VIROLOGY

191,

309-320

(1992)

Alternate

Poliovirus

Nonstructural Protein Processing Cascades Primary Sites of 3C Proteinase Cleavage

Generated

by

MARK A. LAWSON’ AND BERT L. SEMLER’ Department

of Microbiology

and Molecular

Genetics, College of Medicine,

Received March

University

of California.

Irvine, California

92717

19, 1992; accepted July 28, 1992

The post-translational regulation of picornavirus gene expression mediated by the cascade processing of viral proteins is not well understood. Both pulse-chase studies of infected cells and in vitro studies of the translation of poliovirus type 1 RNA transcribed from genomic cDNA clones indicate a specific cascade of polyprotein processing in which the Pi, P2, and P3 precursor proteins are primary products of viral proteinase cleavage. We report the results of a short-time kinetic analysis of poliovirus type 1 protein processing in an in v&o translation system and in infected HeLa cells which indicate the existence of another, rapid pathway of polyprotein processing mediated by the activity of the 3C proteinase. The observed pathway is distinct from and in addition to the one previously known. The potential role of this alternative pathway of processing in the post-translational regulation of viral gene expression is discussed. Q 1992 Academic

Press,

Inc.

INTRODUCTION

fected cells (Summers eta/., 1965; Jacobson and Baltimore, 1968; Holland and Kiehn, 1968) it has been held that the specific cascade of polyprotein processing within the infected cell could serve to regulate viral gene expression. However, only one specific example of processing-mediated regulation of viral protein activity has been described (Jore et al., 1988; Ypma-Wong et a/., 1988). Because in theory picornavirus genomes are committed to direct the synthesis of equimolar amounts of viral protein in the infected cell, alternative processing is a potential avenue for regulating gene function. Such a mechanism of gene regulation has been described for the alphavirus Sindbis virus. Recent studies of the processing ability of various Sindbis virus proteins containing the proteolytic region of nsP2 have made it clear that the differences in the ability of nsP2 and its precursors to recognize other viral proteins as substrates serve to influence the processing cascade and to provide a mechanism of temporal regulation of protein processing (de Groot et al,, 1990). The proteolytic cascade of encephalomyocarditis virus (EMCV), a picornavirus of the related genus cardiovirus, is the best defined among the picornaviruses and has been examined in infected cells (Butterworth and Rueckert, 1972b) as well as in vitro (Jackson, 1986, 1989b). Salient features of the EMCV processing cascade include the rapid, coordinated cleavage by 3C of the 2B/2C and 2C/3A protein junctions and the early appearance of a 3ABC protein prior to any detectable P3 (Jackson 1986, 1989b). The processing patterns reported in the in vitro studies indicate that the proteolytic activity of the EMCV 3C works most efficiently in an intramolecular fashion (Palmenberg

The Picornaviridae represent a large class of clinically and economically important RNA viruses. The picornavirus genome is a single-stranded, positivesense RNA molecule which encodes a large polyprotein that is processed by virus encoded proteolytic enzymes after synthesis by the host-cell translational apparatus. Among the various genera of the Picornaviridae, distinct patterns of viral polyprotein processing are readily discerned in infected cells. The variety of precursor and end-product proteins detected in cells infected with picornaviruses does not vary greatly among individual members of a particular genus (Holland and Kiehn, 1968). In poliovirus type 1 (PVl), the prototypic member of the genus enterovirus, there are potentially 66 distinct proteins produced by cleavage of the polyprotein by the proteolytic activities of the virus-specified 2A and 3C proteinases (refer to Fig. 1). The number of potential proteins is increased to 91 if the rare cleavage events within 3C (mediated by the proteolytic activity of 3C) and 3D (mediated by the proteolytic activity of 2A) are included (Pallansch et a/., 1984; refer to Fig. 1). In contrast to this theoretical potential for cleavage products, only proteins which arise from the Pl , P2, and P3 precursors are normally seen in high levels within cells infected by PVl . Since the first description of the large number of viral proteins and their post-translational processing in poliovirus-in’ Present address: Department of Reproductive Medicine, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093. 2 To whom reprint requests should be addressed. 309

0042.6822/92

55.00

Copyright 0 1992 by Academic Press, Inc All rights of reproduction in any form reserved

310

LAWSON AND SEMLER VP9 PVlRNA

743 I IA l

73To Poly(A) wvvb IB

IC .

ID .

2A 28 n

.

.

