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PubMed1992

Specific inhibitor of human immunodeficiency virus proteinase prevents the cytotoxic effects of a single-chain proteinase dimer and restores particle formation.

The active form of the retroviral proteinase (PR) is a homodimer of monomeric subunits expressed as integral parts of the viral gag-pol precursor polyproteins, and dimerization of polyproteins is presumed to be important for regulation of PR activity. Expression of a single-chain dimer of the human immunodeficiency virus (HIV) type 1 PR as a component of the viral polyprotein has been shown to prevent particle assembly and viral infectivity (H.-G. Kräusslich, Proc. Natl. Acad. Sci. USA 88:3213-3217, 1991). Ro31-8959, a specific inhibitor of HIV PR, blocked proteolysis of polyproteins containing either wild-type or single-chain dimer PR at the same inhibitor concentration. Different inhibitor concentrations gave three phenotypic effects for the linked PR: at a concentration of 10 nM, cytotoxicity was prevented yet viral polyproteins were almost completely processed and no particles were released. The majority of HIV capsid proteins was found in the soluble cytoplasmic fraction, whereas at a concentration of 1 microM inhibitor most HIV gag proteins were associated with an insoluble fraction. Release of particles consisting of partially processed polyproteins was observed at 100 nM Ro31-8959, and polyprotein processing was blocked at 10 microM. Particles derived from the dimer-containing provirus were noninfectious independently of the inhibitor concentration. Production of infectious HIV after transfection of wild-type provirus was abolished at 100 nM and markedly reduced at 10 nM Ro31-8959.

Blotting, Western↗
PubMed1993

Expression of poliovirus P3 proteins using a recombinant vaccinia virus results in proteolytically active 3CD precursor protein without further processing to 3Cpro and 3Dpol.

The expression of the poliovirus genome occurs by the translation of a single open reading frame to generate a long polyprotein which is subsequently processed by viral encoded proteases. The initial proteolytic cleavages result in the production of a P1 polyprotein which contains the capsid proteins, and the P2 and P3 polyproteins which contain proteins required for replication. The P3 polyprotein consists of the 3AB protein (containing the viral genome-linked protein, VPg), the viral protease, 3Cpro, and RNA polymerase, 3Dpol. To further study the expression and proteolytic processing of poliovirus P3 proteins in vivo, we have utilized recombinant vaccinia virus vectors to express nucleotides 5240-7400 containing the P3 region proteins of poliovirus. The P3 protein expressed from the recombinant vaccinia virus VV-P3 exhibited in vivo proteolytic activity as evident by processing of the polyprotein to generate the 3CD protein, consisting of a fusion between the 3Cpro and 3Dpol proteins. Further processing of the 3CD protein to 3Cpro and 3Dpol, however, was not detected in cells infected with VV-P3. Subcellular fractionation of VV-P3-infected cells demonstrated that the 3CD protein was present in both the soluble and membrane fractions. Finally, the 3CD protein expressed from VV-P3 was stable in cells co-infected with VV-P3 and poliovirus and no further processing to 3Dpol was detected. These results are discussed with regards to in vivo studies which suggest that the 3CD polyprotein is not a precursor to 3Dpol in poliovirus-infected cells.

Capsid↗
PubMed2004

Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts.

