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At least 163 records · Page 9Linked to original sources
PubMed1999

Proteolytic processing in infectious bursal disease virus: identification of the polyprotein cleavage sites by site-directed mutagenesis.

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of an immune depressive disease that affects domesticated and wild avian species. The expression strategy of IBDV includes the synthesis of a 110-kDa polyprotein containing the capsid precursor polypeptides. The polyprotein is autocatalitically processed rendering three polypeptides: NH2-VPX-VP4-VP3-COOH. We have carried out a systematic analysis, using a series of plasmids encoding polyproteins containing either deletions or single amino acid substitutions, to identify the processing sites. The results obtained showed the existence of two sites, 511LAA513 and 754MAA756, that are essential for the processing of the VPX-VP4 and VP4-VP3 precursors, respectively. These sequences are highly conserved among IBDV strains form serotypes 1 and 2. A secondary VPX-VP4 processing site was detected in a 19-amino acid stretch located upstream of the 511LAA513 site. Analyses using versions of the 754MAA756 VP4-VP3 processing site containing conservative and nonconservative amino acid substitutions demonstrated that the specificity of the cleavage is dictated by the conserved AA dipeptide.

Amino Acid Sequence↗
PubMed2000

Differential budding efficiencies of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro polyproteins from insect and mammalian cells.

In this study, we examined the ability of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro to assemble immature virus-like particles (VLPs) and bud from insect and mammalian cells. Transmission electron microscopy of insect cells infected with a recombinant baculovirus carrying the entire gag gene revealed that Pr53(Gag) is targeted to the plasma membrane, where it extensively accumulates and forms electron-dense evaginations. However, no particles could be detected either inside the cells or in the culture supernatants. With the Gag-Pro-expressing construct, we observed HTLV-I-specific cytoplasmic proteolysis of the Gag precursor, but again no particle released in the culture supernatants. Transmission electron microscopic analysis of insect cells expressing Gag-Pro polyprotein revealed large vacuoles in the cytoplasm and no budding particles at the plasma membrane. In contrast, human immunodeficiency virus type 1 Gag polyprotein expressed in insect cells is able to release VLPs. These data showed that unlike other retroviruses, Pr53(Gag) is unable to be released as immature VLPs from insect cells. To determine whether the block in particle budding and release is due to an intrinsic property of Pr53(Gag) or the absence of essential cellular factors in insect cells, we expressed Gag and Gag-Pro polyproteins in human 293 cells. The results indicate that Pr53(Gag) and p24 capsid are released within particles into the culture supernatants of human 293 cells. We found that the myristylation of the N-terminal glycine residue is essential for Gag release. Altogether, these results strongly suggest that the proper assembly of HTLV-I particles is dependent on mammalian host cell factors.

Animals↗
PubMed1993

Altered proteolytic processing of the polymerase polyprotein in RNA(-) temperature sensitive mutants of murine coronavirus.

We examined the synthesis and processing of the polymerase polyprotein in RNA(-) temperature sensitive mutant of murine coronavirus strain A59. These temperature sensitive mutants of MHV-A59 synthesize viral RNA at the permissive temperature (33.0 degrees C), but are unable to synthesize viral RNA at the nonpermissive temperature (39.5 degrees C). The ts mutants have been mapped to five different complementation groups in the polymerase gene. The 5'-most complementation groups, Groups A and B, map to a region encoding an autoproteinase responsible for the cleavage of p28, the amino-terminal product of the polymerase polyprotein. We screened six temperature sensitive mutants to determine if there was an alteration in the proteolytic processing of the polymerase polyprotein, particularly in the cleavage of the p28 protein. Two mutants, tsNC9 and tsLA16, had altered proteolytic products at both the permissive and nonpermissive temperatures. One Group B temperature sensitive mutant, designated tsNC11, was defective in the production of p28 protein at the nonpermissive temperature. To further localize the site of the mutation in tsNC11, RNA representing the 5'-most 5.3 kb region of the polymerase gene was transfected into tsNC11-infected cells and virus production monitored. The transfected RNA was able to complement the defect in tsNC11, resulting in viral RNA synthesis and production of viral particles at the nonpermissive temperature. These results indicate that a gene product from the 5.3 kb region of gene 1 is required for coronavirus RNA synthesis.

Genetic Complementation Test↗
PubMed1998

Maturation of the polymerase polyprotein of the coronavirus MHV strain JHM involves a cascade of proteolytic processing events.

