation technology, many thousands of new cases are chronicled annually, especially in developing countries. The unfortunate results produce economic hardships from loss of productive manpower and from the obviously difficult medical burdens incurred in patient care. Animal populations are also susceptible to picornavirus infections. Foot and-mouth disease virus (FMDV) is one of the most virulent pathogens ever described. It infects many varieties of commercial livestock (pigs, sheep, goats, cattle) and causes devastating illness. The diversity of FMDV sero types and lack of effective disease control has resulted in severe agricultural problems in many parts of the world where the virus is enzootic (e. g. Asia, Africa, and South America). Though perhaps somewhat less notorious, other picornaviruses are also of economic and medical importance. These include the human coxsackie viruses (types A and B), hepatitis-A virus, echoviruses (enteric cytopathic human orphan), the human and animal enteroviruses (e.g. swine vesicular disease and bovine enteroviruses), as well as the murine cardioviruses [e.g. Mengovirus, encephalomyocarditis (EMCV) and Theiler's murine encephalomyelitis (TMEV»). For complete review of virus types, see Reference 74.

Family Organizations In spite of the disparate afflictions caused by these agents. all picornaviruses share remarkable similarity in their genome and capsid organization. Com plete or partial nucleotide sequences are now available for more than 90 strains of virus, including representative members from all major and minor classification groups (see 6 1). Resolution of the virion protein structures to atomic level has also been achieved for several of the most interesting types (polio, rhino, Mengo, FMDV) (1, 32, 33, 38, 50, 72). Combination of the sequence and structural information has allowed accurate delineation of the genetic relationships among many picornavirus strains. Figure 1 summarizes these relationships for 25 common serotypes. As a basic pattern, the viruses divide into four main phylogenie branches or genera, designated (arbitrarily) Groups I, II, III, and IV. The Group I viruses include the FMDV strains (aphthoviruses), Group II the murine cardioviruses (EMCV, Mengo, and TMEV), Group III the hepatitis-A isolates, and Group IV the rhino, polio, coxsackie, and other enteroviruses. For convenience (and whimsy) the strains in the latter group are sometimes referred to as "renter oviruses" because they include isolates from the more traditional rhino and

605

PICORNAVIRUS PROCESSING

Genetic Relationship Among Picornaviruses Rhlno-la

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Relationship among picomaviruses according to PI region nucleotide alignments.

Nucleotide alignments for 26 strains of picomaviruses are compared and presented graphically. The different line types allow easy identification of subgroups. This figure is adapted from Reference 61.

entero taxonomic designations. For review of sequence comparison and virus classification, see Reference 61. The capsilds of all picomaviruses are composed of a 60-subunit protein shell (20-30 nm diameter) having intrinsic 5: 3: 2 icosahedral symmetry. Each subunit contains four nonidentical polypeptide chains (VP I, VP2, VP3, and VP4) the largest three of which share, as a common structural motif, a wedge-shaped, eight-stranded, antiparallel f3-barrel configuration (1, 32, 33, 38, 50, 72). The shell encapsidates a single copy of the positive-sense RNA genome, which is released into the cytoplasm of a target cell to initiate the infectious process. The Genomic RNA The 3' ends of all picomaviral RNAs are polyadenylated, as is characteristic of most eukaryotic mRNAs (2). However, the 5' ends are not capped in the usual manner with 5' -5' triphosphate linkages. Instead, these viruses have


606

PALMENBERG

small, viral-coded, genome-linked proteins (VPg) attached by a tyrosine-04phosphodiester bond to the 5' uridylyl nucleotide of the RNA (4, 55, 73). VPg sequences are rich in basic, hydrophilic amino acids and have only one tyrosine residue (the attachment site) at position 3 from the amino end of the peptide. The specific function of VPg linkage to RNA is unknown, although the protein may serve a role in the initiation of RNA synthesis (5, 6, 55, 73). The lengths of genomic RNAs vary from 7,102 bases (rhinovirus type 2) to 8,282 bases (FMDV-OI K) not including the 3' poly(A) tail (50-150 bases). Each encodes a single, giant peptide called the polyprotein, which represents 85-90% of the theoretical coding capacity. The remainder of the bases are distributed (unequally) between the 5' (610-1,194 bases) and 3' (42-126 bases) noncoding regions (Figures 2 and 3) (60). The 5' noncoding regions of cardio- and aphthoviruses are unique in that they contain long homopolymeric polycytidylate (polyC) tracts, whose length (50-200 bases) and exact location rel ative to the 5' end of the genome (150-330 bases) vary with different isolates of virus (12, 15, 30). Experiments with genetically engineered Men govirus suggest these tracts have a bearing on virus pathogenicity (17, 18). Translational control of picornaviral RNA is mediated by the 5' noncoding regions. Several lines of evidence now indicate that unlike 5' capped mRNAs, internal ribosomal entry sites reside within these regions for most, if not all, picornaviruses (3, 77, 8 5) . Initiation of protein synthesis is generally quite efficient and, except for the aphthoviruses, proceeds from a unique start 5,!""__-----------vlral RNA genome

