A mutant poliovirus containing a novel proteolytic cleavage site in VP3 is altered in viral maturation.

A six-amino-acid insertion containing a Q-G amino acid pair was introduced into the carboxy terminus of the capsid protein VP3 (between residues 236 and 237). Transfection of monkey cells with full-length poliovirus cDNA containing the insertion described above yields a mutant virus (Sel-1C-02) in which cleavage occurs almost entirely at the inserted Q-G amino acid pair instead of at the wild-type VP3-VP1 cleavage site. Mutant Sel-1C-02 is delayed in the kinetics of virus production at 39 degrees C and exhibits a defect in VP0 cleavage into VP2 and VP4 at 39 degrees C. Sucrose gradient analysis of HeLa cell extracts prepared from cells infected by Sel-1C-02 at 39 degrees C shows an accumulation of fast-sedimenting replication-packaging complexes and a significant amount of uncleaved VP0 present in fractions containing mature virions. Our data provide in vivo evidence for the importance of determinants other than the conserved amino acid pair (Q-G) for recognition and cleavage of the P1 precursor by proteinase 3CD and show that an alteration in the carboxy terminus of VP3 or the amino terminus of VP1 affects the process of viral maturation.     

A cellular cofactor facilitates efficient 3CD cleavage of the poliovirus P1 precursor.

The production of poliovirus capsid proteins from a capsid protein precursor (P1) is mediated by virus-encoded proteinase 3CD and involves a complicated set of proteinase-substrate interactions. In addition to substrate and enzymatic determinants required for this interaction, we describe a cellular cofactor, which facilitates 3CD recognition of the P1 precursor. Cellular cofactor activity is 3CD dependent and salt dependent. Our analysis shows that proteolytic cleavage of the P1 precursor at the VP0/VP3 cleavage site exhibits a greater dependency on the cellular cofactor than cleavage at the VP3/VP1 site. Such a greater dependency on cellular cofactor activity can be relieved (in part) by the substitution of an Ala residue for the Pro residue at the -4 position of the VP0/VP3 cleavage site. However, mutant viruses containing Pro-to-Ala substitutions at the -4 position of the VP0/VP3 site exhibit defects in viral growth.     

Poliovirus capsid proteins derived from P1 precursors with glutamine-valine cleavage sites have defects in assembly and RNA encapsidation.

Assembly of poliovirus virions requires proteolytic cleavage of the P1 capsid precursor polyprotein between two separate glutamine-glycine (QG) amino acid pairs by the viral protease 3CD. In this study, we have investigated the effects on P1 polyprotein processing and subsequent assembly of processed capsid proteins caused by substitution of the glycine residue at the individual QG cleavage sites with valine (QG-->QV). P1 cDNAs encoding the valine substitutions were created by site-directed mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 precursors. The recombinant vaccinia virus-expressed mutant P1 polyproteins were analyzed for proteolytic processing defects in cells coinfected with a recombinant vaccinia virus (VVP3) that expresses the poliovirus 3CD protease and for processing and assembly defects by using a trans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome that does not express functional capsid proteins (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substituted precursors were proteolytically processed at the altered sites both in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites were slower than those of cleavage at the wild-type QG site in the precursor. Completely processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral structures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substitution at the amino terminus of VP1 (VP1-G001V) assembled empty capsid particles but were deficient in assembling RNA-containing virions. The assembly characteristics of the VP1-G001V mutant were compared with those of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaard and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletion of the first four amino-terminal residues of VP1 (VP1-delta 1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-delta 1-4 precursor also occurred more slowly than complete cleavage of the wild-type precursor, and formation of virions was delayed; however, capsid proteins derived from the VP1-G001V mutant assembled RNA-containing virions less efficiently than those derived from the VP1-delta 1-4 precursor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural domains of the poliovirus polyprotein are major determinants for proteolytic cleavage at Gln-Gly pairs