2C

38 3A 3C I .m..

i 3D n

FIG. 1. Genomic structure and polyprotein processing map of PVl The single strand RNA genome of poliovirus is shown by the solid line flanked by the genome-linked protein VPg (38) at the 5’end of the 743 base non-translated region and the 71 base 3’ non-translated region and polyadenylate tract. The 247.kDa polyprotein encoded by the PVl genomic RNA is shown below the genomic diagram. Map positions of the encoded structural Pl and the non-structural P2 and P3 region proteins are indicated with the cleavage sites. Solid vertical lines indicate sites of cleavage within the polyprotein. A dashed vertical line indicates infrequent cleavage at that site. The solid triangles (A) below the polyprotein indicate Q-G amino acid pairs cleaved specifically by the proteolytic activity of the viral protein 3C. Empty triangles (A) indicate sites cleaved by the Y-G-specific proteolytic activity of the 2A protein. A third proteolytic activity not yet ascribed to any viral or host cell protein mediates the cleavage between the 1A and 1 B proteins.

and Rueckert, 1982) possibly as part of a nascent polyprotein, but that some variation in the primary 3C-catalyzed cleavage event exists. These results are corroborated by the demonstration that BC-containing P3 precursors are efficient processing enzymes compared to 3C alone (Parks et al., 1989). The enterovirus PVl, although not investigated as completely as EMCV with respect to the processing cascade, follows a cascade of processing events which is different in some ways. Both pulse-chase studies of infected cells and in vitro studies of the translation of PVl RNA transcribed from cDNA clones indicate a cascade of polyprotein processing in which the Pl , P2, and P3 precursor proteins are primary products of viral proteinase cleavage (reviewed in Lawson and Semler, 1990). The rapid 2A-mediated cleavage of the Pl-2A junction has been well defined (Toyoda et al., 1986; Ypma-Wong and Semler, 1987a,b). The cleavage of the P2/P3 junction is the primary BC-mediated cleavage event inferred by the rare presence of proteins which can be identified as possessing both P2 and P3 region domains in infected cells or in in vitro translations of RNA transcribed from cDNA clones of PVl (Pallansch et al., 1984; Ypma-Wong and Semler, 1987a). Although the typical conversion of the polyprotein to Pl, P2, and P3 precursors does not allow for proteins of viral origin to possess both P2 and P3 region proteins, the processing of chimeric picornavirus genomes (Dewalt et a/., 1989) of polyproteins possessing chimeric 3C proteinases (Lawson eta/., 1990) and of polyproteins containing mutations of 3C (Lawson and Semler, 1991) has shown that proteins containing both 2C and 3D epitopes can be identified in in vitro translations of transcribed viral cDNAs, and that these proteins are indeed capable of being processed to normal viral proteins by wild-type 3C activity. The presence of proteins found in the above experimental systems of molecular mass less than a full-size P2-P3 fusion protein but which contain 2C and 3D epitopes is explicable by the ability of the PVl polyprotein to be processed in a manner other than through strict Pl,

P2, and P3 precursor generation. A more thorough investigation of the synthesis and processing of the PVl polyprotein both in vitro and in infected cells was thus undertaken. The results of the studies presented in this paper suggest that there are at least two sites in the polyprotein for the 3C proteinase to utilize as the primary site of action and that the initial choice of cleavage generates alternate cascades of polyprotein processing. The mechanisms by which alternative polyprotein processing may serve to regulate viral gene function are discussed. MATERIALS

AND METHODS

Cells and virus The Mahoney strain of poliovirus type 1 was used in pulse-chase and pulse-labeling studies of infected cells. A P4 stock of twice plaque-purified virus was used for all studies described. Uninfected HeLa S3 cells were used to prepare crude cytoplasmic extracts (Brown and Ehrenfeld, 1979) for supplementing rabbit reticulocyte lysate in in vitro translation reactions. In vitro transcription

and translation

Transcription of the PVl cDNA clone pT7-1 (YpmaWong and Semler, 1987a) using bacteriophage T, RNA polymerase and quantitation of product RNA were performed as described (Lawson et a/., 1990). The in vitro translation of transcribed RNA was performed in a rabbit reticulocyte lysate/HeLa cell translation system (Ypma-Wong and Semler, 1987a) as described (Lawson et a/., 1990) with modifications noted in the text and Figure legends. Products of in vitro translation reactions were diluted 1O-fold in Laemmli sample buffer (Laemmli, 1970) and boiled for 3 min prior to analysis on polyacrylamide gels. Products of pulse-labeling, pulse-chase, and in vitro translation experiments were analyzed on 12.5% SDS-polyacrylamide gels. Gels were fluorographed with 2,5-diphenyloxazole (National Diagnostics) and exposed to Kodak XAR-5 film to visu-