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Figure 2

Processing map of a picornavirus genome. Proteins and RNA are drawn to scale

according to the nucleotide sequence of EMCV . The poly(C) tract within the 5' noncoding region and the leader protein, L, are found only in cardio- and aphthoviruses. The polyprotein ofFMDV contains three 3B (VPg) segments, while all other viruses contain only one. The guanidine resistance marker (g') is a genetic locus affecting the action of a drug thought to block initiation of RNA synthesis. Protein 2A has been identified as a protease only in the renterovirus isolates. Nomenclature is that adopted in Reference 75.

607


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Rhlnoviruses Annu. Rev. Microbiol. 1990.44:603-623. Downloaded from www.annualreviews.org Access provided by Mahidol University on 02/07/15. For personal use only.

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Figure 3

3D

Cleavage sequences in picornavirus genomes. Cleavage sequences for prototype

strains (polio-1M, HRV-14, EMCV-R, FMDV-OIK, HAV-LA) are shown in boldface type at appropriate locations in the viral genomes. Strain abbreviations are according to Reference 61.

Alternate cleavage sequences from homologous isolates are indicated in lesser type. Protease 3C-catalyzed r(,actions are shown above each genome. The maturation cleavage is underlined and in italics. The primary reactions are in open faced letters. Sites in parentheses have not been definitively located. Asterisks designate sites cleaved where the identity of the proteolytic agent is

unclear. The L-IA site in aphthoviruses is autocatalyzed by protein L in an unknown mechanism.

site on each RNA. With FMDV, two in-phase AUG codons (separated by bases) initiate synthesis with equal frequency

84

(22).

The Viral Proteins Mature viral proteins are derived by progressive, posttranslational cleavage of the polyprotein (Figure

4). The full-length precursor is only rarely observed


608

PALMENBERG

experimentally because the initial processing events invariably occur while the peptides are still nascent on ribosomes. The individual protein molecular weights can vary somewhat for different isolates, but overall, the proteolytic cascade is quite similar among viruses (see below for exceptions). To simplify homolog identification, the European Study Group on the Molecular Biology of Picomaviruses (75) adopted a uniform nomenclature system, designated L-4-3-4 in 1983. Accordingly, mature proteins and their precursors are subdivided into four groups (L, PI, P2, P3) on the basis of structure, enzymatic function, and position of primary cleavages (refer to Figure 2). The leader or "L" proteins are present only in cardio- and aphthoviruses. The EMCV and Mengo leaders are about 7 kd in molecular weight. FMD viruses have two nested L peptides (16 kd and 23 kd), which share common carboxyl ends, but have different in-phase translational start sites (14, 22, 70). The four P I peptides are the capsid structural proteins, VPI, VP2, VP3, and VP4 ( l D, l B, lC, and lA), named in order of descending molecular weight on polyacrylamide gels (EMCV: 30, 28, 25 and 8 kd). Protein VPO

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Figure 4

Three phases of viral processing. Translation and processing events within the primary, secondary, and maturation stages of a Mengo infection are depicted. Arrows assign proteolytic responsibilities and monomolecular verses bimolecular reactivities.



609

(lAB), tht: uncleaved precursor of VP4+ VP2, can also be detected at trace levels in virions. Cell-free processing experiments have shown that capsid peptides that derive from a common precursor molecule stay together as a protomer unit throughout particle morphogenesis (9, 27, 58). The middle portion of the polyprotein yields peptides 2A, 2B, and 2C (EMCV: 16, 17, and 36 kd). FMDV genomes have very small or deleted 2A sequences when compared to the other viruses. The biological roles of the P2 peptides are currently under examination. The 2A and 2B components are discussed below in conjunction with their activities in the initial steps of polyprotein processing. Protein 2C is the probable genetic locus of the g uanidine resistance marker, a compound that affects the initiation of RNA synthesis (7, 69). However, 2C is not a polymerase, and its contribution to the replication cycle remains unclear. The P3 peptides, 3A, 3B, 3C, and 3D (EMCV: 10, 2, 22, and 51 kd) are more closely associated with genome replication. Purified preparations of 3D can catalyze elongation of nascent RNA chains in primer-dependent reac tions, an activity that identifies this enzyme as the central element of viral polymerase complexes (19, 20, 49, 86). Protein 3B is VPg, the p eptide attached to the 5' end of the genome (57). Aphthoviruses have three tandem I y linked different VPg sequences at this position, making the FMDV P3 segment somewhat longer than in other viruses (21). Initiation of positive and negative-strand RNA synthesis may require VPg, either as free protein, or as part of the donor peptide, 3AB (51). Protein 3C is a viral-specific protease, responsible for many posttranslational cleavage events (see below) (26, 63, 82). -