The processing of poliovirus precursor polypeptides provides a valuable system in which to study the recognition and interaction of a proteolytic enzyme with its substrates. Processing of the poliovirus polyprotein includes cleavage between 9 of 13 available glutamineglycine (Q-G) pairs by the activity of a virally encoded proteinase, 3C. In this study, we assess the importance of primary, secondary, and tertiary structural determinants in the cleavage at two Q-G pairs in the capsid protein precursor, P1. Employing site-directed mutagenesis of cDNA copies of poliovirus RNA, we have made specific alterations in regions of the P1 capsid precursor and have assayed the effect of these alterations on proteinase cleavage at the two Q-G pairs. We have also introduced additional Q-G pairs into P1 and demonstrated that the proteinase can recognize some of the inserted Q-G pairs as cleavage sites. By correlating the predicted three-dimensional structures and the processing phenotypes of several altered P1 precursors, we are able to rank the importance of determinants required for P1 processing. While a Q-G pair appears to be the primary determinant in proteinase recognition, the tertiary location of a Q-G pair in the precursor either allows or prevents processing at that pair. Our results also suggest that the proper folding of at least two of the three P1 beta-barrel structures is required for efficient proteinase cleavage at Q-G pairs.     

Site-specific mutagenesis of cDNA clones expressing a poliovirus proteinase

The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio-specific cleavages occur at glutamine-glycine (Q-G) pairs that are recognized by the viral-encoded proteinase 3C (formerly called P3-7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN-III-C3-7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino- and carboxy-terminal Q-G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN-III-C3-7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site-specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy-terminal Q-G cleavage site. The mutant recombinant plasmid (designated pIN-III-C3-mu 10) directs the synthesis of a bacterial-polio precursor polypeptide that is like the wild-type construct (pIN-III-C3-7c). However, unlike the wild-type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino-terminal Q-G site but not at the carboxy-terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self-cleavage at its carboxy-terminal Q-G pair.     

Folding and processing of the capsid protein precursor P1 is kinetically retarded in neutralization site 3B mutants of poliovirus.

Poliovirus mutants in neutralizing antigenic site 3B were constructed by replacing the glutamic acid residue at amino acid 74 of capsid protein VP2 (VP2074E), using site-specific mutagenesis methods. All viable mutants display small-plaque phenotypes. Characterization of these mutants indicates that capsid assembly is perturbed. Although the defect in capsid assembly reduces the yield of mutant virus particles per cell, the resultant assembled particle is wild-type-like in structure and infectivity. Analyses of capsid assembly intermediates show a transient accumulation of the unprocessed capsid protein precursor, P1, indicating that cleavage of the mutant P1 by the 3CD protease is retarded. The mutant VP0-VP3-VP1 complex generated upon P1 cleavage appears assembly competent, forming pentamer and empty capsid assembly intermediates and infectious virion particles. Although the structure of the infectious mutant virus is virtually identical with that of the wild-type virus, the thermal stability of the mutant virus is dramatically increased over that of the wild-type virus. Thus, mutations at this residue are pleiotropic, altering the kinetics of capsid assembly and generating a virus that is more thermostable and more resistant to neutralization by the site 3B monoclonal antibodies.     

Herpesvirus proteinase: site-directed mutagenesis used to study maturational, release, and inactivation cleavage sites of precursor and to identify a possible catalytic site serine and histidine.