POLIOVIRUS

alize radiolabeled proteins. Fluorograms were scanned with an LKB Ultrascan 2 laser densitometer. Identification proteins

of [YG]methionine-labeled

PVl

Proteins synthesized in vitro or labeled in infected cells with [35S]methionine were immunoprecipitated using rabbit antiserum directed against the viral proteins 2C (Hanecak et a/., 1982) and 3D (Semler et al., 1983) as previously described (Semler et al., 1982, 1983). lmmunoprecipitated products were analyzed on 12.5% SDS-polyacrylamide gels as described above. Quantification of immunoprecipitation efficiency of P2 proteins with the 2C antiserum revealed a decrease in the ability to precipitate P2. The loss of approximately 2096 of the P2 protein in the immunoprecipitation is compensated by the correction factor for methionine content of P2 (18 methionyl residues), 2BC (14 residues), and 2C (13 residues). Thus the ratios of P2:2BC in the immunoprecipitated lanes are representative of the amounts present in the in vitro and infected cell analyses. Pulse-chase cell proteins

and pulse-labeling

analysis

of infected

Pulse-chase and pulse-labeling experiments were performed essentially as described in Blair et al. (1990) with modifications as noted. Briefly, cells were grown to a density of 5-6 X 1O5 cells ml-’ and harvested by centrifugation. The cell pellet was washed once with Earle’s salts and resuspend in methionine-free medium at a density of 5 X 1O6cells ml-‘. Cells were then infected at an m.o.i. of 50. Virus was allowed to adsorb to cells for 30 min at room temperature with stirring prior to the addition of fetal calf serum to lOq/o and incubation at 37” with stirring. At the onset of the experiment cells were pulsed with 100 &i of [35S]methionine (>lOOO Ci/mmol) and approximately 4 X lo6 cells were removed, quickly adjusted to 5 mM ZnSO,, and placed on ice. The preparation of [““S]methionine-labeled subcellular membranous and soluble fractions was performed as described (Takegami et a/., 1983). The membranous pellet derived from subcellular fractionation was resuspended in a volume equal to that of the supernatant soluble fraction from which it was pelleted. Fractions were diluted in Laemmli sample buffer (Laemmli, 1970) before immunoprecipitation according to a previously described method (Semler et a/., 1983). RESULTS Analysis

of processing

311

PROTEIN PROCESSING

in vitro

To investigate the occurrence of a cascade other than the well-described generation of Pl -P2-P3 prod-

ucts in PVl polyprotein processing, a kinetic analysis of the synthesis and processing of the polyprotein encoded by the wild-type cDNA clone pT7-1 was undertaken. ln vitro translation reactions were initiated with RNA transcribed from the wild-type cDNA clone pT7-1 and aliquots of the reaction were removed at the times indicated in Figs. 2 and 3. After removal from the reaction, the aliquots were immediately incubated with RNase A and cycloheximide for three minutes and diluted in Laemmli sample buffer (Fig. 2A). Alternatively, samples were treated as above with cycloheximide and RNAse and then allowed to incubate at 30” until the end of the time course (Fig. 2B). The transition time of ribosomes across the entire PVl RNA is approximately 30 minutes in the assay displayed in Fig. 2, as deduced by the appearance of complete P3 precursor protein in Fig. 2A at that time point. Processing

of Pl in vitro

The Pl capsid precursor protein is produced rapidly during translation of the viral RNA by the activity of the 2A proteinase, which cleaves at the 2A amino terminus while the polyprotein is in the nascent state (Toyoda et a/., 1986). Although Pl precursor is visible at 25 min into the in vitro translation reaction, the processing of Pl is delayed until approximately 70 min (refer to Fig. 2A). This delay is significant considering that 3CD protein [the enzyme responsible for Pl processing in PVl (Jore et al., 1988; Ypma-Wong et al,, 1988)] is detected as early as 50 min into the reaction. The delay in Pl processing is exacerbated in translation reactions which have been treated with the translation initiation inhibitor edeine after an initial incubation time of 20 minutes (Fig. 3A). Although 3CD protein is detected at 55 min and Pl protein is present throughout the time course, no significant Pl processing occurs. Processing

of P2 in vitro

The P2 region of the PVl polyprotein contains the 2A protein, which is partially responsible for the shutoff of host-cell macromolecular synthesis (Krausslich et al., 1987; Lloyd et a/., 1987) and the 2B and 2C proteins which are likely to play a role in replication of the viral genome (Pincus et al., 1986; Li and Baltimore, 1988; Bienz et a/., 1990; Johnson and Sarnow, 1991). The processing of the P2 region proteins in the translation presented in Fig. 2 is very rapid. The P2 and 2BC proteins begin to appear at the 35-min time point, coincident with the appearance of complete P3 products. The appearance of 2C protein is delayed until 50-55 min. Evaluation of the appearance of P2 and 2BC proteins in Fig. 2 was difficult due to the high level of background proteins being synthesized in the ongoing