,

PRIMARY CLEAVAGE EVENTS The primary cleavage event within viral polyproteins is cotranslational, occurring as soon as a ribosome has reached the middle, or P2 region of the genome. Distinct processing sites and catalytic mechanisms are used by the various genera. The most thoroughly studied reactions are those of the polio 2A protease, which cleaves its nascent polyprotein at the PI-P2 junction. A high degree of primary amino acid identity intimates that polio 2A shares its functionality with other members of the renterovirus group, but the dis tinguishing catalytic sequences are not held in common with the aphthovi ruses, cardioviruses, or hepatitis-A viruses (see Figure 3). Rather, the car dioviruses (and probably aphthoviruses) seem to achieve efficient primary scission through use of a unique self-cleavage mechanism, dependent on an usually reactive tetrapeptide sequence spanning the 2A-2B junction. The required sequence is not present in hepatitis-A, and the mechanism and site(s) used for the initial cleavage in this virus have not been determined.

610

PALMENBERG


Primary Cleavage in Entero- and Rhinoviruses THE 2A PROTEASE Rapid, cotranslational scission of renterovirus polypro teins occurs at the (Y, T, H, F, A, V)-G dipeptide pair that marks the junction of the PI and P2 regions. The cleavage is catalyzed by amino acid sequences of the 2A protein as soon as the required elements are synthesized by a ribosome (79, 84). Release of 2A from the polyprotein is not prerequisite for this activity because processing occurs before the sequences of the 3C pro tease (responsible for 2A-2B cleavage) have been translated. Thus, the pri mary . scission is assumed to be monomolecular. When tested in cell-free extracts, however, purified 2A is also capable of trans (bimolecular) reactions on synthetic substrates that mimic the PI-P2 junction, or natural substrates derived from defined, cloned portions of the polyprotein that likewise encom pass this region (53). Antibodies directed against 2A block these in vitro reactions, confirming the identity of the reactive agent (84). Related experiments have also assigned catalytic responsibility for an alternate P3 region cleavage pathway (3C�3C' +3D') to protease 2A. The alternate cleavage, observed as a minor reaction in some renterovirus-infected cells, occurs at a (F, Y)-G dipeptide pair with similar context to the PI-P2 site (84). Because this cleavage can be blocked by site-directed mutagenesis without affecting apparent virus viability (45), the biological function of the alternate cleavage pathway, or its reaction products (3C' + 3D' ), are still indeterminate. Based on inhibitor studies and sequence similarities with viral 3C protease (see below) polio 2A was originally proposed to contain an active-site thiol group and belong, by analogy, to the group of cysteine-type proteases (8, 13, 84). However, further comprehensive analyses by Bazan & Fletterick (11) showed that while 2A-coding sequences are strongly conserved among all renterovrruses and probably do share a catalytic triad (Cys-109, His-20, Asp-38) reminiscent of cysteine proteases, the surrounding sequences more convincingly suggest structural homology with the class of small, bacterial serine-type proteases (e.g. subtilisin). HOST PROTEIN p220 CLEAVAGE Host cell-specific protein synthesis is rapidly shut off during renterovirus infection. The inhibition primarily results from degradative proteolysis of host protein p220, an integral part of the eIF-4F cap-binding complex necessary for translation initiation of eukaryotic capped (e.g. host) mRNA (47). That extracts from polio- or rhino-infected cells can also stimulate this cleavage led to the proposal that a viral-encoded enzyme might be the culpable agent (42). Similar experiments with cell-free extracts, exclusively expressing cloned portions of the P2 region of polio, focused the search upon 2A by demonstrating that engineered insertions or