Alphavirus nonstructural proteins are translated as a polyprotein that is ultimately cleaved into four mature proteins called nsP1, nsP2, nsP3, and nsP4 from their order in the polyprotein. The role of this nonstructural polyprotein, of cleavage intermediates, and of mature proteins in synthesis of Semliki Forest virus (SFV) RNA has been studied using mutants unable to cleave one or more of the sites in the nonstructural polyprotein or that had the arginine sense codon between nsP3 and nsP4 changed to an opal termination codon. The results were compared with those obtained for Sindbis virus (SINV), which has a naturally occurring opal codon between nsP2 and nsP3. We found that (1) an active nonstructural protease in nsP2 is required for RNA synthesis. This protease is responsible for all three cleavages in the nonstructural polyprotein. (2) Cleavage between nsP3 and nsP4 (the viral RNA polymerase) is required for RNA synthesis by SFV. (3) SFV mutants that are able to produce only polyprotein P123 and nsP4 synthesize minus-strand RNA early after infection as efficiently as SF wild type but are defective in the synthesis of plus-strand RNA. The presence of sense or opal following nsP3 did not affect this result. At 30 degrees C, they give rise to low yields of virus after a delay, but at 39 degrees C, they are nonviable. (4) SFV mutants that produce nsP1, P23, nsP4, as well as the precursor P123 are viable but produce an order of magnitude less virus than wild type at 30 degrees C and two orders of magnitude less virus at 39 degrees C. The ratio of subgenomic mRNA to genomic RNA is much reduced in these mutants relative to the parental viruses. (5) At 30 degrees C, the variants containing an opal codon grow as well as or slightly better than the corresponding virus with a sense codon. At 39 degrees C, however, the opal variants produce significantly more virus. These results support the conclusion that SFV and SINV, and by extension all alphaviruses, regulate their RNA synthesis in the same fashion after infection. P123 and nsP4 form a minus-strand replicase that synthesizes plus-strand RNA only inefficiently, especially at the higher temperatures found in mammals and birds. A replicase containing nsP1, P23, and nsP4 can make both plus and minus strands, but prefers the promoter for genomic plus sense RNA to that for subgenomic mRNA. The fully cleaved replicase can make only plus-strand RNA, and prefers the promoter for subgenomic mRNA to that for genomic RNA. Alphaviruses alternate between infection of hematophagous arthropods and higher vertebrates. Although the infection of higher vertebrates is acute and often accompanied by disease, continuing transmission of the virus in nature requires that infection of arthropods be persistent and relatively asymptomatic. We propose that this mechanism for control of RNA synthesis evolved to moderate the pathogenicity of the viruses in their arthropod hosts.

Animals↗
PubMed1997

The cleavage activities of aphthovirus and cardiovirus 2A proteins.

The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theiler's murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-deltaTME2A-GUS] polyprotein was able to mediate cleavage with high (approximately 85%) efficiency--directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT-delta EMC2A-GUS] polyprotein which also mediated cleavage at approximately 85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product.

Amino Acid Sequence↗
PubMed1997

Removal of human immunodeficiency virus type 1 (HIV-1) protease inhibitors from preparations of immature HIV-1 virions does not result in an increase in infectivity or the appearance of mature morphology.

The processing of gag and gag-pol polyproteins by human immunodeficiency virus type 1 (HIV-1) protease is a crucial step in the formation of infectious HIV-1 virions. In this study, we examine whether particles produced in the presence of inhibitors of HIV-1 protease can subsequently undergo gag polyprotein cleavage with restoration of infectivity following removal of the inhibitors. Viral particles produced during 7 days of culture in the presence of the protease inhibitors KNI-272 (10 microM) and saquinavir (5 microM) contained predominantly p55gag polyprotein but little or no p24gag cleavage product. Following resuspension of the particles in medium free of the inhibitor, some gag polyprotein processing was detected in particles produced from the KNI-272-treated cells, but not from the saquinavir-treated cells within the first 3 h. However, the majority of the protein remained as p55gag throughout a 48-h experimental period. The infectivity (50% tissue culture infective dose per milliliter) of the viral particles from KNI-272-treated cells was 10(6)-fold lower than that of control particles and did not significantly increase over the 48 h after the inhibitor was removed, despite the apparent return of protease function in a subset of these virions. This failure to restore infectivity was due neither to a reduction in the number of particles produced by protease inhibitor-treated cells nor to a failure of HIV RNA to be packaged in the virions. These particles also failed to express the mature phenotype by electron microscopy. Thus, while some processing of the gag polyprotein can occur in isolated HIV virions, this does not appear to be sufficient to restore infectivity in the majority of particles. This finding suggests that there may be constraints on postbudding polyprotein processing in the production of viable particles. These results should have positive implications regarding the use of protease inhibitors as anti-HIV drugs.

Blotting, Western↗
PubMed1972

Cleavage of viral precursor proteins in vivo and in vitro.

The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages.

Animals↗
PubMed1988

A nucleotide substitution in the gag N terminus of the endogenous ecotropic DBA/2 virus prevents Pr65gag myristylation and virus replication.