The RNA polymerase gene of the murine coronavirus mouse hepatitis virus (MHV) encodes a polyprotein of greater than 750 kDa. The amino-terminal cleavage product of the MHV polymerase polyprotein, p28, has been shown to be cleaved from the polyprotein by the virus-encoded protease PCP-1. We aim to identify the MHV-JHM proteolytic products downstream of p28 and to determine which viral proteinase domains are responsible for generating each of them. To this end, we have generated antisera directed at specific MHV-JHM ORF1a regions and have used these antisera to identify six viral proteins, representing a large portion of ORF1a, from MHV-JHM-infected cells. These proteins include p28, p72, p65, p250, p210, and p27.

Animals↗
PubMed1992

Characterization of a Euglena gene encoding a polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II.

A 7.4 kb segment of a Euglena nuclear gene encoding a portion of the polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) has been isolated and sequenced. Nine exons can be assembled into a continuous open reading frame encoding 113 amino acids of the C-terminus of an LHCPII, followed by 4 complete LHCPIIs. The intron-exon junctions required to maintain maximum amino acid sequence homology between the LHCPIIs encoded by the Euglena genomic clone and Arabidopsis LHCPII do not conform to the consensus splice site sequences present in most eukaryotic organisms. Three types of LHCPIIs are contained within the polyprotein precursor. There is greater than 90% sequence identity at both the protein and nucleic acid level within a type while between types, there is less than 65% sequence identity. All Euglena LHCPIIs contain three hydrophobic membrane-spanning domains in the same positions as found in other LHCPIIs. Hybridization of genomic Southern blots with a probe encoding LHCPII suggests that the Euglena genome encodes a large number (30-50) of LHCPIIs. A probe derived from the 3' end of the sequenced genomic clone hybridizes with equal intensity to 2-3 genomic fragments on Southern blots. The probe encoding LHCPII hybridizes to RNAs of 9.5 and 6.6 kb on northern blots of total RNA while the 3'-end probe hybridizes only to the 6.6 kb RNA. The 6.6 kb LHCPII band detected on northern blots appears to be composed of transcription products derived from 2-3 LHCPII genes. Euglena appears to be unique in that a large number of LHCPIIs are encoded by a small number (3-5) of polyprotein genes.

Amino Acid Sequence↗
PubMed1980

Characterization of viral polyproteins in cells transformed and producing Moloney murine sarcoma virus-124.

The viral proteins of Moloney murine sarcoma virus-transformed mouse cells (G8-124), that overproduce the sarcoma virus relative to helper leukemia virus, were examined by immunoprecipitation, sizing by gel electrophoresis and peptide mapping. Using antisera to the viral core proteins, a p10-deficient core polyprotein of 63 000 daltons (P63gag) was detected which was found to be a product of the MuSV-124 genome. Helper virus proteins Pr67gag, gPr85gag, Pr200gag-pol, and gPr83env were also detected and characterized. An unusual helper virus precursor polyprotein of 93 000 daltons was also detected and characterized. It was labeled with [3H]fucose, suggesting that it was an intermediate precursor containing gp70 on which further modification of the core oligosaccharide had occurred relative to gPr83env. The usual viral proteins were also found in sarcoma virus particles and they were undistinguishable in size from those of Moloney murine leukemia virus (cl-1 strain). These experiments together with pulse-labeling experiments performed in the presence of the arginine analog canavanine, which prevents proteolytic cleavage, suggest that the product derived from the Mo-MuSV-specific sequences must be expressed as a separate gene product since we were unable to detect in transformed cells a polyprotein containing both viral (e.g., 'gag', 'pol' or 'env') and non-viral components. Cell-free protein synthesis studies performed with Mo-MuSV-124 genomic RNA have confirmed this interpretation and have identified three size classes of polypeptides as translation products of the sarcoma virus genome. They are P63gag, P42-P38 and P23. Intracellular p63gag and cell-free synthesized P63gag, appear to have identical sizes, antigenic determinants and tryptic peptides. The P42-P38 proteins contain core protein determinants. P23 is not related to the products of the replication genes of MuLV. Thus, P23 is a candidate for the 'src' gene product of Mo-MuSV-124.

Animals↗
PubMed1986

The effect of cerulenin on the synthesis of the precursor gag polyprotein in defective murine leukemia and sarcoma virus producing cell lines.

