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PubMed1992

Processing of avian retroviral gag polyprotein precursors is blocked by a mutation at the NC-PR cleavage site.

The avian sarcoma and leukosis viruses (ASLV) encode a protease (PR) at the C terminus of gag which in vivo catalyzes the processing of both gag and gag-pol precursors. The studies reported here were undertaken to determine whether PR is able to cleave these polyproteins while it is still part of the gag precursor or whether the release of its N terminus to form free PR is necessary for full proteolytic activity. To address this question, we created a mutation that disrupts the PR cleavage site between the NC and PR coding regions of the gag gene. This mutation was introduced into a eukaryotic vector that expresses only the gag precursor and into an otherwise infectious clone of ASLV that carries the neo gene as a selectable marker. These constructs were expressed in monkey COS cells or in quail QT35 cells, respectively. Processing was impaired in both systems. Mutant particles were formed, but they contained no mature processed gag proteins. We observed only the uncleaved gag precursor polypeptide Pr76 in one case or Pr76 and a cleaved product of about 60 kDa in the other. Processing of the mutant gag precursor could be complemented in trans by from a wild-type construct, suggesting that the mutation did not induce gross structural alterations in its precursor. Our results suggest that the PR first must be released from its precursor before it can attack other sites in the gag and gag-pol polyproteins and that cleavage at the NC-PR boundary is a prerequisite for the initiation of the PR-directed processing.

Amino Acid Sequence↗
PubMed1993

Identification of proteolytic processing sites within the Gag and Pol polyproteins of feline immunodeficiency virus.

N-terminal amino acid sequencing, ion spray mass spectrometry, and cleavage of synthetic peptide substrates were used to identify the N and C termini of the mature Gag and Pol proteins of feline immunodeficiency virus (FIV). The Gag polyprotein encodes matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. The Gag-Pol polyprotein encodes, in addition to the above proteins, protease (PR), reverse transcriptase (RT), dUTPase (DU), and integrase (IN). Secondary cleavage of RT at Trp-595-Tyr-596 of Pol yields a truncated form lacking the C-terminal RNase H domain. The observed and expected molecular masses of the viral proteins were in agreement, with three exceptions. (i) The molecular mass of MA was 14,735 Da, compared with a predicted mass of 14,649 Da, based on a single cleavage at Tyr-135-Pro-136 of Gag. The observed molecular mass is consistent with myristoylation of MA, which was confirmed by metabolic labeling of FIV MA with [3H]myristic acid. (ii) The N terminus of the NC protein is generated via cleavage at Gln-366-Val-367 of Gag, which predicts a mass of 25,523 for CA and 9,101 for the major form of NC. The observed mass of CA was 24,569, consistent with loss of nine C-terminal amino acids by a second cleavage of CA at Leu-357-Leu-358. Synthetic FIV protease accurately cleaved synthetic peptide substrates containing this site. (iii) The actual mass of NC (7,120 Da) was approximately 2 kDa smaller than the mass predicted by synthesis to the stop codon at the end of Gag (9,101 Da). Experiments are in progress to characterize additional cleavage(s) in NC.

Amino Acid Sequence↗
PubMed1994

The Cys-His motif of Ty3 NC can be contributed by Gag3 or Gag3-Pol3 polyproteins.

The major structural proteins capsid and nucleocapsid (NC) of the Saccharomyces cerevisiae retroviruslike element Ty3 are produced as domains within the Gag3 and Gag3-Pol3 precursor polyproteins. Ty3 NC contains one copy of the conserved motif CX2CX4HX4C found in most retroviral NC proteins. We show here that NC proteins derived by processing of these different precursor species differ at their carboxyl termini. To determine whether the Cys-His motifs of these nascent NC domains contribute differently to replication, Gag3 and Gag3-Pol3 fusion proteins containing wild-type or mutant Cys-His domains were expressed from separate constructs. Although the Cys-His box was shown to be essential for polyprotein processing of a wild-type Ty3 element, this domain could be contributed from Gag3 or as part of Gag3-Pol3. These data suggest that the functions of the retroviral NC Cys-His domain contributed from Gag and Gag-Pol are redundant.

Amino Acid Sequence↗
PubMed1995

3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.

Amino Acid Sequence↗
PubMed1996

Processing of rabbit hemorrhagic disease virus polyprotein.

Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lysates and in Escherichia coli have been used to study the proteolytic processing of RHDV polyprotein encoded by ORF1. An epitope tag was used for monitoring the gene products by a specific antibody. We have identified four gene products with molecular masses of 80, 43, 73, and 60 kDa, from the amino to the carboxy terminus of the polyprotein. The amino-terminal sequences of the 43- and 73-kDa products were determined and indicated that RHDV 3C proteinase cleaved Glu-Gly peptide bonds.

Animals↗
PubMed1998

Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites.

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.

Amino Acid Sequence↗
PubMed1999

Translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 Gag polyprotein.

Human immunodeficiency virus type 1 (HIV-1) gag-encoded proteins play key functions at almost all stages of the viral life cycle. Since these functions may require association with cellular factors, the HIV-1 matrix protein (MA) was used as bait in a yeast two-hybrid screen to identify MA-interacting proteins. MA was found to interact with elongation factor 1-alpha (EF1alpha), an essential component of the translation machinery that delivers aminoacyl-tRNA to ribosomes. EF1alpha was then shown to bind the entire HIV-1 Gag polyprotein. This interaction is mediated not only by MA, but also by the nucleocapsid domain, which provides a second, independent EF1alpha-binding site on the Gag polyprotein. EF1alpha is incorporated within HIV-1 virion membranes, where it is cleaved by the viral protease and protected from digestion by exogenously added subtilisin. The specificity of the interaction is demonstrated by the fact that EF1alpha does not bind to nonlentiviral MAs and does not associate with Moloney murine leukemia virus virions. The Gag-EF1alpha interaction appears to be mediated by RNA, in that basic residues in MA and NC are required for binding to EF1alpha, RNase disrupts the interaction, and a Gag mutant with undetectable EF1alpha-binding activity is impaired in its ability to associate with tRNA in cells. Finally, the interaction between MA and EF1alpha impairs translation in vitro, a result consistent with a previously proposed model in which inhibition of translation by the accumulation of Gag serves to release viral RNA from polysomes, permitting the RNA to be packaged into nascent virions.

Amino Acid Sequence↗
PubMed2000

The rice tungro bacilliform virus gene II product interacts with the coat protein domain of the viral gene III polyprotein.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.

Amino Acid Sequence↗
PubMed2000

Four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly.

The replicase gene (gene 1) of the coronavirus mouse hepatitis virus (MHV) encodes two co-amino-terminal polyproteins presumed to incorporate all the virus-encoded proteins necessary for viral RNA synthesis. The polyproteins are cotranslationally processed by viral proteinases into at least 15 mature proteins, including four predicted cleavage products of less than 25 kDa that together would comprise the final 59 kDa of protein translated from open reading frame 1a. Monospecific antibodies directed against the four distinct domains detected proteins of 10, 12, and 15 kDa (p1a-10, p1a-12, and p1a-15) in MHV-A59-infected DBT cells, in addition to a previously identified 22-kDa protein (p1a-22). When infected cells were probed by immunofluorescence laser confocal microscopy, p1a-10, -22, -12, and -15 were detected in discrete foci that were prominent in the perinuclear region but were widely distributed throughout the cytoplasm as well. Dual-labeling experiments demonstrated colocalization of the majority of p1a-22 in replication complexes with the helicase, nucleocapsid, and 3C-like proteinase, as well as with p1a-10, -12, and -15. p1a-22 was also detected in separate foci adjacent to the replication complexes. The majority of complexes containing the gene 1 proteins were distinct from sites of accumulation of the M assembly protein. However, in perinuclear regions the gene 1 proteins and nucleocapsid were intercalated with sites of M protein localization. These results demonstrate that the complexes known to be involved in RNA synthesis contain multiple gene 1 proteins and are closely associated with structural proteins at presumed sites of virion assembly.

Cell Line↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes.

