Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus.

Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence. The 5'-proximal open reading frame, A, of the prototypical hypovirulence-associated viral dsRNA, L-dsRNA, present in hypovirulent strain EP713, was recently shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase (G. H. Choi and D. L. Nuss, EMBO J. 11:473-477, 1992). We mapped this suppressive activity to the autocatalytic papain-like protease, p29, present within the amino-terminal portion of open reading frame A-encoded polyprotein p69. Mutational analysis revealed that the ability of p29 to alter fungal phenotype is dependent upon release from the polyprotein precursor but is independent of intrinsic proteolytic activity. Deletion of the p29-coding domain within the context of an infectious L-dsRNA cDNA clone resulted in a replication-competent viral dsRNA that exhibited intermediate suppressive activity while retaining the ability to confer hypovirulence. Thus, p29 is necessary but not sufficient for the level of virus-mediated suppression of fungal pigmentation, sporulation, and laccase accumulation observed for wild-type hypovirulent strain EP713 and is nonessential for viral RNA replication and virulence attenuation. These results also illustrate the feasibility of engineering infectious viral cDNA for construction of hypovirulent fungal strains with specific phenotypic traits.     

Characterization of the rubella virus nonstructural protease domain and its cleavage site.

The region of the rubella virus nonstructural open reading frame that contains the papain-like cysteine protease domain and its cleavage site was expressed with a Sindbis virus vector. Cys-1151 has previously been shown to be required for the activity of the protease (L. D. Marr, C.-Y. Wang, and T. K Frey, Virology 198:586-592, 1994). Here we show that His-1272 is also necessary for protease activity, consistent with the active site of the enzyme being composed of a catalytic dyad consisting of Cys-1151 and His-1272. By means of radiochemical amino acid sequencing, the site in the polyprotein cleaved by the nonstructural protease was found to follow Gly-1300 in the sequence Gly-1299-Gly-1300-Gly-1301. Mutagenesis studies demonstrated that change of Gly-1300 to alanine or valine abrogated cleavage. In contrast, Gly-1299 and Gly-1301 could be changed to alanine with retention of cleavage, but a change to valine abrogated cleavage. Coexpression of a construct that contains a cleavage site mutation (to serve as a protease) together with a construct that contains a protease mutation (to serve as a substrate) failed to reveal trans cleavage. Coexpression of wild-type constructs with protease-mutant constructs also failed to reveal trans cleavage, even after extended in vitro incubation following lysis. These results indicate that the protease functions only in cis, at least under the conditions tested.     

Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein define a requirement for dibasic residues for intracellular cleavage.

We investigated the amino acid sequence requirements for intracellular cleavage of the Rous sarcoma virus glycoprotein precursor by introducing mutations into the region encoding the cleavage recognition site (Arg-Arg-Lys-Arg). In addition to mutants G1 (Arg-Arg-Glu-Arg) and Dr1 (deletion of all four codons) that we have reported on previously (L. G. Perez and E. Hunter, J. Virol. 61:1609-1614, 1987), we constructed two additional mutants, AR1 (Arg-Arg-Arg-Arg), in which the highly conserved lysine is replaced by an arginine, and S19 (Ser-Arg-Glu-Arg), in which no dibasic pairs remain. The results of these studies demonstrate that when the cleavage sequence is deleted (Dr1) or modified to contain unpaired basic residues (S19), intracellular cleavage of the glycoprotein precursor is completely blocked. This demonstrates that the cellular endopeptidase responsible for cleavage has a stringent requirement for the presence of a pair of basic residues (Arg-Arg or Lys-Arg). Furthermore, it implies that the cleavage enzyme is not trypsinlike, since it is unable to recognize arginine residues that are sensitive to trypsin action. Substitution of the mutated genes into a replication-competent avian retrovirus genome showed that cleavage of the glycoprotein precursor was not required for incorporation into virions but was necessary for infectivity. Treatment of BH-RCAN-S19-transfected turkey cells with low levels of trypsin resulted in the release of infectious virus, demonstrating that exogenous cleavage could generate a biologically active glycoprotein molecule.     