611


deletions within 2A selectively eliminated an extract's potential for p220 cleavage (42). 2A itself, however, was clearly not the direct catalytic agent (46, 48). Rather, 2A is now suspected to induce p220 scission through activation .of one or more latent cellular proteases whose normal metabolic roles may include regulation of protein synthesis levels through controlled p220 prote:olysis (E. Wyckoss, D. Croall, & E. Ehrenfeld, submitted for publication). Specific identification of this (these) agents and the mechanism by which 2A can effect activation await further experimentation. Primary Cleavage in Cardio- and Aphthoviruses EVIDENCE FOR A UNIQUE EVENT Two lines of evidence indicate that cardioviral and aphthoviral 2A peptides are not functionally equivalent to the renteroviruses, and that analogous nascent cleavage activity for these isolates is necessarily located elsewhere in their genomes. First, the 2AB region of aphthoviruses is much shorter than in entero- or rhinoviruses, and sequence comparisons strongly suggest that the missing or deleted segment(s) corre sponds to peptide 2A (14,60,70,76). The remaining 15- (or 16--) amino acid segment of FMDV 2A does share a strong degree of sequence similarity with the carboxyl portions of the cardioviral 2A proteins, but these regions do not bear any visible relationship to the required and conserved catalytic triad of the renterovirus 2A protease (Figure 5) (A. Palmenberg, unpublished data). The second rational for an alternative mechanism is that nascent cleavage within cardio- and aphthoviral polyproteins actually occurs at a different site than in renteroviruses (2A-2B versus Pl-2A), and releases a much larger primary pro�cursor (L-Pl -2A versus PI) (28, 78). By analogy with polio, the first picornavirus to be sequenced, the primary site for EMCV cleavage was initially placed at a Q-G dipeptide pair located midway within the 2AB segment. The placement was consistent with dansylation experiments that suggested glycine as the amino-terminal residue of the EMCV 2B protein (62). However, while subsequent sequencing confirmed these amino acids to be conservl�d among other cardioviruses, their identity as the authentic 2A-2B cleavage site was incongruous with parallel data from the aphthoviruses. For FMDV, placement of the primary cleavage site at the homologous L-V sequence (see Figure 5) would necessarily create a 2A fragment much larger than the one actually observed (14, 70, 76). Instead, microsequencing more definitively located the primary site of FMDV-AI2 at an R-P sequence, 16 amino acids downstream from the ID-2A junction (70). Most other sequenced cardio- and aphthovirus strains maintain a G-P pair in the equivalent position. A NOVEL SELF-CLEAVAGE MECHANISM Still, relocation to the R-P (or G-P) site dlid little to clarify the primary mechanism used by these viruses.


RBNTEllOVIRUS 2A PROTEASE SEQUENCES

*

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tv

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Rhino-2

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Rhino-l4

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Polio-lS

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Cox-A2l

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lD / 2A

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Rhino-2

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Rhino-l4

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CAlmIO-

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AND

APBTBOVIRUS 2A SEQtlENCES

FMD-AlO

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FMD-Al2

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FMD-Cl

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FMD-Olk

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EMC-R

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Mengo

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TME-Da

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FMD-AlO

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*

FMD-AI2

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FMD-Cl

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FMD-Olk

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TME-Da

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2A / 2B

5

Primary processing reagents (Top) An amino acid alignment for prototype renterovirus 2A proteins is presented. Gaps ( ... ) have been inserted to maximize similarity among aligned residues according to the methods of Reference 61. Upper case letters denote positions where a majority of the sequences share the same or a closely related amino

acid. Locations of the ID-2A and 2A-2B cleavage sites are indicated. Putative catalytic residues are highlighted in boldface and emphasized with asterisks. (Bottom) Equivalent 2A alignments are presented for typical cardio- and aphthovirus sequences. The 2A-2B junction cleaved during primary scission is placed between the G-P residues, as suggested by the FMDV sequencing experiments described in the text. The N-P-G-P sequences upon which the synthetic peptides were modeled are highlighted in boldface.

.....

� � �



613

Unlike the renteroviruses, where 2A sequences are recognizable as those of a proteolytic enzyme, extensive pattern searches of cardio- and aphthovirus 2AB segments failed to detect identifiable catalytic motifs (A. Palmenberg, unpublished data). Even more puzzling, cell-free translation experiments showed that large segments of the 2A and 2B coding regions could be entirely deleted from engineered EMCV and FMDV RNA transcripts without effect ing cleavage activity in the remaining expressed peptide. The L, PI, 2C, and P3 regions of the genome are likewise dispensable for nascent activity in extracts from a wide variety of cells (e.g. rabbit reticulocyte, HeLa, wheat germ, and insect) (76; A. Palmenberg, unpublished data). The logical alternatives, that (a) some ubiquitous host protease was responsible for primary scission, or (b) that a small common portion of cardio- and aphthovirus polyproteins was autonomously catalytic, were puta tively resolved by the recent creation and testing of synthetic peptide se quences. By focusing on the (relatively) conserved segment spanning the 2A-2B junction, Peter Pallai (personal communication) and colleagues demonstrated that synthetic tetrapeptides containing the viral Asn-Pro-Gly Pro sequence (N-P-G-P) were spontaneously cleaved to Asn-Pro and Gly-Pro when incubated only in buffer. Although detailed mechanistic studies are still incomplete:, the astonishing simple autocatalytic reaction seems to be carried out most efficiently in slightly basic reaction mixtures (i.e. pH 8.0), as might be expected for an authentic physiological event. Considering this intrinsic activity, not surprisingly, N-P-G-P sequences prove rare amino acid combinations in nature. In picornaviruses, they are found at the 2A-2B junction of cardio- and aphthoviruses, but nowhere else. The pattern is uncommon even for published cellular databases (A. Palmen berg, unpublished data). Therefore, should further experiments confirm the autocatalytic properties of this unusual segment, a novel and probably unique self-cleavage mechanism can be proposed for primary scission in cardio- and aphthoviruses. Namely, during translation, these polyproteins literally break themselves at the 2A-2B junction by virtue of the inherently active N-P-G-P sequences. Clearly, further study is warranted before general establishment of this proposal. In addition to testing potential contributions of other adjacent sequences, it will be necessary to carefully determine whether the primary site in vivo is indeed at the P-G pair, or one residue away (G-P) as was measured for FMDV-A12. SECONDARY CLEAVAGE EVENTS Release of PI or L-PI-2A from the growing peptide chain is only the first step in picornaviral processing. Most subsequent or secondary cleavages are effected by viral protease 3C, the central enzyme in the cleavage cascade.