312

LAWSON AND SEMLER

B M

PlP33CD-

PlP33CD-

P2-

P2-

2BC-

2BC-

lAB-

1 AB-

25

30

35

40

45

50

55

60

70

60

90

120 180

1 c-

2A2A-

3AB3AB-

FIG. 2. Kinetics of pT7-1 -directed protein synthesis and processing in v&o. ln vitro translation reactions were conducted as described (Lawson et al., 1990). Aliquots of an ongoing translation reaction were removed at the times indicated above the lanes, treated with RNase A at 500 pg ml-’ and cycloheximide at 5 pg ml-’ for 3 min and further treated as follows: (A) Aliquots were diluted immediately in Laemmli sample buffer. This treatment will show the extent of protein synthesis at the indicated time points. (B) Aliquots were allowed to incubate for the duration of the time course and then diluted in Laemmli sample buffer. This analysis will allow the proteolytic activity present in the reaction at the time the aliquot was removed to fully process any polyprotein. The poliovirus marker proteins (M) were generated by pulse-labeling HeLa cells infected at an m.o.i. of 50 from 4 to 6 hr postinfection with [35S]methionine.

translation reaction and the presence of processed Pl-derived proteins. However, the addition of the translation initiation inhibitor edeine after allowing translation to take p,lace for 20 min increased resolution by allowing only those protein products initiated during that initial 20-min period to continue to be elongated and processed (Fig. 3). The edeine treatment also inhibited Pl processing. The visualization of P2 proteins was further enhanced by immunoprecipitation of 2C-containing proteins from the translation reaction with 2C-directed antiserum, thereby removing the background of non-P2 proteins. The P2, 2BC, and 2C proteins as well as any precursor proteins containing 2C epitopes are detected by this treatment (Fig. 3B). The fluorogram of such an immunoprecipitation presented in Fig. 3B shows that the 2BC protein is present at 35-40 min into the reaction but detectable levels of both the P2 and 2C proteins are delayed by an additional 5 min. The delay in the appearance of P2 protein is not consistent with the classical Pl -P2-P3 cascade of processing assumed for PVl . If P2 was rapidly processed upon

synthesis, P2 protein would not be detected at significant levels at any time during translation. If 2BC was produced only as a direct product of P2, it would not be detected prior to P2. Densitometric analysis of fluorograms of anti-2C immunoprecipitated products reveals that the initial production of 2BC is three times greater than the production of P2. The half-life of P2 in the in vitro translation system used in these studies has been found to be approximately 65 min (Lawson and Semler 199 1). The low processing rate of P2 in vitro suggests that the high level of 2BC detected early in the kinetic assay is due to the initial production of 2BC rather than production of 2BC from P2. Processing

of P3 in vitro

It was of interest that the processing of P3 proteins in the short-time analysis presented here is not substantially different than that observed in previous in vitro analyses (Ypma-Wong and Semler, 1987a; Lawson and Semler, 1991). The P3 protein is predominantly processed to 3CD and 3AB in the in vitro translation

POLIOVIRUS

PR(ITE IIN PR:OCESSING

M

20

25

313

30

35

40

45

50

55

60

SO

-F

PiP33CDP2PZ2BC-

ZBC1 AB-

2c-

2cID-

lC-

2A-

3AB-

FIG. 3. Single-round translation of pT7-1 directed protein synthesis and processing in vitro. Translation reactions programmed with RNA transcribed from pT7-1 were performed as in Fig. 2 with the exception that the translation initiation inhibitor edeine (a gift of Richard Jackson) was added to a final concentration of 8 m/L?. (A) Aliquots were removed from the reaction at the times indicated and treated as in Fig. 2A. (B) Aliquots of the translation reaction were immunoprecipitated according to the method described in Lawson eta/. (1990) using antiserum directed against the viral protein 2C. A marker lane (M) of PVl proteins prepared as described in the legend to Fig. 2 is displayed in the first lane of each panel. A translation reaction not treated with edeine (-E) is also shown

assay. The production of 3D and 3C occurs slowly in this assay and is not detected. The P3 protein exhibits a half-life of approximately 50 min in the in vitro translation system (Lawson and Semler, 1991) and a similar half-life is observed in the edeine-inhibited translation presented in Fig. 3A. With respect to the alternate pathway of P2 protein generation, it should be noted that proteins of molecular mass and antigenic composition indicating the presence of both 2C and 30 epitopes can be immunoprecipitated from translation reactions (Lawson and Semler, unpublished observations) and in infected cells (Figs. 6B and 6C). No such proteins have been absolutely identified as processing intermediates or as incompletely translated products. The appearance of P3 concurrent with that of 2BC suggests the rapid cleavage of any 2BC-3ABCD proteins. Analysis

of processing

in PVl -infected

cells

The processing of non-structural proteins in infected cells was examined to determine whether a similar pat-

