Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.     

Foot-and-mouth disease virus 2A protease mediates cleavage in attenuated Sabin 3 poliovirus vectors engineered for delivery of foreign antigens.

Poliovirus vectors are being studied as potential vaccine delivery systems, with foreign genetic sequences incorporated as part of the viral genome. The foreign sequences are expressed as part of the viral polyprotein. Addition of proteolytic cleavage sites at the junction of the foreign polypeptide and the viral proteins results in cleavage during polyprotein processing. The ability of foot-and-mouth disease virus (FMDV) 2A to mediate proteolytic cleavage in the context of poliovirus vectors was studied. The results demonstrate that FMDV 2A is able to generate cleavage of the foreign antigen from the viral polyprotein. A second cleavage event between the FMDV 2A peptide and the foreign protein was also observed.     

Flavivirus enzyme-substrate interactions studied with chimeric proteinases: identification of an intragenic locus important for substrate recognition.

The proteins of flaviviruses are translated as a single long polyprotein which is co- and posttranslationally processed by both cellular and viral proteinases. We have studied the processing of flavivirus polyproteins in vitro by a viral proteinase located within protein NS3 that cleaves at least three sites within the nonstructural region of the polyprotein, acting primarily autocatalytically. Recombinant polyproteins in which part of the polyprotein is derived from yellow fever virus and part from dengue virus were used. We found that polyproteins containing the yellow fever virus cleavage sites were processed efficiently by the yellow fever virus enzyme, by the dengue virus enzyme, and by various chimeric enzymes. In contrast, dengue virus cleavage sites were cleaved inefficiently by the dengue virus enzyme and not at all by the yellow fever virus enzyme. Studies with chimeric proteinases and with site-directed mutants provided evidence for a direct interaction between the cleavage sites and the proposed substrate-binding pocket of the enzyme. We also found that the efficiency and order of processing could be altered by site-directed mutagenesis of the proposed substrate-binding pocket.     

A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein

The poliovirus polyprotein is cleaved at three different amino acid pairs. Viral polypeptide 3C is responsible for processing at the most common pair (glutamineglycine). We have found that a cDNA fragment encoding parts of the capsid protein region (P1) and the nonstructural protein region (P2), and including the P1-P2 processing site (tyrosine-glycine), can be expressed in E. coli. The translation product was correctly processed. Disruption of the coding sequence of 2A, a nonstructural polypeptide mapping carboxy-terminal to the tyrosine-glycine cleavage site, by linker mutagenesis or deletion, prevented processing. Deletion of the adjacent polypeptide 2B had no such effect. Antibodies against 2A specifically inhibited processing at the 3C'-3D' processing site (tyrosine-glycine) in vitro. We conclude that poliovirus encodes the second proteinase 2A, which processes the polyprotein at tyrosine-glycine cleavage sites.     

The foot-and-mouth disease virus leader proteinase gene is not required for viral replication.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase has only two known functions: (i) autocatalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. Cleavage of p220 appears to be important for picornavirus replication, since rhinoviruses and enteroviruses utilize a different proteinase (2A) to cleave p220. To explore the role of L in FMDV replication, we generated synthetic FMDV genomes lacking the L gene and tested their viability in cells. Genomes were constructed with the N-terminal Gly codon of VP4 positioned directly following either the first (Lab) or second (Lb) Met codon of the L protein. Cells transfected with synthetic RNAs lacking L and initiating with the Lab Met codon failed to produce viable virus, but cells transfected with RNAs that utilized the second AUG to drive translation of the viral polyprotein produced viable viruses. These leader-deleted viruses produced plaques on BHK cells that were slightly smaller than those produced by wild-type (WT) virus, grew to slightly lower titers than WT virus in BHK cells, shut off host protein synthesis more slowly than WT virus, and were slightly attenuated in mice. These studies indicate that the L proteinase is not essential for FMDV replication and show that in the cells and animals tested the L gene has a limited effect on virus replication.     

Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein.     

Foot-and-mouth disease virus leader proteinase: involvement of C-terminal residues in self-processing and cleavage of eIF4GI.