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This enzyme catalyzes a specific and complicated series of monomolecular and bimolecular scissions within its own polyprotein to the (near) exclusion of exogenous reactions with cellular proteins. In FMDV however, at least one secondary cleavage is carried out by another viral protease, the amino tenninal leader (L) protein. The eventual result of the secondary cascade is release of all mature viral proteins necessary for establishment and completion of a successful infectious cycle.


The 3C Protease 3c ACTIVITY

With three exceptions (primary cleavage, maturation cleav age, and leader-catalyzed cleavage in FMDV), 3C is responsible for all defined picornavirus processing reactions. The enzyme was originally identi fied by its activity in reticulocyte extracts programmed with EMCV RNA (26, 63, 68, 82), but has since been isolated for many viruses from diverse natural and synthetic sources (e.g. infected cells or genetically engineered expression systems). The strong pattern of similarities shared by all viral strains suggests that 3C mechanisms are probably universal, even for those isolates where the enzyme has not been specifically studied. But since 3Cs do tend to exhibit marked preference for homologous substrates, individual peculiarities of these cleavages may also be unique for each virus. Excellent references summarizing 3C isolation and activities have recently been published (31, 41,

44).

As a common scheme, 3C frees itself and its related precursors through progressive autocatalytic reactions. The order and rate of these events in vivo has not been precisely measured, but as the observation of an intact P2-P3 protein is always rare, the 2B-2C and 2C-3A bonds are probably particularly susceptible to catalysis and are cleaved shortly after synthesis (35, 78). Scission at the 2A-2B site to release protease 2A in renteroviruses is also carried out by 3C, most probably at a time point similarly early in the series (29). After separation from P2, the P3 precursor is further cleaved into smaller, more stable products. Numerous experiments have confmned these reactions to depend on the availability of active 3C. In cell-free extracts programmed with EMCV RNA, P3 reactions are dilution insensitive, implying a mono molecular mechanism (64). But if the internal catalytic sequences are de stroyed through mutation, processing can also occur in a bimolecular pathway when functional 3C is exogenously supplied (65, 87). The cleavage between 3A-3B is only rarely observed in vitro, in either monomolecular or bimolecu lar assays, unless all other processing routes are blocked (65). Presumably in vivo, these reactions take place in conjunction with VPg addition to RNA during replication initiation, and under these conditions may be facilitated by the presence of other proteins, or by particular configurations of the partici pants.



615

The PI and L-PI-2A precursors released by primary cleavage are also substrates for 3C processing. These reactions are efficient, and commonly used in cell-free assays to monitor protease activity. All EMCV 3C containing P3 proteins (i.e. P3, 3ABC, 3CD, 3C) possess PI reactivities (65). They process the substrate sequentially at L-VPO, VP I-2A, VP3-VPl , and then VPO-VP3, although blockage at any point in the series does not necessar ily inhibit subsequent steps (63, 67). In contrast, responsibility for renterovi rus PI sites appears to reside with different forms of the enzyme. The VP3-VPl junction in polio, for example, can be processed by purified 3C, but cleavage between VPO-VP3 of the same precursor requires 3CD (37, 87). The mUltiple activities of P3 precursors intimate that 3C and its zymogens should be viewed as a family of protease species rather than a single entity. Perhaps by apportioning cleavage responsibilities to different enzyme forms, picoma viruses can prudently and efficiently modulate the processing cascade to release th{� desired proteins at the required times throughout the infectious cycle. 3C CLEAVAGE SPECIFICITY Natural 3C reactions occur only at certain sites in the polyprotein. Completion of the first polio sequence (type 1 Mahoney) conveyed the relatively simple impression that acceptable locations might merely be defined by the position of Q-G pairs (29, 39). As other viruses were sequenced, however, this stringency became exceptional. Sites with a variety of other amino acids are commonly found in cardio-, aphtho-, and rhinovirus es and specific substitution experiments established the cleavable repertoire to be even larger than appears in a given polyprotein (see Figure 3) (44, 60). Besides the natural Q-G, Q-S, and E-S pairs, the EMCV 3C can process Q-A and Q-C sequences. While other sites have proven inactive for this particular enzyme (Q-T, Q-I, Q-V, Q-E, Q-Y, K-G, K-A, K-V, K-E, R-G, L-G, and P-G), prot eases from different viruses can apparently recognize and cleave them (e.g. Q-T and Q-I are authentic sites in FMDV) (14, 65, 70). To compound the confusion over selection criteria, not all apparently correct sequences within any virus are actually recognized and processed (29). What then, constitutes an appropriate 3C site? Understandably, primary sequence does play a major role. Most sites are of the pattern.: (E, Q)-(G, S, A), although there are many exceptions. Specificity clearly also extends to elements from the surrounding sequences. Renterovi rus polyproteins usually have aliphatic residues (e.g. Ala) in the P4 position, while cardioviruses seem to prefer Pro in the P2 or P2' location (54, 62). Even within these contexts, a site must additionally be correctly displayed. Extrapolations from structural data suggest that authentic 3C sites are typical ly presented in flexible, tum-coil surface configurations (9, 52, 56). Nonsur face sequelllces buried in beta sheets or helices are not as efficiently recog nized (32). Synthetic insertions or deletions in proteins can destroy a site's