tern of processing as that observed in the in vitro analyses could be detected. The processing of the PVl polyprotein has been analyzed using pulse-chase and pulse-labeling approaches (Rueckert et a/., 1979; Dewalt and Semler, 1987; Blair et a/., 1990; Kean et al., 1990). The processing of some non-structural proteins, notably the P2 precursor, occurred slowly in the previous analyses. However, in the initial labeling of viral protein in most of the previous pulse-chase experiments, considerable product proteins are detected, indicating that the majority of nonstructural protein processing has occurred during the time of labeling. A processing cascade characterized by the primary generation of 2A, 2BC, and P3 predicts at least a transient existence of a 2BC-3ABCD protein precursor which contains all of the viral polypeptides thought to be associated with virus replication (reviewed in Richards and Ehrenfeld, 1990). The association of replication complexes with intracellular membranes as well as the association of viral mRNA-containing polyribosomes with intracellular membranes made it of interest to examine the synthesis and processing of viral proteins in

LAWSON AND SEMLER

314

both the membranous and soluble fractions of infected cells. The pattern of non-structural protein processing during the initial period of viral protein synthesis was then determined. Infected cells were fractionated (after labeling proteins with [36S]methionine) into membranous and soluble fractions according to the protocol described in Takegami et al. (1983) which separates crude membrane-bound viral replication complexes from other cytosolic components.

Pulse-labeling proteins

of membrane-bound

and soluble PVl

The analysis of the production of PVl proteins in membrane-bound and soluble fractions of infected cells was conducted by pulse-labeling HeLa cells for 20 min at 2.5 hr postinfection. Aliquots of the pulse-labeled cells were removed at the times indicated, adjusted to 5 mMZnS0, to inhibit further protein processing, and placed on ice. After fractionation into membranous and soluble components, proteins were then examined on 12.5% SDS-polyacrylamide gels or immunoprecipitated with anti-2C or anti-3D rabbit serum before gel analysis to identify proteins over the background of host-cell proteins (at 2.5 hr postinfection the host-cell macromolecular synthesis has not been completely shut-off, nor do cells show significant cytopathic effect, an indication of lost membrane integrity). The Pl precursor accumulates in the soluble fraction although considerable processed 1AB, lC, and 1 D can be seen in both fractions (Fig. 4A). A rapid accumulation of processed P2 proteins is seen in the membrane fraction, whereas only the P2 protein itself accumulates to significant levels in the soluble fraction (Fig. 4B). The lack of processed P2 proteins present in the soluble fraction is surprising considering that large amounts of P3 proteins were present in the same fraction (Fig. 4C). The stability of P2 suggests that P3 and 3CD are not capable of efficient P2 processing activity or that processing of P2 is membrane-associated. The strong association of P2-derived proteins with membranous structures has been previously described (Bienz et a/., 1983, 1987, 1990). The sequence of 3A has been shown to confer an affinity of the 3AB protein for cellular membranes (Semler et a/., 1982; Giachetti and Semler, 1991). The approximately equal partitioning of P3 protein in both the membrane and soluble fractions implies that the hydrophobic 3A sequence is apparently not capable of firmly anchoring the entire P3 protein in the membranous cellular fraction, suggesting that no interaction or an only transient interaction of 3A sequences and membrane surfaces is possible while 3A is maintained as part of the P3 precursor.

Pulse-chase

analysis of PVl proteins in infected cells

The accumulation of processed P2 proteins in the pulse-labeling experiment suggested that P2 processing occurs very rapidly on membrane surfaces in infected cells. To determine if the kinetics of processing in infected cells were similar to those seen in vitro, a pulse-chase analysis of protein processing was performed (Fig. 5). Each aliquot of the time course was subjected to immunoprecipitation with 2C- and 3D-antiserum as above to separate proteins from the hostcell background. The first time point taken from the membranous fraction displayed in Fig. 5B shows that, after corrections for methionine content are made (see Materials and Methods), the 2BC protein was initially produced in a greater relative amount than the P2 protein. It is not likely that the initial amount of 2BC arose from P2 protein because the 2BC protein is processed to 2C and 2B at a rate 2.5 times the rate that P2 is processed to 2BC and 2A in this analysis. The increased level of 2BC detected is consistent with the non-structural protein processing observed in vitro. No significant differences in the processing of P3 proteins in membranous or soluble fractions were detected in the pulse-chase analysis although very low levels of proteins were found to accumulate in the soluble fraction during this short time course (Fig. 5C). The production of viral proteins at late times during infection was also examined. Rather than examining production of proteins before host protein synthesis was completely halted, the analysis in Fig. 6 shows protein production 3.5 hr postinfection, well beyond the time at which inhibition of host-cell protein synthesis is complete and CPE are readily detected. A pulsechase analysis like that described at 2.5 hr postinfection was conducted and the results are displayed in Fig. 6. The accumulation of Pl and P3 protein was seen in both the soluble fraction and the membrane fraction (Fig. 6A). Of the major P2-derived proteins, only the P2 protein itself was seen to be present at significant levels in the soluble fraction (Fig. 6B). The lack of incompletely translated products in the soluble fraction, which are readily detected in the membrane fraction in Fig. 6A, suggests that proteins appearing in the soluble fraction are first synthesized in the membranous fraction and then diffuse to the soluble fraction. It should also be noted that the apparent differential production of P2 and 2BC visualized in the previous analysis performed early during infection is not evident at late times during infection, as shown by the anti-2C immunoprecipitation in Fig. 6B. The processing of the P3 protein can be more clearly seen in the 3.5-hr postinfection pulse-chase experiment. The production of 3AB and 3A (Fig. 6A) as well as the production of 3D