The cytomegalovirus maturational proteinase is synthesized as a precursor that undergoes at least three processing cleavages. Two of these were predicted to be at highly conserved consensus sequences--one near the carboxyl end of the precursor, called the maturational (M) site, and the other near the middle of the precursor, called the release (R) site. A third less-well-conserved cleavage site, called the inactivation (I) site, was also identified near the middle of the human cytomegalovirus 28-kDa assemblin homolog. We have used site-directed mutagenesis to verify all three predicted sequences in the simian cytomegalovirus proteinase, and have shown that the proteinase precursor is active without cleavage at these sites. We have also shown that the P4 tyrosine and the P2 lysine of the R site were more sensitive to substitution than the other R- and M-site residues tested: substitution of alanine for P4 tyrosine at the R site severely reduced cleavage at that site but not at the M site, and substitution of asparagine for lysine at P2 of the R site reduced M-site cleavage and nearly eliminated I-site cleavage but had little effect on R-site cleavage. With the exception of P1' serine, all R-site mutations hindered I-site cleavage, suggesting a role for the carboxyl end of assemblin in I-site cleavage. Pulse-chase radiolabeling and site-directed mutagenesis indicated that assemblin is metabolically unstable and is degraded by cleavage at its I site. Fourteen amino acid substitutions were also made in assemblin, the enzymatic amino half of the proteinase precursor. Among those tested, only 2 amino acids were identified as essential for activity: the single absolutely conserved serine and one of the two absolutely conserved histidines. When the highly conserved glutamic acid (Glu22) was substituted, the proteinase was able to cleave at the M and I sites but not at the R site, suggesting either a direct (e.g., substrate recognition) or indirect (e.g., protein conformation) role for this residue in determining substrate specificity.     

Myristylation of poliovirus capsid precursor P1 is required for assembly of subviral particles.

The poliovirus capsid precursor polyprotein, P1, is cotranslationally modified by the addition of myristic acid. We have examined the importance of myristylation of the P1 capsid precursor during the poliovirus assembly process by using a recently described recombinant vaccinia virus expression system which allows the independent production of the poliovirus P1 protein and the poliovirus 3CD proteinase (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). We constructed a site-directed mutation in the poliovirus cDNA encoding an alanine at the second amino acid position of P1 in place of the glycine residue required for the myristic acid addition and isolated a recombinant vaccinia virus (VVP1myr-) that expressed a nonmyristylated form of the P1 capsid precursor. The 3CD proteinase expressed by a coinfecting vaccinia virus, VVP3, proteolytically processed the nonmyristylated precursor P1 expressed by VVP1myr-. However, the processed capsid proteins, VP0, VP3, and VP1, did not assemble into 14S or 75S subviral particles, in contrast to the VP0, VP3, and VP1 proteins derived from the myristylated P1 precursor. When cells were coinfected with VVP1myr- and poliovirus type 1, the nonmyristylated P1 precursor expressed by VVP1myr- was processed by 3CD expressed by poliovirus, and the nonmyristylated VP0-VP3-VP1 (VP0-3-1) protomers were incorporated into capsid particles and virions which sedimented through a 30% sucrose cushion. Thus, the nonmyristylated P1 precursor and VP0-3-1 protomers were not excluded from sites of virion assembly, and the assembly defects observed for the nonmyristylated protomers were overcome in the presence of myristylated capsid protomers expressed by poliovirus. We conclude that myristylation of the poliovirus P1 capsid precursor plays an important role during poliovirus assembly by facilitating the appropriate interactions required between 5S protomer subunits to form stable 14S pentamers. The results of these studies demonstrate that the independent expression of the poliovirus P1 and 3CD proteins by using recombinant vaccinia viruses provides a unique experimental tool for analyzing the dynamics of the poliovirus assembly process.     

Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.

Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.     

The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, deletions of the domain 442 to 487 or 502 to 512 did not allow virus recovery. Several amino acids of the peptide 502 to 512 appeared essential for virus viability. Substitutions of the P1 and/or P1" position were engineered at each of the cleavage sites (P1-P1": 441-442, 487-488, 494-495, 501-502, and 512-513). Most substitutions at the pVP2-VP4 junction (512-513) and at the final VP2 maturation cleavage site (441-442) were lethal. Mutations of intermediate cleavage sites (487-488, 494-495, and 501-502) led to viable viruses showing different but efficient pVP2 processing. Our data suggested that while peptides 488 to 494 and 495 to 501 play an accessory role, peptides 442 to 487 and 502 to 512 have an unknown but important function within the virus cycle.     

Cleavage specificity of purified recombinant hepatitis A virus 3C proteinase on natural substrates.