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activity, or alternatively, create a new site elsewhere. Since most cellular proteins are left untouched during viral infection (40, 54), the 3C enzyme's finicky requirements for substrate sequence and topology undoubtedly create its high degree of tropism for homologous polyproteins. 3C STRUCTURE All 3C processing reactions are sensitive to thiol inhibitors (e.g. N-ethylmaleimide, Zn+, iodoacetamide). Thus, the protease and its precursors were originally categorized as cysteine-type agents (8, 25, 68). Nevertheless, as with the renterovirus 2A proteases, refined sequence com parisons now show that picornavirus 3Cs are probably more closely homologous to serine proteases and, more specifically, bear the strongest putative structural resemblance to the large class of serine enzymes like trypsin and chymotrypsin (11, 13, 23, 24). Genetic engineering (34) and crystallographic models (23) anticipate a catalytic triad composed of Cys-147, His-40, and D-85 (in polio), though confirmation of this active site must necessarily await actual resolution of an atomic structure.

Leader-Catalyzed Cleavage in FMDV Leader proteins, preceding the PI region, are unique to cardio- and aphthovi ruses. In EMCV and TMEV, scission between L and PI is catalyzed by protease 3C as the first step in the L-Pl -2A cascade (66, 71). With FMDV, however, "the equivalent cleavage is carried out by the leader protein itself, in a still undefined reaction (76, 80). This occurrence is the only picornaviral secondary processing event that is not catalyzed by 3C or by one of its precursors. Evidence has also been presented that the autonomous FMDV L activity may additionally serve as a p220ase enhancer in a manner analogous to renterovirus 2A (16). Details of these reactions remain unclear.

MATURATION CLEAVAGE The final cleavage within picornaviral polyproteins, maturation processing of the lAB peptide (also called VPO), is not catalyzed by any of the other identified viral proteases. The phenomenon is considered universal among picornaviruses, though a putative hepatitis-A VP4 peptide (only 23 amino acids long) has never been specifically isolated (10, 61, 83). Maturation reactions are normally observed in vivo only during the final stages of virion morphogenesis and are believed to be concomitant with RNA association into large capsid assembly structures (36). An agent responsible for VPO pro teolysis has never been isolated, but crystallographic resolution of several virion structures has permitted speculation on the nature of this event (9, 59, 72). For example, within each capsid subunit of rhinovirus-14, the side chain of a VP2 ( l B) serine residue is positioned near, and points toward the carboxyl

617



VP4 FMD-AIO FMD-Olk FMD-Sat3 EMC Mengo TME-BeAn HA-LA Polio-IS Cox-A2l Cox-B1 Cox-B4 Ev-70 BEV SVD Rhino-2 Rhino-14

Structure

Figure 6

••••COOH

tntqnndwFsklassaFtglfgalLa tntqnndwFsklassaFsglfgalLa nntqnndwFsklaqsaIsglfgalLa yg.qfsnlFsg.avnaFsnml.plLa yg.qfsnlLsg.avnaFsnml.plLa ng.qlsniLgg.aanaFatma.plLl ........................ La . . . . . . • kFte.pikdVliktspmLn ....... kFte.pvkdLmlktapaLn ....... kFte.pvkdlmiksmpaLn ....... kFte.pvkdVmikslpaLn ....... kFte.pvaealkagapvLk .......kFtq.piadViketavpLk .......kFte.pvkdlmvksmpaLn .......kFtd.pvkdVlekgiptLq ....... kFte.pvkdLmlkgapaLn