We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11 to 53 amino acids in length. In the replicase polyprotein, a total of eight ES were identified, six of which localized to the Nsp2 replicase polyprotein processing end product. In the structural proteins, a total of two ES were identified, in the ORF3 and ORF4 minor envelope glycoproteins. The ORF4 ES was previously identified by monoclonal antibody mapping (J. J. M. Meulenberg, A. P. van Nieuwstadt, A. van Essen-Zandenbergen, and J. P. M. Langeveld, J. Virol. 71:6061-6067, 1997), but its immunogenicity had not been examined in pigs. We found that six experimentally PRRSV-infected pigs consistently had very high antibody titers against the ORF4 ES. In some animals, sera diluted 1:62,500 still gave weak positive enzyme immunoassay reactivity against the ORF4 ES. This hitherto unrecognized immunodominance likely caused phages displaying the ORF4 ES to outcompete phages displaying other ES during library screening with porcine sera and accounted for our failure to identify more than two ES in the structural genes of PRRSV. Genetic analysis showed that variable ES were also the most immunogenic in vivo. Serological analysis indicated differences in the immunoglobulin A responses between short-term and longer-term viremic pigs towards some ES. The implications of these findings for PRRSV diagnostics and immunopathogenesis are discussed.

Amino Acid Sequence↗
PubMed2003

Purification, identification, and biochemical characterization of a host-encoded cysteine protease that cleaves a leishmaniavirus gag-pol polyprotein.

Leishmania RNA virus (LRV) is a double-stranded RNA virus that infects some strains of the protozoan parasite leishmania As with other totiviruses, LRV presumably expresses its polymerase by a ribosomal frameshift, resulting in a capsid-polymerase fusion protein. We have demonstrated previously that an LRV capsid-polymerase polyprotein is specifically cleaved by a Leishmania-encoded cysteine protease. This study reports the purification of this protease through a strategy involving anion-exchange chromatography and affinity chromatography. By using a Sepharose-immobilized lectin, concanavalin A, we isolated a fraction enriched with LRV polyprotein-specific protease activity. Analysis of the active fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoreses and silver staining revealed a 50-kDa protein that, upon characterization by high-pressure liquid chromatography electrospray tandem mass spectrometry (electrospray ionization/MS/MS), was identified as a cysteine protease of trypanosomes. A partial amino acid sequence derived from the MS/MS data was compared with a protein database using BLAST software, revealing homology with several cysteine proteases of Leishmania and other trypanosomes. The protease exhibited remarkable temperature stability, while inhibitor studies characterized the protease as a trypsin-like cysteine protease-a novel finding for leishmania. To elucidate substrate preferences, a panel of deletion mutations and single-amino-acid mutations were engineered into a Gag-Pol fusion construct that was subsequently transcribed and translated in vitro and then used in cleavage assays. The data suggest that there are a number of cleavage sites located within a 153-amino-acid region spanning both the carboxy-terminal capsid region and the amino-terminal polymerase domain, with LRV capsid exhibiting the greatest susceptibility to proteolysis.

Animals↗
PubMed2005

Characterization of the protease domain of Rice tungro bacilliform virus responsible for the processing of the capsid protein from the polyprotein.

BACKGROUND: Rice tungro bacilliform virus (RTBV) is a pararetrovirus, and a member of the family Caulimoviridae in the genus Badnavirus. RTBV has a long open reading frame that encodes a large polyprotein (P3). Pararetroviruses show similarities with retroviruses in molecular organization and replication. P3 contains a putative movement protein (MP), the capsid protein (CP), the aspartate protease (PR) and the reverse transcriptase (RT) with a ribonuclease H activity. PR is a member of the cluster of retroviral proteases and serves to proteolytically process P3. Previous work established the N- and C-terminal amino acid sequences of CP and RT, processing of RT by PR, and estimated the molecular mass of PR by western blot assays. RESULTS: A molecular mass of a protein that was associated with virions was determined by in-line HPLC electrospray ionization mass spectral analysis. Comparison with retroviral proteases amino acid sequences allowed the characterization of a putative protease domain in this protein. Structural modelling revealed strong resemblance with retroviral proteases, with overall folds surrounding the active site being well conserved. Expression in E. coli of putative domain was affected by the presence or absence of the active site in the construct. Analysis of processing of CP by PR, using pulse chase labelling experiments, demonstrated that the 37 kDa capsid protein was dependent on the presence of the protease in the constructs. CONCLUSION: The findings suggest the characterization of the RTBV protease domain. Sequence analysis, structural modelling, in vitro expression studies are evidence to consider the putative domain as being the protease domain. Analysis of expression of different peptides corresponding to various domains of P3 suggests a processing of CP by PR. This work clarifies the organization of the RTBV polyprotein, and its processing by the RTBV protease.

Amino Acid Sequence↗
PubMedDate not supplied

[Inhibition of nuclear import of HIV-1 Vpr protein by Gag polyprotein in the process of virus-like particles formation].