POLIOVIRUS

PROTEIN PROCESSING

Pl-

P33CD-

P33CD-

PZ-

P23DBABC2BC-

2BC-

1 AB2c-

:;=

3D’-

lC-

2A-

3AB-

3A-

FIG. 4. Pulse-labeling of PVlinfected HeLa cells at 2.5 hr postinfection. HeLa cells in spinner culture were infected with PVl as described under Materials and Methods. At 2.5 hr postinfection, cells were treated with [%S]methionine at a final concentration of 20 &i ml-‘. Aliquots of the infection were removed at the times indicated, adjusted to a 5 mn/l final concentration of ZnSO,, and placed on ice until the end of the time course. Aliquots were then fractionated according to the method described by Takegami eta/. (1983). (A) Electrophoretic analysis of labeled viral and cellular proteins during the pulse-labeling. Membranous (M) and soluble (S) subcellular fractions of cells removed at the time points indicated are shown by the designation above the lanes. Viral proteins are identified on the left. (6) lmmunoprecipitation of pulse-labeled proteins using 2C antiserum. lmmunoprecipitates obtained from the membranous and soluble cellular fractions in (A) are designated accordingly. (C) tmmunoprecipitation of pulse-labeled proteins prepared as in (B) using 3D antiserum.

(Fig. 6C) is readily detected and is restricted to the membranous fraction of the infected cells, suggesting that complete P3 processing is membrane-associated. DISCUSSION This investigation consists of two sets of general observations of the processing of poliovirus polypeptides. The first set of observations, derived from the examination of viral protein processing in vitro, provides evidence that alternative pathways of non-structural protein processing are possible in PVl, and these are illustrated in Fig. 7. The observations of the differential appearance of 2BC and P2 can be explained by the following model. The nascent polyprotein containing the P2 region could be alternately cleaved at either the 2A/2B cleavage site or the 2CY3A site. One pathway is characterized by the rapid processing of P2 proteins by 3C activity, generating the 2A, 2BC, and P3 proteins as

primary cleavage products without the primary generation of P2. The second method of processing is characterized by a slower generation of P2 and P3 proteins. The P2 and P3 products are then slowly processed to their constituent non-structural proteins. Why such a dichotomy of processing occurs is not clear. However, the consideration of the known roles of several picornavirus proteins does allow some conclusions from the reported observations. The non-structural polyproteins, 2B-3D, have all been implicated in the replication of the viral genome (for review see Richards and Ehrenfeld, 1990). It is known that the replication of the viral genome occurs on host-cell membranes, particularly on vesicles induced during infection by an as yet unknown mechanism (Penman et al., 1964; Tershak 1984; Bienz et al., 1990). The high affinity of the 2BC and 2C proteins for membranes has also been described (Bienz et al., 1983). The rapid processing pathway evident in vitro offers a method by which all of the

316

LAWSON AND SEMLER

PlP33CDP2-

P3, 3CD. P2-

3D. BABC’.

2BC3C’ 2c1 D-

2c3w

lC-

2A-

3AB-

3A.

FIG. 5. Pulse-chase analysis of PVl -infected cells at 2.5 hr postinfection. Pulse-chase analysis of PVl -infected HeLa cells was conducted as described in Blair er al. (1990) with the exception that [%]methionine was used at a concentration of 20 pCi ml-‘. The pulse time was 2 min followed by a chase with cold methionine. Aliquots were removed at the times indicated and processed as described in the legend to Fig. 4. (A) Direct analysis of viral proteins from the membranous (M) and soluble (S) subcellular fraction of cells removed at the times indicated. (B) lmmunoprecipitated proteins from the fractionated aliquots in (A) using anti-2C serum. (C) lmmunoprecipitated proteins from the fractionated aliquots in (A) using anti-3D serum.