NH2•• • •

*

vp2

*

dkktEettlleDRIlttrnGHttstTQsavgv dkktEettlleDRIlttrnGHttstTQsavgv dkktEetthleDRIlttrhntttstTQsavgv dqntEemenlaDRVsqdtaGNtvtnTQstvgr dqntEemenlaDRVsqdtaGNtvtnTQstvgr dqntEemenlaDRVasdkaGNsatnTQstvgr dieeEqrniqsvDRtavtgasyftsvdQssvht spniEa.cgyaDRVlqltlGNstitTQeaans spnvEa.cgyaDRVrqitlGNstitTQeaana spsaEe.cgyaDRVrsitlGNstitTQecanv sptvEe.cgyaDRVrsitlGNstitTQecanv spsaEa.cgyaDRVlqlklGNssivTQeaani spsaEa.cgysDRvaqltlGNstitTQeaani spsaEe.cgyaDRVrsitlGNstitTQecanv sptvEa.cgyaDRliqitrGDstitsQdvana spnvEa.cgyaDRVqqitlGNstitTQeaana

lA / IB

bbbAIbbbbbbbbA2bb

The: VPO cleavage site. (Top) Amino acid sequences near the VP4/vP2 cleavage site

for 17 strains of picomaviruses have been aligned by computer analysis as described in Refer ences 61 and 59. The bottom line (bbb) shows beta-Allbeta-A2 amino acids of rhino-14. Serines (S) in boldface are putative catalytic residues. (Bottom) The probable conformation near the cleavage site between VP4 and VP2 of rhino-I 4 is depicted schematically. The complete structure is described in Reference 72. This figure is adapted from Reference 59.

end of its homologous VP4 ( l A) peptide chain. Refined crystallographic data suggest that these amino acids are linked by hydrogen bonds (9). Aspartate and arginim: residues are located near the serine and an adjacent beta-pleated sheet (beta- Allbeta-A2) helps orient and stabilize the region. Figure 6 illus trates the relevant portion of the rhinovirus structure. The spacial arrangement evokes analogy to the active site conformations found in normal serine proteases such as trypsin and chymotrypsin (43, 81). However, while these enzymes usually also have a proton-accepting histidine residue located near


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the active serine, this feature is mlssmg from the viral structures. The histidine-equivalent position in the serine protease-like active site is instead occupied by viral RNA, packaged inside the capsid. This configuration led to the hypothesis that during virion assembly, the RNA may act in place of histidine, accepting protons, and thereby help to catalyze VPO cleavage (9, 59, 72). A proposed mechanism for VPO processing needs to be compatible with and accountable for differences (sequence or structural) among the viruses, but the hypothetically active serine at the amino terminus of the hairpin is found only in the rentero- and cardiovirus strains. The first VP2 serine residue in FMDV is part of a Gln-Ser-Ser-Val sequence, which is common also to the hepatitis-A strains. The equivalent rhino-14 sequence, Gln-Glu-Ala-Ala, is spatially located at the carboxyl end of the beta-Allbeta-A2 structure. In other words, every picornavirus does display a serine at either the extreme carboxyl or amino end of the VP2 beta-hairpin. If these amino acid positions (bold faced in Figure 5) could be considered functionally equivalent in a proteolytic mechanism, either could theoretically catalyze the VPO cleavage because the processing site between VP4 and VP2 would always be in direct proximity of one of these relevant side chains. Though a RNA-aided mechanism for VPO cleavage is compelling in its simplicity, there is still very little experimental evidence to support this hypothesis. The resolved virion structures of polio and FMDV, unlike those of rhino and Mengo, place the respective carboxyl ends of VP4 at some distance from the putative catalytic serines (1, 32). Though these arrange ments could potentially have been created after VPO cleavage by shifting of the respective termini, it is also possible that a heretofore undetected mech anism is actually responsible for maturation processing before, during, or after particle assembly.

REPASS AND SUMMARY Proteolytic processing is a distinguishing feature of the picornavirus life cycle. Though the RNA genomes are effectively monocistronic, the three tiered cascade of primary, secondary, and maturation cleavages efficiently produces the spectrum of viral proteins necessary for a productive infection. The proteases themselves represent an eclectic collection of catalytic entities. Some, like 3C, or the renterovirus 2A proteins, may be adaptations from cellular proteases. Sequence comparisons, tracing the footprints of evolution, suggest these may have derived from the large and small classes of serine proteases, respectively. Likewise, the L protease of FMDV, whatever its mechanism, may eventually prove to be some captured host enzyme, trans formed to the needs of the virus. The putative RNA-aided VPO catalysis, and


PICORNAVIRUS PROCESSiNG

619

the autocatalytically reactive N-P-G-P sequences of cardio- and aphthovirus es, however, have no known counterparts in other eukaryotic or prokaryotic biological systems. Whether these activities were invented by picomaviruses, or whether they, too, represent gross modifications of (primitive?) cellular enzymes must await further resolution of the mechanisms. Nevertheless, it is interesting that all the reactive agents definitively associ ated with picomaviral processing are encoded by viral genomes (2A, N-P-G P, L, 3C, and VPO). No cellular components are required, or at least, none have yet been isolated. Enzymatic self-sufficiency must clearly be an advan tage to a highly mobile pathogen, and may explain in part why the picomavi ruses can efficiently infect a diverse variety of hosts with virulence and such obvious success. On the other hand, the finely tailored catalytic mechanisms upon which infectivity depends may ultimately provide the very weapons with which to fight these virus diseases. Anti-protease therapies would clearly have unique targets in the highly adapted and specific viral activities. Un fortunately, the prophylactic value of these approaches is still highly specula tive. The above-cited work is intended to outline the principal elements of the viral processing scheme and the mechanistic constituents by which proteolysis contributes to regulation of the viral life cycle. Who knows? With further advances there may yet be a cure for the common cold.