For studies of intracellular Vpr transport and the effect of the HIV-1 Gag polyprotein on the process, a recombinant baculovirus strain was constructed, which directs the synthesis of Vpr fused with the baculovirus secretory polypeptide. During infection the majority of Vpr has been observed in the cell nuclear fraction. These data suggest that Vpr nucleophilic signal is more active than the secretory one. However, during Vpr and Gag co-expression in the baculovirus expression system, Vpr content in the nuclei is decreased, since this protein incorporates effectively into virus-like particles and forms stable complexes with Gag polyprotein. Presumably, the Vpr-Gag post-translational interactions are needed for the Vpr incorporation into virions and suppress the nuclear import of this protein.

Animals↗
PubMed1994

A polyprotein precursor of two mitochondrial enzymes in Neurospora crassa. Gene structure and precursor processing.

N-Acetylglutamate kinase (AGK) and N-acetyl-gamma-glutamyl-phosphate reductase (AGPR) function as two separate mitochondrial enzymes, but are encoded by a single nuclear gene in several fungi. The Neurospora crassa arg-6 gene encoding these enzymes has been cloned and sequenced, and the enzymes responsible for processing the polyprotein precursor have been identified. The 871-amino acid precursor contains a normal N-terminal mitochondrial targeting sequence, an internal connecting region (approximately 200 amino acids) upstream of the distal reductase domain, and coding regions with N-terminal amino acid sequences identical with those of purified N-acetylglutamate kinase and N-acetyl-gamma-glutamyl-phosphate reductase. Sequence comparisons of the coding regions indicate high levels of conservation between prokaryotic and fungal proteins. Regions suggesting ancestral relationships to N-acetylglutamate synthase and aspartate beta-semialdehyde dehydrogenase have been identified. Both the N-terminal targeting sequence and the connecting region contain consensus sites for cleavage by the mitochondrial processing peptidase and processing enhancing protein. In vitro processing assays with intact mitochondria, solubilized mitochondria, and purified enzymes have shown that the mitochondrial processing peptidase and processing enhancing protein cleave not only the N-terminal mitochondrial targeting sequence but also process the polyprotein precursor into the two mature enzymes.

Aldehyde Oxidoreductases↗
PubMed1995

Proteolytic processing of the MHV polymerase polyprotein. Identification of the P28 cleavage site and the adjacent protein, P65.

The polymerase gene of Mouse Hepatitis Virus strain JHM (MHV-JHM) encodes a polyprotein larger than 750 kilodaltons. This polyprotein is proposed to be processed by several viral proteinases into functional subunits. The amino-terminal subunit is p28, which is cleaved by the first viral papain-like proteinase domain. In this study, we identified the cleavage site of this papain-like cysteine proteinase by amino acid sequencing of radiolabeled polypeptide adjacent to p28. Proteolysis occurs between the glycine-247 and valine-248 dipeptide bond. To determine which amino acid residues are critical for proteolysis, we preformed site-directed mutagenesis on the coding sequences surrounding the cleavage site and assayed for the efficiency of cleavage of p28 in an in vitro transcription and translation system. We report that glycine-247 and arginine-246 are the most critical residues for efficient processing of p28.

Amino Acid Sequence↗
PubMedDate not supplied

[Interaction of gag polyprotein-precursors p55 and p48 with p160gag-pol during formation of virus-like particles of HIV-1 with recombinant strains of vaccinia virus].

The polypeptide composition of HIV-I virus-like particles produced by CV-I cells during mono- and coinfection with recombinant vaccinia virus (rVV) strains containing the whole (p55) and carboxyterminal truncated (p48) gag genes and gag-pol sequence is studied. In monoinfection both the gag-strains actively produced virus-like particles consisting of non-processed p55Gag and p48Gag polyprotein without p6 domain. In case of a coinfection of the cells with one of these strains and the rVV producing p160Gag-Pol polyprotein the virus-like particles consisted of p24 protein and a negligible amount of non-processed Gag precursors. The share of p24 protein increased in proportion to the duration of coinfection and decreased with a reduction of multiplicity of infection with rVV carrying p160Gag-Pol. Hence, the absence of p6 domain does not influence the processing of Gag proteins during virus-like particles assembly and budding. In contrast to the natural systems of HIV-I development, in the rVV expression system the p6Gag domain virtually does not contribute to reactions between Gag and Gag-Pol precursors and to the particles' morphogenesis.

Animals↗