Identification and characterization of a protein destruction signal in the encephalomyocarditis virus 3C protease

The amino acid sequence LLVRGRTLVV, which is probably located in a strand-turn-strand structure, has been identified as a protein destruction signal in the rapidly degraded encephalomyocarditis virus 3C protease. Mutations within this sequence reduced the susceptibility of the 3C protease toward ubiquitination and degradation in rabbit reticulocyte lysate. This signal is transferable, since poliovirus 3C protease, which is a poor ubiquitin-mediated proteolytic system substrate, was found to be ubiquitinated and degraded when the signal sequence was either generated at an internal location in the protein or fused to the N terminus. An evaluation of the behavior of 3C protease proteins containing mutations in the signal region indicates that considerable variability in the primary structure is tolerated, although the conservation of certain features appears to be required for signal function. Two E3 ubiquitin-protein ligases that recognize the encephalomyocarditis virus 3C protease as a substrate were also partially purified. One of these was identified as the previously described E3alpha, and this was shown to require the destruction signal sequence to catalyze efficiently the ubiquitination of the 3C protease. The other is a Ubc5-dependent E3 that appears to recognize a different, unidentified feature of the 3C protease.     

Stimulation of encephalomyocarditis virus RNA translation in wheat embryo extracts by rabbit reticulocyte ribosomal wash.

Encephalomyocarditis virus RNA induced a low level of amino acid incorporation in wheat embryo extracts, but the incorporation did not result in the formation of any viral proteins. However, if wheat embryo extracts were supplemented with a ribosomal wash fraction from rabbit reticulocytes, a large stimulation of amino acid incorporation and the formation of encephalomyocarditis virus-specific proteins were obtained. Among the proteins synthesized were capsid precursor proteins which can be processed by an encephalomyocarditis virus-coded protease to produce the viral capsid proteins.     

Evidence for intramolecular self-cleavage of picornaviral replicase precursors.

It has previously been shown that when encephalomyocarditis viral RNA is translated in cell-free extracts of rabbit reticulocytes, it synthesizes a virus-coded protease, p22, which is derived by cleavage of a precursor protein, C. Protein C is shown here to be cleaved by two different mechanisms, which were distinguished by their sensitivity to dilution. One mechanism was sensitive to dilution; the other was not. The biphasic cleavage behavior was unchanged by diluting incubation mixtures with untranslated reticulocyte extract instead of buffer, suggesting that both types of cleavage were mediated by virus translation products. It is proposed that the dilution-sensitive cleavage of protein C is due to a virus-coded protease, probably p22 itself, and that the dilution-independent cleavage is due to intramolecular self-cleavage of protein C.     

Processing of the Encephalomyocarditis Virus Capsid Precursor Protein Studies in Rabbit Reticulocyte Lysates Incubated with N-Formyl-[35S]Methionine-tRNAfMet

Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates in the presence of N-formyl-[(35)S]methionine-tRNA(f) (Met) revealed that a small polypeptide is cleaved from the N-terminus of the capsid protein precursor, preA, by virus-coded protease activity. Therefore, this N-terminal segment comprising the translation initiation site is not conserved in any of the mature capsid proteins.     

Proteolytic cleavage of encephalomyocarditis virus capsid region substrates by precursors to the 3C enzyme.

Picornavirus protease 3C is normally released from its P3 precursor by two successive self-cleavage reactions. The free enzyme can then catalyze most of the remaining processing events within the viral polyprotein. To investigate the role of the 3C precursors in the processing cascade, we constructed cDNA clones which expressed genetically altered forms of the encephalomyocarditis P3 region in vitro. Site-specific substitutions were introduced into the Gln-Gly residues at the 3B-3C and 3C-3D junctions, and the resulting proteins were tested for their ability to self-process and to catalyze cleavage of viral capsid precursors in cell-free protease assays. We determined that three P3 region precursor proteins (3ABC, 3CD, and P3), harboring inactive cleavage sites, were as active as the free enzyme (3C) in processing assays with capsid substrates. Further, we found that in addition to the naturally occurring Gln-Gly and Gln-Ser amino acid pairs, the encephalomyocarditis 3C enzyme was able to process Gln-Cys but not Gln-Thr, Gln-Ile, Gln-Tyr, Arg-Gly, or Leu-Gly combinations when these residues were substituted into normal cleavage site contexts.     

Site-specific mutations at a picornavirus VP3/VP1 cleavage site disrupt in vitro processing and assembly of capsid precursors.

Most proteolytic cleavages within the picornavirus polyproteins are carried out by viral protease 3C. For encephalomyocarditis virus, the protease 3C-catalyzed processing occurs between Gln-Gly or Gln-Ser amino acid pairs which are flanked by proline residues, but the sequence-specific constraints on recognition and cleavage by the enzyme are not completely understood. To examine alternative cleavage site sequences, we constructed a cDNA plasmid which expresses the viral L-P1-2A capsid precursor in vitro and introduced site-specific mutations into the Gln-Gly pair at the VP3/VP1 junction. The altered protein substrates were tested for cleavage activity in assays with protease 3C. The encephalomyocarditis virus 3C processed Gln-Ala as efficiently as its natural sites but did not cleave Gln-Val, Gln-Glu, Lys-Gly, Lys-Ala, Lys-Val, Lys-Glu, or Pro-Gly combinations. Displacement of the flanking proline residue by an engineered insertion slowed but did not prevent cleavage at this site. Also, a mutant defective in processing at the VP3/VP1 junction was unable to form 14S pentameric assembly intermediates in vitro.     