ACKNOWLEDGMENTS I thank my many colleagues in the laboratories of Drs. Eckard Wimmer, Bert Semler, Ellie Ehrenfeld, Andrew King, and Ben Dunn for supplying me with preprints and reviews of their work before publication. Special thanks to Dr. Peter Pallai for allowing me to cite his novel and exciting N-P-G-P ex periments. This work is supported by NIH grant AI-I733I to A. C. Palmen berg. Literature Cited I. Acharya, R., Fry, E., Stuart, D., Fox,

G., Rowlands, D., Brown, F. 1989. The three-dimensional structure of foot-and mouth disease virus at 2.9 A resolution. Nature 337:709-15 2. Ahlquist, P., Kaesberg, P. 1979. De termination of the length distribution of poly(A) at the 3' terminus of the virion RNAs of EMC virus, poliovirus, rhino virus, RAV-61 and CPMV and of mouse globin mRNA. Nucleic Acids Res. 7:1195-1204 3. Alsaadi, S., Hassard, S., Stanway, G. 1989. Sequences in the 5 ' non-coding region of human rhinovirus 14 RNA that affect in vitro translation. J. Gen. Viral. 70:2799-2804

4. Ambros, V., Baltimore, D. 1978. Pro tein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine. J. Bioi. Chem. 253:526366 5. Ambros, V., Baltimore, D. 1980. Purification and properties of a HeLa cell enzyme able to remove the 5' ter minal protein from polioviral RNA. J. Bioi. Chem. 255:6739-44 6. Ambros, V., Pettersson, R. F., Balti more, D. 1978. An enzymatic activity in uninfected cells that cleaves the linkage between poliovirion RNA and the 5' ter minal protein. Cell 5:1439-46 7. Anderson-Sillman, K . , Bartal, S., Ter shak, D. 1984. Guanidine-resistant

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24. Gorbalenya, A. E., Dochenko, A. P., Blinov, V. M . , Koonin, E. V. 1989. Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases: A distinct protein su perfamily with a common structural fold. FEBS Lett. 243 : 103-1 4 2 5 . Gorbalenya, A. E., Svitkin, Y . V. 1983. Protease of encephalomyocarditis virus: purification and role of the SH groups in processing of the structural proteins pre cursor. Biochemistry (USSR) 48:385-95 26. Gorbalenya, A. E . , Svitkin, Y. V., Kazachkov, Y . A., Agol, V. I . 1979. Encephalomyocarditis virus-specific polypeptide p22 is involved in the pro cessing of the viral precursor polypeptides. FEBS Lett. 108: 1-5 27. Grigera, P., Vasquez, C., Palmenberg, A. C. 1 985. Foot-and-mouth disease virus capsid proteins VPO, VPl and VP3 synthesized by in vitro translation are the major components of 14S particles. Acta Viral. 29:449-54 28. Grubman, M. J . , Baxt, B. 1 982. Translation of foot-and-mouth disease virion RNA and processing of the pri mary cleavage products in a rabbit retic ulocyte lysate. Virology 1 16: 1 9-30 29. Hanecak, R . , Semler, B . L . , Anderson, C. W., Wimmer, E. 1 982. Proteolytic processing of poliovirus polypeptides: Antibodies to a polypeptide P3-7c in hibit cleavage at glutamine-glycine


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Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity.

Spleen necrosis virus gag polyprotein is necessary for particle assembly and release but not for proteolytic processing.

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Proteolytic processing of human lysosomal arylsulfatase A.

Sequence-derived structural features driving proteolytic processing.

Protease required for processing picornaviral coat protein resides in the viral replicase gene.

Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line.

Acidotropic amines inhibit proteolytic processing of flavivirus prM protein.

Proteolytic processing of thyroglobulin by extracts of thyroid lysosomes.

Proteolytic events in the processing of secreted proteins in fungi.

Proteolytic processing of chromogranin A in cultured chromaffin cells.

ERVK polyprotein processing and reverse transcriptase expression in human cell line models of neurological disease.

Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing.

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit.

Citrullination and proteolytic processing of chemokines by Porphyromonas gingivalis.

MCP-1 by meprins.

The VE-cadherin cytoplasmic domain undergoes proteolytic processing during endocytosis.

Monitoring proteolytic processing events by quantitative mass spectrometry.

Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor.

Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5.

Cell-free translational analysis of the processing of infectious pancreatic necrosis virus polyprotein.

Triatoma virus structural polyprotein expression, processing and assembly into virus-like particles.

The 5'-untranslated region of picornaviral genomes.

Prokaryotic polyprotein precursors.