Hepatitis A virus (HAV) 3C proteinase expressed in Escherichia coli was purified to homogeneity, and its cleavage specificity towards various parts of the viral polyprotein was analyzed. Intermolecular cleavage of the P2-P3 domain of the HAV polyprotein gave rise to proteins 2A, 2B, 2C, 3ABC, and 3D, suggesting that in addition to the primary cleavage site, all secondary sites within P2 as well as the 3C/3D junction are cleaved by 3C. 3C-mediated processing of the P1-P2 precursor liberated 2A and 2BC, in addition to the structural proteins VP0, VP3, and VP1-2A and the respective intermediate products. A clear dependence on proteinase concentration was found for most cleavage sites, possibly reflecting the cleavage site preference of 3C. The most efficient cleavage occurred at the 2A/2B and 2C/3A junctions. The electrophoretic mobility of processing product 2B, as well as cleavage of the synthetic peptide KGLFSQ*AKISLFYT, suggests that the 2A/2B junction is located at amino acid position 836/837 of the HAV polyprotein. Furthermore, using suitable substrates we obtained evidence that sites VP3/VP1 and VP1/2A are alternatively processed by 3C, leading to either VP1-2A or to P1 and 2A. The results with regard to intermolecular cleavage by purified 3C were confirmed by the product pattern derived from cell-free expression and intramolecular processing of the entire polyprotein. We therefore propose that polyprotein processing of HAV relies on 3C as the single proteinase, possibly assisted by as-yet-undetermined viral or host cell factors and presumably controlled in a concentration-dependent fashion.     

Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase.

Processing of the hepatitis C virus polyprotein is accomplished by a series of cotranslational and posttranslational cleavages mediated by host cell signalases and two virally encoded proteinases. Of these the NS3 proteinase is essential for processing at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions. Processing between NS3 and NS4A occurs in cis, implying an intramolecular reaction mechanism, whereas cleavage at the other sites can also be mediated in trans. Sequence analysis of the amino termini of mature cleavage products and comparisons of amino acid residues around the scissile bonds of various hepatitis C virus isolates identified amino acid residues which might contribute to substrate specificity and processing efficiency: an acidic amino acid at the P6 position, a Thr or Cys at the P1 position, and a Ser or Ala at the P1' position. To study the importance of these residues for NS3-mediated cleavage we have undertaken a mutational analysis using an NS3'-5B polyprotein expressed by recombinant vaccinia viruses in mammalian cells. For all NS3-dependent cleavage sites P1 substitutions had the most drastic effects on cleavage efficiency, showing that amino acid residues at this position are the most critical substrate determinants. Since less drastic effects were found for substitutions at the P1' position, these residues appear to be less important for proper cleavage. For all cleavage sites the P6 acidic residue was dispensable, suggesting that it is not essential for substrate recognition and subsequent cleavage. Analysis of a series of mutations at the NS3/4A site revealed great flexibility for substitutions compared with more stringent requirements at the trans cleavage sites. On the basis of these results we propose a model in which processing in cis is determined primarily by polyprotein folding, whereas cleavage in trans is governed not only by the structure of the polyprotein but also by specific interactions between the proteinase and the polyprotein substrate at or around the scissile bond.     

Cleavage sites within the poliovirus capsid protein precursors.

Partial amino-terminal sequence analysis was performed on radiolabeled polio-virus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.     

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.     

Amino acid substitutions in the poliovirus maturation cleavage site affect assembly and result in accumulation of provirions.