viral replicative functions can be localized on a membrane surface via 2C. The affinity of a transiently existing 2BC-BABCD protein for cellular membranes would then serve to localize the individual protein products on or near a membrane surface after the post-translational processing by 3C activity. The pathway of rapid processing of non-structural proteins can be accounted for by the division of the polyprotein into replicative and non-replicative functional domains. The identification of a second pathway of non-structural processing in vitro which associates replicative proteins with known membrane affinities in a transiently-existing polypeptide, predicts that viral proteins must be distributed unequally within the infected cell. This prompted an investigation into viral protein production in infected cells from which a second set of observations was obtained. To define the characteristics of the short-term viral protein content of subcellular membrane structures, three separate analyses of proteins present in soluble and membranous fractions were undertaken. In the first analysis, infected cells

were pulse-labeled for 20 min to monitor the accumulation of proteins in both fractions (Fig. 4). From this analysis, it is obvious that processed non-structural P2 proteins accumulated rapidly in the membranous fraction whereas P3 proteins and the Pl and P2 precursors were capable of diffusion into the soluble fraction. The presence of P2 and P3 proteins concurrently in the soluble fractions without observation of further P2 processing suggests that the P3 or 3CD proteins may not be active in P2 processing in trans (Figs. 4B and 4C), although considerable Pl processing is detected in that same fraction. The lack of trans processing of P2 argues strongly for a preference of c&mediated processing of P2 as observed in vitro. In the second analysis (pulse-chase analysis of protein synthesis at 2.5 hr postinfection), it is apparent that in the membrane fraction the 2BC protein was produced in greater relative amounts than the P2 protein, and the P2 protein was cleaved at a slower rate than 2BC (Fig. 5). The different cleavage rates of P2 and 2BC argue that the 2BC protein present initially did not

PO~LIOVIRUS PROTEIN PROCESSING

B

S

M 0

12

PlP33CD-

30

12

M 0

3

1

S 230123

P33CDP23D-

2BC-

3ABC2BC-

1 AB2c1 D-

3c2c-

2A-

3AB-

3A-

FIG. 6. Pulse-chase analysis of PVl-infected cells at 3.5 hr postinfection. PVl infected HeLa cells were pulsed with [35S]methionine at 3.5 hr postinfection for 2 min followed by a cold methionine chase. Samples were removed at the times indicated and processed as described in the legend to Fig. 4. (A) Direct analysis of proteins labeled in the pulse-chase experiment at 3.5 hr postinfection. Lanes containing proteins derived from the membranous and soluble subcellular fractions at each time point are indicated by the M and S designation above the lanes, respectively. (B) lmmunoprecipitation of proteins derived from the aliquots indicated from (A) using anti-2C serum. (8) lmmunoprecipitation of proteins derived from the aliquots indicated from (A) using anti-3D serum.

arise from P2. The non-structural proteins synthesized in the short 2-minute pulse period did not migrate to the soluble fraction (Figs. 5B and 5C), although the Pl protein did (Fig. 5A). The processing of proteins of P2 and P3 origin was very rapid and membrane-associated, suggesting that the majority of protein processing occurs in a &-dependent fashion. The third analysis of infected cells (a pulse-chase analysis at 3.5 hr postinfection) presents a different picture of non-structural protein processing (Fig. 6). At 3.5 hr postinfection, protein synthesis is almost exclusively virus-directed. It can be clearly seen that Pl , P3, 3CD, and P2 appear in the soluble fraction upon their synthesis but that only Pl was further processed in that fraction. The processing of P3 in vivo was slow, and no P3 processing other than that initially occurring in the membranous fraction is detected over the 3-min chase period. The processing of P2 proteins is markedly different than in the previous analyses in that there is no striking disparity in the relative amounts of P2 and 2BC proteins early in the chase. The rapid production

of 2C, which occurs in greater amounts than can be accounted for by the degradation of P2 and 2BC, suggests that the processing rate of 2BC to 2C has been increased, presumably through the higher levels of Vans-acting proteolytic activity present at this late time during infection. The detection of an alternative pathway of processing allows several conclusions which are supported by analyses in infected cells. The rapidly processed nonstructural proteins found in the membranous fraction indicate that protein processing is highly organized and may occur in a c&-like fashion. The most expedient way to conduct processing may be to maintain 3C activity as an integral part of the polyprotein during processing. This is suggested by the alternate pathway described here. The observed processing of in vitro synthesized polyprotein may over-emphasize the rapid cascade of cleavage due to the low level of protein synthesized in the in vitro system, which would limit diffusible, trans-like proteolytic activity. The processing activity of 3C protein purified from Escherichia co/i ex-

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IC .