Translation of encephalomyocarditis virus RNA in vitro yields an active proteolytic processing enzyme.

In contrast to other cell-free translation systems, the mRNA-dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome.     

Cleavage of the capsid protein precursors of encephalomyocarditis virus in rabbit reticulocyte lysates

The cleavage pathway of the three capsid protein precursors of encephalomyocarditis virus, proteins A1, A, and B, was studied in rabbit reticulocyte lysates. Kinetic data suggested that the three proteins were cleaved through a sequential of A1 to A to B.     

Encephalomyocarditis Virus 3C Protease Relieves TRAF Family Member-associated NF-κB Activator (TANK) Inhibitory Effect on TRAF6-mediated NF-κB Signaling through Cleavage of TANK

TRAF family member-associated NF-κB activator (TANK) is a negative regulator of canonical NF-κB signaling in the Toll-like receptor- and B-cell receptor-mediated signaling pathways. However, functions of TANK in viral infection-mediated NF-κB activation remain unclear. Here, we reported that TANK was cleaved by encephalomyocarditis virus 3C at the 197 and 291 glutamine residues, which depends on its cysteine protease activity. In addition, encephalomyocarditis virus 3C impaired the ability of TANK to inhibit TRAF6-mediated NF-κB signaling. Interestingly, we found that several viral proteases encoded by the foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, and equine arteritis virus also cleaved TANK. Our results suggest that TANK is a novel target of some viral proteases, indicating that some positive RNA viruses have evolved to utilize their major proteases to regulate NF-κB activation.     

Characterization of the Large Picornaviral Polypeptides Produced in the Presence of Zinc Ion

Zinc ion inhibits the posttranslational cleavages of human rhinovirus-1A, encephalomyocarditis virus, and poliovirus polypeptides. Each virus displayed a different susceptibility to zinc. However, in each case the cleavages of the capsid precursor and the cleavages analogous to the C --> D --> E conversion in encephalomyocarditis virus were most sensitive to zinc. Higher concentrations of zinc resulted in the buildup of even larger precursor polypeptides of a size between 106,000 and 214,000 daltons. The sizes of these polypeptides and the relative position of their gene loci on the viral RNA were determined. These data were used to place these polypeptides in the over-all scheme of viral protein processing.     

Picornaviral VPg sequences are contained in the replicase precursor.

It has previously been shown that the RNA replicase of encephalomyocarditis virus contains two virus-coded proteins, D and E, which are produced in two successive proteolytic steps: (i) C leads to D + ?; and (ii) D leads to p22 + E. It is here shown (i) that virus protein H (molecular weight, 12,000) is the previously unidentified product of the first step and (ii) that VPg, a protein linked covalently to the virion RNA, yields two tryptic peptides found in protein C but not in protein D. The results suggest that VPg is derived by cleavage of protein C and that protein H may be intermediate. Preliminary experiments with VPg sequences in polioviral noncapsid protein 1b, the counterpart of encephalomyocarditis viral protein C, were inconclusive.     

Virus-induced diabetes mellitus: mengovirus infects pancreatic beta cells in strains of mice resistant to the diabetogenic effect of encephalomyocarditis virus.

In mice, Mengovirus produces a fatal encephalitis. Plaque purification of the virus resulted in the isolation of a clone (Mengo- 2T ), which in addition to encephalitis caused diabetes. Microscopic examination of pancreases from infected mice revealed necrosis in the islets of Langerhans and infiltration of inflammatory cells. By immunofluorescence viral antigens were found in the islets, and radioimmunoassays demonstrated a substantial decrease in pancreatic immunoreactive insulin. Studies on susceptibility among inbred strains of mice showed that whereas the D variant of encephalomyocarditis virus caused diabetes only in SJL/J mice, Mengo- 2T caused diabetes in strains of mice resistant to encephalomyocarditis-induced diabetes (i.e., CBA/J, C3H/HeJ, CE/J, AKR/J, C57BL/6J). The ability of Mengo- 2T to induce diabetes in encephalomyocarditis-resistant mice was found to be due to the greater capacity of Mengo- 2T as compared to the D variant of encephalomyocarditis virus to replicate in and destroy the islets of these animals. Although Mengo- 2T and the D variant of encephalomyocarditis virus are antigenically indistinguishable by hyperimmune sera, our studies show that these viruses have different host ranges and tissue tropisms .     

Kinetic analysis of the conjugation of ubiquitin to picornavirus 3C proteases catalyzed by the mammalian ubiquitin-protein ligase E3alpha

The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E2(14Kb), and human E3alpha. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K(m) = 42 +/- 11 microm and V(max) = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K(m) = 20 +/- 5 microm and V(max) = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The K(m) and V(max) values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a k(cat) effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K(m) and V(max) values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.     