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain A59.

The 21.7-kb replicase locus of mouse hepatitis virus strain A59 (MHV-A59) encodes several putative functional domains, including three proteinase domains. Encoded closest to the 5' terminus of this locus is the first papain-like proteinase (PLP-1) (S. C. Baker et al., J. Virol. 67:6056-6063, 1993; H.-J. Lee et al., Virology 180:567-582, 1991). This cysteine proteinase is responsible for the in vitro cleavage of p28, a polypeptide that is also present in MHV-A59-infected cells. Cleavage at a second site was recently reported for this proteinase (P. J. Bonilla et al., Virology 209:489-497, 1995). This new cleavage site maps to the same region as the predicted site of the C terminus of p65, a viral polypeptide detected in infected cells. In this study, microsequencing analysis of the radiolabeled downstream cleavage product and deletion mutagenesis analysis were used to identify the scissile bond of the second cleavage site to between Ala832 and Gly833. The effects of mutations between the P5 and P2' positions on the processing at the second cleavage site were analyzed. Most substitutions at the P4, P3, P2, and P2' positions were permissive for cleavage. With the exceptions of a conservative P1 mutation, Ala832Gly, and a conservative P5 mutation, Arg828Lys, substitutions at the P5, P1, and P1' positions severely diminished second-site proteolysis. Mutants in which the p28 cleavage site (Gly247 / Val248) was replaced by the Ala832 / Gly833 cleavage site and vice versa were found to retain processing activity. Contrary to previous reports, we determined that the PLP-1 has the ability to process in trans at either the p28 site or both cleavage sites, depending on the choice of substrate. The results from this study suggest a greater role by the PLP-1 in the processing of the replicase locus in vivo.     

Molecular analysis of the feline immunodeficiency virus protease: generation of a novel form of the protease by autoproteolysis and construction of cleavage-resistant proteases.

The feline immunodeficiency virus (FIV) protease is essential for virion maturation and subsequent viral replication in that it cleaves the Gag and Gag/Pol polyproteins at eight sites to release the respective structural proteins and enzymes. During purification of a recombinant FIV protease (PR), we noted that it underwent autoproteolysis (autolysis) to give discrete cleavage products. These additional PR cleavage sites were defined using N-terminal amino acid sequence analysis and mass spectrometry. Protease breakdown products were also found in FIV virions and were of the same apparent molecular weights as the in vitro autolysis products. Four primary PR autolysis sites were blocked via substitution of either the P1 amino acid with a beta-branched amino acid or the P1' amino acid with lysine. Cleavage-resistant PRs which had Km and k(cat) values similar to those of FIV PR were constructed. An autolysis time course determined that blocking all four primary autolysis sites yielded a cleavage-resistant PR which was enzymatically stable. Concomitant with autolysis is the generation of an N-terminally truncated form of the PR (Thr6/PR) which has enhanced stability with respect to that of FIV PR. A structural basis for the Thr6/PR activity is presented, as are the possible roles of autolysis in the viral replication cycle.     

Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3/ICE-like protease.

Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolished P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.     

Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition.

The leader protease of foot-and-mouth disease virus, as well as cleaving itself from the nascent viral polyprotein, disables host cell protein synthesis by specific proteolysis of a cellular protein: the eukaryotic initiation factor 4G (eIF4G). The crystal structure of the leader protease presented here comprises a globular catalytic domain reminiscent of that of cysteine proteases of the papain superfamily, and a flexible C-terminal extension found intruding into the substrate-binding site of an adjacent molecule. Nevertheless, the relative disposition of this extension and the globular domain to each other supports intramolecular self-processing. The different sequences of the two substrates cleaved during viral replication, the viral polyprotein (at LysLeuLys/GlyAlaGly) and eIF4G (at AsnLeuGly/ArgThrThr), appear to be recognized by distinct features in a narrow, negatively charged groove traversing the active centre. The structure illustrates how the prototype papain fold has been adapted to the requirements of an RNA virus. Thus, the protein scaffold has been reduced to a minimum core domain, with the active site being modified to increase specificity. Furthermore, surface features have been developed which enable C-terminal self-processing from the viral polyprotein.     

Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight.

The complete nucleotide sequence of the largest double-stranded (ds) RNA present in hypovirulent strain EP713 of the chestnut blight pathogen, Cryphonectria parasitica, was determined and the predicted genetic organization was confirmed by translational mapping analysis. The deduced RNA sequence was 12 712 bp in length, excluding the terminal poly(A):poly(U) homopolymer domain. The strand terminating with 3'-poly(A) contained two contiguous large open reading frames (ORF A and ORF B) beginning at nucleotide residues 496-498 and extending to nucleotide positions 11 859-11 861. The junction between ORF A and ORF B consisted of the sequence 5'-UAAUG-3', where UAA served as the termination codon for ORF A and AUG was the 5'-proximal initiation codon within ORF B. ORF A (622 codons in length, excluding the termination codon) was recently shown to encode two polypeptides, p29 and p40, which were generated from a nascent polyprotein by an autocatalytic event mediated by p29 (Choi et al., 1991). A similar autocatalytic event was observed during in vitro translation of ORF B (3165 codons in length) resulting in the release of a 48 kd polypeptide from the amino-terminal portion of the ORF B-encoded polyprotein. These results are discussed in terms of the opportunities they provide for elucidating the molecular basis of transmissible hypovirulence and possible origins of hypovirulence-associated dsRNAs.     

DNA recombination and RNA cleavage activities of the Flp protein: roles of two histidine residues in the orientation and activation of the nucleophile for strand cleavage.

Using a combination of DNA and hybrid DNA-RNA substrates, we have analyzed the mechanism of phosphoryl transfer by the Flp site-specific recombinase in three different reactions: DNA strand breakage and joining, and two types of RNA cleavage activities. These reactions were then used to characterize Flp variants altered at His309 and His345, amino acid residues that are in close proximity to two key catalytic residues (Arg308 and Tyr343). These histidine residues are important for strand cutting by Tyr343, the active-site nucleophile of Flp, but neither residue contributes to the type II RNA cleavage activity or to the strand-joining reaction in a pre-cleaved substrate. Strand cleavage reactions using small, diffusible nucleophiles indicate that this histidine pair contributes to the correct positioning and activation of Tyr343 within the shared active site of Flp. The implications of these results are evaluated against the recently solved crystal structure of Flp in association with a Holliday junction.     

Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease.

In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation.     

Functional correlation between a novel amino acid insertion at codon 19 in the protease of human immunodeficiency virus type 1 and polymorphism in the p1/p6 Gag cleavage site in drug resistance and replication fitness

Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1' position (Leu to Pro) and the P5' position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.     

Pearl millet cysteine protease inhibitor. Evidence for the presence of two distinct sites responsible for anti-fungal and anti-feedent activities.