The endogenous ecotropic provirus Emv-3 present in DBA/2 mice is poorly expressed in the animal, as well as in cell cultures. Transfection of proviral DNA into NIH 3T3 cells localized the expression defect to the 5' region of the viral genome, spanning the untranslated region and the N-terminal part of the gag gene. Comparison of the nucleotide sequence of the Emv-3 provirus with the sequence of the highly infectious Akv murine leukemia virus revealed three nucleotide differences within the gag coding region. One of these differences was found in codon 3 of the gag polyprotein, where a Gln codon is seen in Akv and a Pro codon is differences was found in codon 3 of the gag polyprotein, where a Gln codon is seen in Akv and a Pro codon is seen in Emv-3. By site-directed mutagenesis, we showed that the defect of Emv-3 expression indeed is localized to codon 3 of the gag gene. The gag polyprotein of mammalian type C retrovirus contains myristic acid covalently linked to the N-terminal glycine. This myristylation is not seen in the Emv-3-coded gag polyprotein. We showed that the in vitro-mutagenized Emv-3 genome containing a Gln codon at position 3 of the gag gene yields a myristylated gag polyprotein. Thus, it seems most likely that the defect of expression of the Emv-3 provirus is due to the presence of a proline is position 3 of the gag polyprotein, preventing the myristylation.

Animals↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1994

Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants.

The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its N- and C-terminal regions, respectively. We reported previously that a 30-kDa protease is derived by autoproteolytic processing of a polyprotein which is the translation product of the entire UL80 open reading frame (E. Z. Baum, G. A. Bebernitz, J. D. Hulmes, V. P. Muzithras, T. R. Jones, and Y. Gluzman, J. Virol. 67:497-506, 1993). Three autoproteolytic cleavage sites within the UL80 polyprotein were characterized; site 143 is within the protease domain and inactivates the protease. In this article, we report (i) expression analyses of UL80 in infected cells, including the processing kinetics of the UL80 polyprotein; (ii) the existence of an additional cleavage site (site 209) within the protease domain of the UL80 polyprotein; and (iii) the effect of mutagenesis at each of the cleavage sites upon proteolytic activity and steady-state levels of the UL80 processing products. During the course of infection, UL80 polyprotein processing begins at cleavage site 643 and follows at sites 256 and 143. Cleavage at site 643 and/or 256 within the polyprotein is not a prerequisite for efficient protease activity, since all three proteases (85-, 80-, and 30-kDa proteins) were equally active in cleaving the assembly protein precursor to its mature form. Inhibition of cleavage at site 143 resulted in a three- to sixfold increase in the steady-state level of the 30-kDa protease, supporting the hypothesis that cleavage at this site may represent a mechanism by which cytomegalovirus regulates the level of active protease.

Amino Acid Sequence↗
PubMed1995

The p6gag domain of human immunodeficiency virus type 1 is sufficient for the incorporation of Vpr into heterologous viral particles.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a virion-associated regulatory protein. Mutagenesis has shown that the virion association of Vpr requires sequences near the C terminus of the HIV-1 Gag polyprotein Pr55gag. To investigate whether Vpr incorporation is mediated by a specific domain of Pr55gag, we examined the ability of chimeric HIV-1/Moloney murine leukemia virus (MLV) Gag polyproteins to direct the incorporation of Vpr. Vpr expressed in trans did not associate with particles formed by the authentic MLV Gag polyprotein or with particles formed by chimeric Gag polyproteins that had the matrix (MA) or capsid (CA) domain of MLV precisely replaced by the corresponding domain of HIV-1HXB2. By contrast, Vpr was efficiently incorporated upon replacement of the C-terminal nucleocapsid (NC) domain of the MLV Gag polyprotein with HIV-1 p15 sequences. Vpr was also efficiently incorporated into particles formed by a MLV Gag polyprotein that had the HIV-1 p6 domain fused to its C terminus. Furthermore, a deletion analysis revealed that a conserved region near the C terminus of the p6 domain is essential for Vpr incorporation, whereas sequences downstream of the conserved region are dispensable. These results show that a virion association motif for Vpr is located within residues 1 to 46 of p6.

Amino Acid Sequence↗
PubMed1995

Domains upstream of the protease (PR) in human immunodeficiency virus type 1 Gag-Pol influence PR autoprocessing.