The leader proteinase (L(pro)) of foot-and-mouth disease virus frees itself from the nascent polyprotein, cleaving between its own C terminus and the N terminus of VP4 at the sequence Lys-Leu-Lys- downward arrow-Gly-Ala-Gly. Subsequently, the L(pro) impairs protein synthesis from capped mRNAs in the infected cell by processing a host protein, eukaryotic initiation factor 4GI, at the sequence Asn-Leu-Gly- downward arrow-Arg-Thr-Thr. A rabbit reticulocyte lysate system was used to examine the substrate specificity of L(pro) and the relationship of the two cleavage reactions. We show that L(pro) requires a basic residue at one side of the scissile bond to carry out efficient self-processing. This reaction is abrogated when leucine and lysine prior to the cleavage site are substituted by serine and glutamine, respectively. However, the cleavage of eIF4GI is unaffected by the inhibition of self-processing. Removal of the 18-amino acid C-terminal extension of L(pro) slowed eIF4GI cleavage; replacement of the C-terminal extension by unrelated amino acid sequences further delayed this cleavage. Surprisingly, wild-type L(pro) and the C-terminal variants all processed the polyprotein cleavage site in an intermolecular reaction at the same rate. However, when the polyprotein cleavage site was part of the same polypeptide chain as the wild-type Lb(pro), the rate of processing was much more rapid. These experiments strongly suggest that self-processing is an intramolecular reaction.     

Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation.

Proteolytic processing of the Rous sarcoma virus (RSV) Gag precursor was altered in vivo through the introduction of amino acid substitutions into either the polyprotein cleavage junctions or the PR coding sequence. Single amino acid substitutions (V(P2)S and P(P4)G), which are predicted from in vitro peptide substrate cleavage data to decrease the rate of release of PR from the Gag polyprotein, were placed in the NC portion of the NC-PR junction. These substitutions do not affect the efficiency of release of virus-like particles from COS cells even though recovered particles contain significant amounts of uncleaved Pr76gag in addition to mature viral proteins. Single amino acid substitutions (A(P3)F and S(P1)Y), which increase the rate of PR release from Gag, also do not affect budding of virus-like particles from cells. Substitution of the inefficiently cleaved MA-p2 junction sequence in Gag by eight amino acids from the rapidly cleaved NC-PR sequence resulted in a significant increase in cleavage at the new MA-p2 junction, but again without an effect on budding. However, decreased budding was observed when the A(P3)F or S(P1)Y substitution was included in the NC-PR junction sequence between the MA and p2 proteins. A budding defect was also caused by substitution into Gag of a PR subunit containing three amino acid substitutions (R105P, G106V, and S107N) in the substrate binding pocket that increase the catalytic activity of PR. The defect appears to be the result of premature proteolytic processing that could be rescued by inactivating PR through substitution of a serine for the catalytic aspartic acid residue. This budding defect was also rescued by single amino acid substitutions in the NC-PR cleavage site which decrease the rate of release of PR from Gag. A similar budding defect was caused by replacing the Gag PR with two PR subunits covalently linked by four glycine residues. In contrast to the defect caused by the triply substituted PR, the budding defect observed with the linked PR dimer could not be rescued by NC-PR cleavage site mutations, suggesting that PR dimerization is a limiting step in the maturation process. Overall, these results are consistent with a model in which viral protein maturation occurs after PR subunits are released from the Gag polyprotein.     

Characterization of the Epstein-Barr virus proteinase and comparison with the human cytomegalovirus proteinase.

The BVRF2 gene of Epstein-Barr virus (EBV) shows homology to the UL26 and UL80 genes of herpes simplex virus type 1 (HSV-1) and cytomegalovirus (CMV), respectively. These genes are believed to provide a scaffold protein for the assembly of capsids leading to the formation of infectious viral particles. We have cloned the BVRF2 gene from the B95.8 strain of EBV and shown that the BVRF2 gene product is a polyprotein capable of autoproteolytic cleavage. Two Ala-Ser-containing recognition sequences were identified in the BVRF2 polyprotein at amino acid positions 568/569 and 570/571 where this cleavage was expected to occur. Here, we show that EBV proteinase is capable of cleaving at the first Ala-Ser bond but not the second. Comparison of the processing of the EBV and human CMV assembly domains in vitro by either EBV or human CMV proteinase revealed that, while both proteinases could cleave their native assembly domain, only EBV proteinase was able to cleave the assembly domain of the other virus.     

Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease.

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.     

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.     

VPg gene amplification correlates with infective particle formation in foot-and-mouth disease virus.