Recently, pearl millet cysteine protease inhibitor (CPI) was, for the first time, shown to possess anti-fungal activity in addition to its anti-feedent (protease inhibitory) activity [Joshi, B.N. et al. (1998) Biochem. Biophys. Res. Commun. 246, 382-387]. Characterization of CPI revealed that it has a reversible mode of action for protease inhibition. The CD spectrum exhibited a 35% alpha helix and 65% random coil structure. The intrinsic fluorescence spectrum was typical of a protein devoid of tryptophan residues. Demetallation of Zn2+ resulted in a substantial change in the secondary and tertiary structure of CPI accompanied by the complete loss of anti-fungal and inhibitory activity indicating that Zn2+ plays an important role in maintaining both structural integrity and biological function. The differential response of anti-fungal and inhibitory activities to specific modifiers showed that there are two different reactive sites associated with anti-fungal and anti-feedent activity in CPI located on a single protein as revealed from its N-terminal sequence data (AGVCYGVLGNNLP). Modification of cysteine, glutamic/aspartic acid or argnine resulted in abolition of the anti-fungal activity of CPI, whereas modification of arginine led to an enhancement of the inhibitory activity in solution. Modification of histidine resulted in a twofold increase in the protease inhibitory activity without affecting the anti-fungal activity, whereas modification of serine led to selective inhibition of the protease inhibitory activity. The differential nature of the two activities was further supported by differences in the temperature stabilities of the anti-fungal (60 degrees C) and inhibitory (40 degrees C) activities. Binding of papain to CPI did not abolish the anti-fungal activity of CPI, supporting the presence of two active sites on CPI. The differential behavior of CPI towards anti-fungal and anti-feedent activity cannot be attributed to changes in conformation, as assessed by their CD and fluorescence spectra. The interaction of CPI modified for arginine or histidine with papain resulted in an enhancement of CPI activity accompanied by a slight decrease in fluorescence intensity of 15-20% at 343 nm. In contrast, modification of serine resulted in inhibition of CPI activity with a concomitant increase of 20% in the fluorescence intensity when complexed by the enzyme. This implies the involvement of enzyme-based tryptophan in the formation of a biologically active enzyme-inhibitor complex. The presence of anti-fungal and anti-feedent activity on a single protein, as evidenced in pearl millet CPI, opens up a new possibility of raising a transgenic plant resistant to pathogens, as well as pests, by transfer of a single CPI gene.     

Norwalk virus nonstructural protein p48 forms a complex with the SNARE regulator VAP-A and prevents cell surface expression of vesicular stomatitis virus G protein

Norwalk virus (NV), a reference strain of human calicivirus in the Norovirus genus of the family Caliciviridae, contains a positive-strand RNA genome with three open reading frames. ORF1 encodes a 1,789-amino-acid polyprotein that is processed into nonstructural proteins that include an NTPase, VPg, protease, and RNA-dependent RNA polymerase. The N-terminal protein p48 of ORF1 shows no significant sequence similarity to viral or cellular proteins, and its function in the human calicivirus replication cycle is not known. The lack of sequence similarity to any protein in the public databases suggested that p48 may have a unique function in the NV replication cycle or, alternatively, may perform a characterized function in replication by a unique mechanism. In this report, it is shown that p48 displays a vesicular localization pattern in transfected cells when fused to the fluorescent reporter EYFP. A predicted transmembrane domain at the C terminus of p48 was not necessary for the observed localization pattern, but this domain was sufficient to redirect localization of EYFP to a fluorescent pattern consistent with the Golgi apparatus. A yeast two-hybrid screen identified the SNARE regulator vesicle-associated membrane protein-associated protein A (VAP-A) as a binding partner of p48. Biochemical assays confirmed that p48 and VAP-A interact and form a stable complex in mammalian cells. Furthermore, expression of the vesicular stomatitis virus G glcyoprotein on the cell surface was inhibited when cells coexpressed p48, suggesting that p48 disrupts intracellular protein trafficking.     

Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus.

The ribonucleoprotein transfection system for influenza virus allowed us to construct two influenza A viruses, GP2/BIP-NA and HGP2/BIP-NA, which contained bicistronic neuraminidase (NA) genes. The mRNAs derived from the bicistronic NA genes have two different open reading frames (ORFs). The first ORF encodes a foreign polypeptide (GP2 or HGP2) containing amino acid sequences derived from the gp41 protein of human immunodeficiency virus type 1. The second ORF encodes the NA protein; its translation is achieved via an internal ribosomal entry site which is derived from the 5' noncoding region of the human immunoglobulin heavy-chain-binding protein mRNA. The GP2 (79 amino acids) and HGP2 (91 amino acids) polypeptides are expressed in cells infected with the corresponding transfectant virus. The HGP2 polypeptide, which contains transmembrane and cytoplasmic domains identical to those of the hemagglutinin (HA) protein of influenza A/WSN/33 virus, is packaged into virus particles. This novel influenza virus system involving an internal ribosomal entry site element may afford a way to express a variety of foreign genes in mammalian cells.