The effect of cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, on the synthesis of the precursor gag polyprotein, Pr65gag in a defective murine leukemia virus (334C) producing murine cell line (3JE) and a defective murine sarcoma virus (Gazdar) producing hamster cell line (HTG-2) was examined. In contrast to Moloney murine leukemia virus (M-MuLV) producing cell lines (MJD-54, clone 2) the amount of the Pr65gag remaining in the presence of cerulenin (20 micrograms/ml) was greatly reduced in both defective virus-infected cells. This effect appears specific for the Pr65gag polyprotein, since the env precursor polyprotein Pr80env was normally synthesized and remained undegraded in cerulenin-treated 3JE-infected cells. Thin-section electron micrographs showed an increased accumulation of virion particles in vesicles of treated HTG-2 cells.

Antifungal Agents↗
PubMed1989

Molecular genetic analysis of a plant virus polyprotein cleavage site: a model.

The RNA genome of tobacco etch virus (TEV) is expressed as a polyprotein which is co- and post-translationally processed by viral encoded proteinases. The TEV 49,000 dalton (49-kDa) proteinase cleaves the polyprotein at five positions each defined by the seven amino acid consensus sequence, (formula; see text) One of the cleavage sites, the 58-kDa nuclear inclusion/30-kDa capsid protein junction was altered by site-directed mutagenesis and the effects of these alterations on cleavage were determined. Polyprotein precursors were synthesized by translation of T7 polymerase-derived transcripts and processed in a cell-free system using TEV nuclear inclusion bodies as a source of 49-kDa proteolytic activity. A wild-type cleavage site and 61 substrates containing site-directed amino acid replacements at the nonconserved P7, P5, P4, P2, and P'2 positions were examined. Amino acid replacements flanking the putative TEV cleavage sequence at the P7 and P'2 positions had minimal effects on cleavage. Amino acid substitutions at positions P5, P4, and P2 resulted in substrates which were processed by the 49-kDa TEV proteinase, albeit generally at reduced rates. No substitution at any of these five positions resulted in total elimination of cleavage. A model is presented which proposes different roles for conserved and variable positions in the TEV heptapeptide cleavage sequence.

Amino Acid Sequence↗
PubMed1991

Functional domains of HIV-1 gag-polyprotein expressed in baculovirus-infected cells.

Seven recombinants of AcNPV harboring various forms of complete or truncated gag gene from HIV-1 were constructed to determine which functional domains of the gag polyprotein are implicated in its self-assembly and cellular localization. The p6 carboxy-terminal portion of the p15 NCgag domain appeared to be dispensable for assembly, budding, and release of gag particles by insect cells. However, all the morphopoietic information was not entirely confined to the p9 NC domain, as N-myristylation could compensate for p15 NC deletion in gag assembly and the budding process. The two consensus karyophilic signals situated in the p17 MAgag domain were inefficient for targeting nonmyristylated forms of gag polyprotein to the nucleus when the p6 NC domain was deleted. In the presence of p6, or with a third, baculovirus-specific, karyophilic signal added at its N-terminus, gag particles relocated in the nucleus. These data suggested that p6 played a critical role in the conformation of gag polyprotein.

Amino Acid Sequence↗
PubMed1992

Proteolytic processing of the plum pox potyvirus polyprotein by the NIa protease at a novel cleavage site.

The expression of potyvirus genomic RNA takes place through translation of its unique long and functional open reading frame into a large polyprotein that undergoes extensive proteolytic processing. Most of the cleavages are performed by the virus-encoded NIa protease, which cuts the polyprotein at defined sites that are characterized by conserved heptapeptide sequences. We have demonstrated in vitro cleavage activity by the plum pox potyvirus (PPV) NIa protease at a novel site, previously identified by sequence analysis, thus allowing a further refinement of the potyviral genetic map. This novel site is located 52 amino acids upstream from the site corresponding to the N-terminus of the CI protein (the NIa cleavage site previously considered the closest to the beginning of the polyprotein). The specificity of the processing was demonstrated by its abolishment when the Gln at position -1 of the cleavage site was changed to His. This novel NIa cleavage site was only partially processed, a characteristic that was not altered when its heptapeptide sequence was modified to become that of the efficiently cleaved NIb-CP junction. On the contrary, substitutions at the nonconserved position +3 had notable effects, positive or negative, on the efficiency of processing. These results show the relevance of sequence and/or conformational context outside the conserved heptapeptide for modulating the cleavage reaction catalyzed by the NIa protease.

Amino Acid Sequence↗
PubMed1986

Synthesis and processing of Semliki forest virus polyprotein in Saccharomyces cerevisiae: a yeast type glycosylation of E1 envelope protein.