In order to analyze the function of VPg amplification in aphthoviruses, we have undertaken the first mutational analysis of the repetitive VPg-coding region using an improved foot-and-mouth disease virus (FMDV) cDNA clone from which infective viral RNA was synthesized. A set of VPg mutants was constructed by site-directed mutagenesis which includes different VPg deletion mutations, a VPg insertion mutation, and amino acid residue replacement mutations that interfere with binding of the VPg protein to the viral RNA and with its proteolytic processing. Our results revealed that an amazing flexibility in the number of VPgs is tolerated in FMDV. Optimal viability is given when three VPgs are encoded. Deletion as well as insertion of one VPg gene still resulted in infective particle production. Infective particle formation was observed as long as one VPg remained intact. No obvious differences in the individual VPg molecules with regard to their promoting viral RNA synthesis were observed, indicating that all three VPgs can act equally in FMDV replication. Mutant polyprotein processing was comparable to that of the wild-type virus. However, VPg mutants showed reduced viral RNA synthesis levels after infection. The levels of viral RNA synthesis and infective particle formation were found to correlate with the number of functional VPgs left in the mutant virus. These findings suggest a direct VPg gene dosage effect on viral RNA synthesis, with a secondary effect on infective particle formation.     

Picornaviral processing: some new ideas

Mature picornaviral proteins are derived by progressive, post-translational cleavage of a giant precursor polyprotein. At least three viral-encoded proteolytic activities are involved in the processing. The first cleavage takes place while the polyprotein is still nascent on a ribosome. In poliovirus, this event is probably catalyzed by peptide 2A, a protein from the middle portion of the genome. Most subsequent processing is effected by viral protease 3C, a thiol-type enzyme, responsible for eight to ten self-cleaving and autocatalytic reactions within the polyprotein. The final proteolytic processing event, maturation of the VPO peptide, may occur by a novel, autocatalytic, serine-type mechanism, where viral RNA serves as proton-acceptor during the cleavage reaction.     

Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products

The coronavirus Avian infectious bronchitis virus (IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identified the first cleavage event as proteolysis at the Gly(673)-Gly(674) dipeptide bond mediated by the first papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this report, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mutagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly(2265)-Gly(2266) dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleavage product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in trans when expressed in intact cells. Both the N- and C-terminal cleavage products were detected in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modified by N-linked glycosylation at the Asn(2313) residue encoded by nucleotides 7465 to 7467. By using a region-specific antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifically immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a previously predicted cleavage site (Q(2583)-G(2584)) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product.     

Regulation of the sequential processing of Semliki Forest virus replicase polyprotein

The replication of most positive-strand RNA viruses and retroviruses is regulated by proteolytic processing. Alphavirus replicase proteins are synthesized as a polyprotein, called P1234, which is cleaved into nsP1, nsP2, nsP3, and nsP4 by the carboxyl-terminal protease domain of nsP2. The cleavage intermediate P123+nsP4 synthesizes minus-strand copies of the viral RNA genome, whereas the completely processed complex is required for plus-strand synthesis. To understand the mechanisms responsible for this sequential proteolysis, we analyzed in vitro translated Semliki Forest virus polyproteins containing noncleavable processing sites or various deletions. Processing of each of the three sites in vitro required a different type of activity. Site 3/4 was cleaved in trans by nsP2, its carboxyl-terminal fragment Pro39, and by all polyprotein proteases. Site 1/2 was cleaved in cis with a half-life of about 20-30 min. Site 2/3 was cleaved rapidly in trans but only after release of nsP1 from the polyprotein exposing an "activator" sequence present in the amino terminus of nsP2. Deletion of amino-terminal amino acids of nsP2 or addition of extra amino acid residues to its amino terminus specifically inhibited the protease activity that processes the 2/3 site. This sequence of delayed processing of P1234 would explain the accumulation of P123 plus nsP4, the early short-lived minus-strand replicase. The polyprotein stage would allow correct assembly and membrane association of the RNA-polymerase complex. Late in infection free nsP2 would cleave at site 2/3 yielding P12 and P34, the products of which, nsP1-4, are distributed to the plasma membrane, nucleus, cytoplasmic aggregates, and proteasomes, respectively.     

A cellular J-domain protein modulates polyprotein processing and cytopathogenicity of a pestivirus

Pestiviruses are positive-strand RNA viruses closely related to human hepatitis C virus. Gene expression of these viruses occurs via translation of a polyprotein, which is further processed by cellular and viral proteases. Here we report the formation of a stable complex between an as-yet-undescribed cellular J-domain protein, a member of the DnaJ-chaperone family, and pestiviral nonstructural protein NS2. Accordingly, we termed the cellular protein Jiv, for J-domain protein interacting with viral protein. Jiv has the potential to induce in trans one specific processing step in the viral polyprotein, namely, cleavage of NS2-3. Efficient generation of its cleavage product NS3 has previously been shown to be obligatory for the cytopathogenicity of the pestiviruses. Regulated expression of Jiv in cells infected with noncytopathogenic bovine viral diarrhea virus disclosed a direct correlation between the intracellular level of Jiv, the extent of NS2-3 cleavage, and pestiviral cytopathogenicity.