A critical step in the formation of infectious retroviral particles is the activation of the virally encoded protease (PR) and its release from the Gag-Pol precursor polyprotein. To identify factors that influence this step, the maturation of human immunodeficiency virus type 1 PR from various Gag-PR polyproteins was assayed in vitro by a using rabbit reticulocyte lysate as a coupled transcription-translation-autoprocessing system. Highly efficient autoprocessing was detected with polyproteins containing the viral nucleocapsid (NC) domain. In contrast, polyproteins consisting of only p6 and PR domains or containing a truncated NC domain exhibited no autoprocessing activity. Experiments designed to test the dimerization capability of short PR polyproteins revealed that precursors containing the NC domain exhibited very efficient homotypic protein-protein interactions while PR precursors consisting of only p6 and PR did not interact efficiently. The strong correlation between autoprocessing activity and PR polyprotein precursor dimerization suggests that NC and p6* domains play a role in PR activation by influencing the dimerization of the PR domain in the precursor.

Amino Acid Sequence↗
PubMed1995

Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase.

The genome of the hepatitis C virus directs the synthesis of a single polyprotein, which is proteolytically cleaved into at least nine functional proteins. The amino-terminal portion of the polyprotein forms the structural proteins, while the carboxy-terminal region constitutes a variety of viral enzymes. The nonstructural 3 (NS3) protein, consisting of amino acids 1027-1657 of the polyprotein, is believed to be a multifunctional protein with an amino-terminal serine protease domain, which is involved in polyprotein processing, and a carboxy-terminal ATPase/RNA helicase domain, presumably involved in viral replication. We have assembled an expression vector which directs the synthesis of residues 1207-1612 of the polyprotein with an amino-terminal polyhistidine purification tag. This portion of the NS3 protein contains the putative ATPase/helicase domain. The protein has been purified to yield 30-50 mg of enzymatically active protein per liter of culture. The purified NS3 protein has both NTPase and RNA helicase activities. ATP is the preferred substrate for the NTPase; GTP is also utilized; however, UTP is a very poor substrate and CTP is not utilized. The RNA helicase activity is dependent on ATP and divalent cation. Either manganese or magnesium can serve as the divalent cation.

Adenosine Triphosphatases↗
PubMed1994

Determinants of the p28 cleavage site recognized by the first papain-like cysteine proteinase of murine coronavirus.

The murine coronavirus polymerase gene is 22 kb in length with the potential to encode a polyprotein of approximately 750 kDa. The polyprotein has been proposed to encode three proteinase domains which are responsible for the processing of the polyprotein into mature proteins. The proteolytic activity of the first proteinase domain has been characterized and resembles the papain family of cysteine proteinases. This proteinase domain acts autoproteolytically to cleave the amino terminal portion of the polymerase polyprotein, releasing a 28-kDa protein designated p28. To identify the cleavage site of this papain-like cysteine proteinase, we isolated the peptide adjacent to p28 and determined the amino terminus sequence by Edman degradation reaction. We report that proteolysis occurs between the Gly-247 and Val-248 dipeptide bond. To determine the role of the amino acid residues surrounding the cleavage site, we introduced a total of 42 site-specific mutations at the residues spanning the P5 to P3' positions and assessed the effects of the mutations on the processing of p28 in an in vitro transcription and translation system. The substitutions of Gly-247 at the P1 position or Arg-246 at the P2 position resulted in a dramatic decrease of proteolytic activity, and the mutations of Arg-243 at P5 position also led to considerable reduction in p28 cleavage. In contrast, the substitutions of amino acids Gly-244 (P4), Tyr-245 (P3), Val-248 (P1'), Lys-249 (P2'), and Pro-250 (P3') had little or no effect on the amount of p28 that was released. This work had identified Gly-247-Val-248 as the cleavage site for the release of p28, the amino-terminal protein of the murine coronavirus polymerase polyprotein. Additionally, we conclude that the Gly-247 and Arg-246 are the major determinants for the cleavage site recognition by the first papain-like cysteine proteinase of murine coronavirus.

Amino Acid Sequence↗
PubMed1997

Sequence and phylogenetic analyses of highly virulent infectious bursal disease virus.

The nucleotide sequences of the genome segments A and B encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3039 nucleotides (1012 deduced amino acids) and 2640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV.

Amino Acid Sequence↗
PubMed1991

Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo.