Processing of a pestivirus protein by a cellular protease specific for light chain 3 of microtubule-associated proteins

The genome of the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) JaCP contains a cellular insertion coding for light chain 3 (LC3) of microtubule-associated proteins, the mammalian homologue of yeast Aut7p/Apg8p. The cellular insertion induces cp BVDV-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease Aut2p/Apg4p. Three candidate bovine protease genes were identified on the basis of the sequence similarity of their products with the Saccharomyces cerevisiae enzyme. The search for a system for functional testing of these putative LC3-specific proteases revealed that the components involved in this processing have been highly conserved during evolution, so that the substrate derived from a mammalian virus is processed in cells of mammalian, avian, fish, and insect origin, as well as in rabbit reticulocyte lysate, but not in wheat germ extracts. Moreover, two of these proteases and a homologous protein from chickens were able to rescue the defect of a yeast AUT2 deletion mutant. In coexpression experiments with yeast and wheat germ extracts one of the bovine proteases and the corresponding enzyme from chickens were able to process the viral polyprotein containing LC3. Northern blots showed that bovine viral diarrhea virus infection of cells has no significant influence on the expression of either LC3 or its protease, bAut2B2. However, LC3-specific processing of the viral polyprotein containing the cellular insertion is essential for replication of the virus since mutants with changes in the LC3 insertion significantly affecting processing at the LC3/NS3 site were not viable.     

Inducible expression of encephalomyocarditis virus 3C protease activity in stably transformed mouse cell lines.

An inducible expression vector system has been developed to facilitate the study of the effects of individual virus gene products on cell function. The vector utilizes the mouse metallothionein promoter carried on the bovine papillomavirus genome. Conditions which optimize the induced expression of open reading frames inserted downstream from the mouse metallothionein promoter have recently been described. In this communication we describe the use of this system to clone and express the encephalomyocarditis virus 3C protease in cultured mouse cells. Stably transformed cell lines could be induced to produce levels of 3C protease activity comparable to those observed during normal virus infection. In spite of this, no effects on cellular protein synthesis rate or membrane permeability were observed. It was also discovered that 3C protease as well as 3C protease-containing polyproteins are turned over. This was true not only in the induced cell clones, but also during the normal course of encephalomyocarditis virus infection, as well as in translation systems in vitro. This phenomenon was highly specific for this family of polypeptides, perhaps explaining their apparent lack of cytotoxic effects.     

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus.

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38 degrees C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavage-defective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wild-type capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38 degrees C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38 degrees C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cell-free translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration.     

Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.     

Characterization of the proteins of intracisternal type A and extracellular oncornavirus-like particles produced by MOPC-460 myeloma cells.

A number of translation inhibitors were tested for their effects on both control and encephalomyocarditis virus-infected mouse 3T6 cells. The virus-infected cells were specifically inhibited by gougerotin, edeine, and blasticidin S, whereas these drugs failed to penetrate into uninfected cells. Inhibition of infected cells by gougerotin became apparent when the synthesis of viral proteins commenced, suggesting that the latter process is accompanied by a permeability change in the cells that allows uptake of the drug. This permeability change was not observed in cells treated with cycloheximide soon after viral infection, although treatment with actinomycin D did not prevent inhibition of gougerotin. It is possible, therefore, that a specific viral protein is involved in the permeability change of the plasma membrane. Moreover, gougerotin was unable to inhibit protein synthesis in the presence of zinc ions, thus preventing gougerotin from entering into the infected cell. Membrane leakiness was not restricted to the encephalomyocarditis virus-3T6 system; it was also observed in mengovirus-infected 3T6 cells, Semliki Forest virus-infected BHK cells, and simian virus 40-infected CVI1 cells at the time in which the synthesis of late proteins is maximal.     

A new splice variant of the human guanylate-binding protein 3 mediates anti-influenza activity through inhibition of viral transcription and replication

Guanylate-binding proteins (GBPs) belong to the family of large GTPases that are induced in response to interferons. GBPs contain an N-terminal globular GTPase domain and a C-terminal α-helical regulatory domain that are connected by a short middle domain. Antiviral activity against vesicular stomatitis virus and encephalomyocarditis virus has been shown for hGBP-1; however, no anti-influenza virus properties for GBPs have been described to date. Here we show that hGBP-1 and hGBP-3 possess anti-influenza viral activity. Furthermore, we have identified a novel splice variant of hGBP-3, named hGBP-3ΔC, with a largely modified C-terminal α-helical domain. While all three GBP isoforms were up-regulated on influenza virus infection, hGBP-3ΔC showed the most prominent antiviral activity in epithelial cells. Mutational analysis of hGBPs revealed that the globular domain is the principal antiviral effector domain, and GTP-binding, but not hydrolysis, is necessary for antiviral action. Furthermore, we showed that hGBP-3ΔC strongly represses the activity of the viral polymerase complex, which results in decreased synthesis of viral vRNA, cRNA, mRNA, and viral proteins, as well.