ID A

2A 28 A AA

ID

2A 28

2C

4 IA

IB

1c

MEMBRANE

38 3A 3C AM .A

:3D

3B

2C

3A

3c

130

\TIOLUBLE

FIG. 7. Alternate processing cascades of poliovirus non-structural proteins. The two alternate processing cascades detected in the present study are shown diagrammatically. The cascade presented on the left is the rapid cascade detected at early times in PVl infection of HeLa cells or in synchronized translation of RNA derived from the wild-type PVl cDNA clone pT7-1. This cascade is the result of the primary BC-mediated cleavage occurring at the 2AJ2B protein junction. The cascade presented on the right shows the slower processing cascade of the Pl, P2, and P3 proteins that occurs through the frans-like pathway and is visualized in the soluble fraction of the infected cell cytoplasm. The cascade on the left shows the rapid, cis-like processing cascade visualized in vitro and in the membranous fraction of the infected cell cytoplasm. The BCD-mediated processing of Pl to 1AB, 1C and 1 D is not illustrated. The wavy horizontal lines represent the noncoding regions at the 5’and 3’ ends of viral genomic RNA. The open triangles (A) denote 2A-mediated cleavage sites. The filled triangles (A) denote 3C- or 3CD-mediated cleavage sites. The thick arrows indicate rapid or readily-detectable cleavage pathways while the thin arrows indicate slow or less-frequent cleavage pathways.

pressing a subgenomic cDNA clone of PV2 3C is very low, even when tested on in vitro synthesized protein substrates such as 2BC (Hammerle et al., 1991). The low level of 3C activity on non-structural substrates in trans assays strongly points to the preference of nonstructural processing to occur through a c&-like mechanism. The affinity of the rapidly processed viral proteins for subcellular membranes suggests that the rapid cascade is utilized to associate proteins with replicative function on the membrane surface. This conclusion is supported by the observation that the production of 3D, 3AB, and 3A, proteins intimately involved in viral RNA replication, is detected primarily in the membranous fraction (Fig. 6). The need for a slower processing cascade is less clear. The processing of Pl may provide some insight. The processing of Pl by 3CD is inherently diffusion-dependent. The detection of proteins in both membrane and soluble fractions suggests that Pl processing may depend on the formation of a processing complex by the association of the 3CD and Pl proteins. The delayed processing of Pl proteins in vitro (Fig. 2) and the apparent dependence on 3CD concentration for Pl processing (Fig. 3) can be interpreted to mean that steps other than the production of the enzyme and substrate may be required for Pl processing. It is possible that the low rate of P3 processing observed both in

vitro and in infected cells is due to the use of the slower method of processing to generate a diffusible proteolytic activity in the form of 3CD that is necessary for capsid production. The use of 3CD generated by this method of processing would be distinct from the rapid processing of P3 proteins required forthe replication of the viral genome. The 3CD protein generated in or diffused to the soluble fraction would not then be an authentic precursor of the 3C and 3D proteins, but rather would be an end product necessary for Pl processing. The apparent regulation of 3C activity through the use of 3CD as the enzyme processing Pl is characteristic of enteroviruses and may be true for the rhinoviruses (Jore et a/., 1988; Ypma-Wong et al., 1988; Dewalt et al,, 1989) but is not found in cardiovirus or aphthovirus protein processing (Parks et al,, 1989; Vakharia et al., 1987; Clarke and Sangar, 1988). It is not clear why the enteroviruses utilize this additional regulation of 3C activity with respect to virus particle formation. In summary, PVl appears to utilize two processing cascades in the production of viral proteins, the first of which is rapid and operates through a cis-like mechanism, and a second slower method which is vans-like, i.e., diffusion-dependent. The dichotomy of the processing cascade is based on the ability of 3C to recognize the 2A/2B or the 2B/2C cleavage site as the primary site of action. Densitometric analysis of Fig. 3

POLIOVIRUS

PROTEIN PROCESSING

indicates that the 2N2B cleavage site is preferred approximately three times over the 2C/3A site. In addition, a separate study from this laboratory on the in vitro processing of poliovirus polyproteins containing lethal lesions in protein 3AB provides support for the 2N2B cleavage as a preferred primary event in protein processing (Giachetti et al., 1992). Such a pathway of processing may provide PVl with a way of locating replicative proteins on the membrane surface to establish a replication complex(the rapid cascade) orto alternatively generate diffusible, or rrans-acting, proteins which may then serve to perform functions related to the production of virus particles or the inhibition of host-cell protein synthesis (mediated by 3CD and 2A, respectively). Poliovirus is thus capable of alternatively regulating the many functions of viral gene products by controlling the manner in which discrete proteins are released from the parent polyprotein. ACKNOWLEDGMENTS We thank Stephen J. Sharp and Wade S. Blair for critical reading of the manuscript. This work was supported by U.S. Public Health Service Grant Al22693 from the National Institutes of Health. M.A.L. was a trainee with Public Health Service Grant CA09054.

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