The production, from polyprotein precursors, of two hydrophobic nonstructural proteins of dengue 2 (DEN2) virus, NS4A and NS4B, was analyzed both in cell-free systems and in infected cells. In DEN2-infected cells, NS4B is first produced as a peptide of apparent size 30 kDa; NS4B is then post-translationally modified, in an unknown way, to produce a polypeptide of apparent size 28 kDa. The rate and extent of NS4B modification was found to be cell-dependent; in BHK cells the half-time for the conversion of the 30-kDa form to the 28-kDa form was 90 min. N-terminal sequence analysis of NS4B suggests that the N-terminus is produced by an enzyme with a specificity similar to that of signalase. Low levels of a putative polyprotein, NS4AB, were also found in mammalian cells, but not mosquito cells, infected with DEN2, suggesting that a small proportion of DEN2 4A/4B cleavage can occur post-translationally or that some nonstructural polyproteins escape normal processing. Cleavage of the 4A/4B bond in infected cells required expression of DEN2 sequences in addition to those in NS4A and NS4B, as NS4AB produced in cells by a vaccinia expression system was not cleaved. NS4AB produced in cells by a vaccinia expression system was modified post-translationally, presumably in the same way as NS4B. We show that upon translation of DEN2 polyproteins in a cell-free system, the N-terminus of NS4A is generated by cleavage by the viral nonstructural proteinase NS3 and that processing of DEN2 polyproteins occurs with a preferred, but nonobligatory order.

Amino Acid Sequence↗
PubMed1992

Characterization of the potyviral HC-pro autoproteolytic cleavage site.

The helper component-proteinase (HC-Pro) encoded by potyviruses functions to cleave the viral polyprotein by an autoproteolytic mechanism at the HC-Pro C-terminus. This protein belongs to a group of viral cysteine-type proteinases and has been shown previously to catalyze proteolysis between a Gly-Gly dipeptide. The amino acid sequence requirements surrounding the HC-Pro C-terminal cleavage site of the tobacco etch virus polyprotein have been investigated using site-directed mutagenesis and in vitro expression systems. A total of 51 polyprotein derivatives, each differing by the substitution of a single amino acid between the P5 and P2' positions, were tested for autoproteolytic activity. Substitutions of Tyr (P4), Val (P2), Gly (P1), and Gly (P1') were found to eliminate or nearly eliminate proteolysis. Substitutions of Thr (P5), Asn (P3), and Met (P2'), on the other hand, were permissive for proteolysis, although the apparent processing rates of some polyproteins containing these alterations were reduced. These results suggest that auto-recognition by HC-Pro involves the interaction of the enzymatic binding site with four amino acids surrounding the cleavage site. Comparison of the homologous sequences of five potyviral polyproteins revealed that the residues essential for processing are strictly conserved, whereas the nonessential residues are divergent. The relationship between HC-Pro and other viral and cellular cysteine-type proteinases is discussed.

Amino Acid Sequence↗
PubMed1992

Mutational analysis of the tobacco etch potyviral 35-kDa proteinase: identification of essential residues and requirements for autoproteolysis.

The tobacco etch potyvirus (TEV) polyprotein is processed by three virus-encoded proteinases, termed Nla, HC-Pro, and the 35-kDa proteinase. The 35-kDa proteinase is derived from the amino-terminal region of the polyprotein. Analysis of polyproteins containing beta-glucuronidase fused to the expected carboxy terminus of the 35-kDa proteinase confirmed the previously identified Tyr304-Ser305 dipeptide as the cleavage site between the 35-kDa proteinase and HC-Pro. The 35-kDa proteinase of TEV was unable to catalyze proteolysis when synthetic substrate polyproteins were supplied in a bimolecular or trans reaction, suggesting that processing occurs by an autolytic mechanism. The results of a mutational analysis within the 35-kDa proteolytic domain indicated that His214, Asp223, Ser256, and Asp288 were required for optimal autoproteolytic activity. Replacement of Ser256 with either Thr or Cys resulted in low but detectable proteinase activity, as did substitution of Asp223 and Asp288 with Glu. These results are consistent with the hypothesis that the 35-kDa proteinase resembles cellular serine-type proteinases, with Ser256 functioning as the nucleophilic residue within the active site. Cleavage mediated by the 35-kDa proteinase has been shown previously to occur after polyprotein synthesis in wheat germ extracts and transgenic plants, but not in rabbit reticulocyte lysate. We were able to demonstrate that processing in vitro may require a heat-labile factor present in wheat germ extracts.

Amino Acid Sequence↗
PubMed2006

The role of the S-S bridge in retroviral protease function and virion maturation.

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

Amino Acid Sequence↗