The assembly of infectious poliovirus virions requires a proteolytic cleavage between an asparagine-serine amino acid pair (the maturation cleavage site) in VP0 after encapsidation of the genomic RNA. In this study, we have investigated the effects that mutations in the maturation cleavage site have on P1 polyprotein processing, assembly of subviral intermediates, and encapsidation of the viral genomic RNA. We have made mutations in the maturation cleavage site which change the asparagine-serine amino acid pair to either glutamine-glycine or threonine-serine. The mutations were created by site-directed mutagenesis of P1 cDNAs which were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses. The P1 polyproteins expressed from the recombinant vaccinia viruses were analyzed for proteolytic processing and assembly defects in cells coinfected with a recombinant vaccinia virus (VV-P3) that expresses the poliovirus 3CD protease. A trans complementation system using a defective poliovirus genome was utilized to assess the capacity of the mutant P1 proteins to encapsidate genomic RNA (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The mutant P1 proteins containing the glutamine-glycine amino acid pair (VP4-QG) and the threonine-serine pair (VP4-TS) were processed by 3CD provided in trans from VV-P3. The processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor VP4-QG were unstable and failed to assemble into subviral structures in cells coinfected with VV-P3. However, the capsid proteins derived from VP4-QG did assemble into empty-capsid-like structures in the presence of the defective poliovirus genome. In contrast, the capsid proteins derived from processing of the VP4-TS mutant assembled into subviral intermediates both in the presence and in the absence of the defective genome RNA. By a sedimentation analysis, we determined that the capsid proteins derived from the VP4-TS precursor encapsidated the defective genome RNA. However, the cleavage of VP0 to VP4 and VP2 was delayed, resulting in the accumulation of provirions. The maturation cleavage of the VP0 protein containing the VP4-TS mutation was accelerated by incubation of the provirions at 37 degrees C. The results of these studies demonstrate that mutations in the maturation cleavage site have profound effects on the subsequent capability of the capsid proteins to assemble and provide evidence for the existence of the provirion as an assembly intermediate.     

Identification of the murine coronavirus MP1 cleavage site recognized by papain-like proteinase 2

The replicase polyprotein of murine coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a trans-cleavage assay and showed that PLP2 cleaves the replicase polyprotein between p210 and membrane protein 1 (MP1) (A. Kanjanahaluethai and S. C. Baker, J. Virol. 74:7911-7921, 2000). Here, we report the results of our studies identifying and characterizing this cleavage site. To determine the approximate position of the cleavage site, we expressed constructs that extended various distances upstream from the previously defined C-terminal end of MP1. We found that the construct extending from the putative PLP2 cleavage site at glycine 2840-alanine 2841 was most similar in size to the processed MP1 replicase product generated in a trans-cleavage assay. To determine which amino acids are critical for PLP2 recognition and processing, we generated 14 constructs with amino acid substitutions upstream and downstream of the putative cleavage site and assessed the effects of the mutations in the PLP2 trans-cleavage assay. We found that substitutions at phenylalanine 2835, glycine 2839, or glycine 2840 resulted in a reduction in cleavage of MP1. Finally, to unequivocally identify this cleavage site, we isolated radiolabeled MP1 protein and determined the position of [(35)S]methionine residues released by Edman degradation reaction. We found that the amino-terminal residue of MP1 corresponds to alanine 2841. Therefore, murine coronavirus PLP2 cleaves the replicase polyprotein between glycine 2840 and alanine 2841, and the critical determinants for PLP2 recognition and processing occupy the P6, P2, and P1 positions of the cleavage site. This study is the first report of the identification and characterization of a cleavage site recognized by murine coronavirus PLP2 activity.     

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.     

Analysis of N-terminal processing of hepatitis C virus nonstructural protein 2.

We determined the partial amino (N)-terminal amino acid sequence of hepatitis C virus p21 (nonstructural protein 2 [NS2]). Cleavage at the p21 (NS2) N terminus depended on the presence of microsomal membranes. The amino-terminal position of p21 (NS2) was assigned to amino acid 810 of the hepatitis C virus strain IIJ precursor polyprotein. Mutation of the alanine residue at position P1 of the putative cleavage site inhibited membrane-dependent processing. This alteration in processing together with the fact that hydrophobic amino acid residues are clustered upstream of the putative cleavage site suggested the involvement of a signal peptidase(s) in the cleavage. Furthermore, mutation analysis of this possible cleavage site revealed the presence of another microsome membrane-dependent cleavage site upstream of the N terminus of p21 (NS2).