A cDNA coding for the structural proteins of Semliki Forest virus (SFV) was ligated between the ADC1 promoter and terminator in a yeast expression vector, pAAH5. Synthesis of the SFV-specific proteins in Saccharomyces cerevisiae transformed with this vector was shown by immunoblotting and immunoprecipitation. Detection of the N-terminal and the C-terminal components of the viral polyprotein, capsid protein and E1 envelope protein, respectively, suggested that the entire polyprotein was translated in yeast. The capsid protein was effectively released from the polyprotein as a normal size polypeptide, but the following protein, p62 (E3, E2 precursor) was not detected, suggesting that it was rapidly degraded. Electrophoretic analyses indicated that the final protein, E1, entered the secretory pathway, the signal sequence was cleaved off and the protein became extensively and heterogeneously glycosylated. These data suggest that E1 was transported to the Golgi complex and that yeast-characteristic outer-chain glycans were added to the protein.

Capsid↗
PubMed1988

Biosynthesis and analysis of a genetically engineered HIV-1 reverse transcriptase/endonuclease polyprotein in Escherichia coli.

Elimination of the protease domain from the polymerase open reading frame (pol) of the human immunodeficiency virus type 1 (HIV-1) leads, in Escherichia coli, to synthesis and accumulation of a non-processed 98-kDa reverse transcriptase/endonuclease (RT/ENDO) polyprotein. A partially purified preparation of this reverse RT/ENDO polyprotein displays little or no RT activity. Introduction of the pol protease domain as a separate transcriptional unit on the same plasmid restores the processing program, generating correctly sized RT and ENDO polypeptides. Concomitant with restoration of processing is the reappearance of RT activity. These results suggest that for HIV-1 RT to be active, it must be matured from the pol polyprotein.

Endodeoxyribonucleases↗
PubMed2005

Localization of self-interacting domains within betaretrovirus Gag polyproteins.

The Betaretrovirus genus is characterized by the ability to preassemble immature capsids within the cytoplasm. For Mason-Pfizer monkey virus (M-PMV) this ability depends in part upon the unique Internal Scaffold Domain (ISD) within the p12 region of Gag. In this study, we have further characterized the ability of M-PMV p12 to promote Gag-Gag interaction and have examined the Gag polyprotein of the related mouse mammary tumor virus (MMTV) to potentially identify a region with equivalent function. Using the yeast two-hybrid system, we confirmed that both Gag polyproteins strongly interact, primarily through the CA-NC regions, but also through additional domains N-terminal to CA. For M-PMV, this auxiliary interaction domain was p12. For MMTV, no single strongly self-interacting protein was identified. Instead, MMTV Gag appears to utilize the weak contributions of several protein domains to support the main interaction of its CA-NC. Our findings suggest that, in addition to the canonical NC "I-domain" interaction, MMTV Gag self-association results from the concerted action of multiple regions of the polyprotein while M-PMV Gag relies mainly on its p12 domain.

Amino Acid Sequence↗
PubMed1991

A sequence related to the human gonadoliberin precursor near the N-termini of HIV and SIV gag polyproteins.

A highly conserved sequence near the N-terminus of all human (HIV) and simian (SIV) immunodeficiency virus gag polyproteins appears to be a precursor for a viral mimic of the amidated C-terminus of human gonadoliberin. The gag polyproteins are known to be myristylated; processing of the amidation site would yield myristylated 23-residue peptides whose C-terminal sequences mimic gonadoliberin and presumably behave as ligands for the gonadoliberin receptor. This paper describes the discovery of conserved gonadoliberin-precursor-related sequences in HIV and SIV gag polyproteins and in the p-17 core proteins derived from them. Arguments are presented that the conserved precursor structure requires post-translational processing to a peptide amide derivative which is a ligand for the gonadoliberin receptor. A model has been developed for entry of the viral genomic RNA into host cells through the gonadoliberin receptor and experiments are suggested to confirm or refute the model. This proposed mechanism for entry of HIV genomic RNA into host cells, if it proves to be substantially correct, suggests several new approaches to prevention and treatment of acquired immunodeficiency syndrome (AIDS).

Amino Acid Sequence↗
PubMed1980

Feline sarcoma virus polyprotein P115 binds a host phosphoprotein in transformed cells.

Several independent isoltes of feline sarcoma virus (FeSV) have been described. Such viruses are apparently derived by genetic recombination between feline leukaemia virus (FeLV) genomic RNA and host cellular genetic sequences with transforming potential. Two FeSV isolates, one originally described by Gardner and the second by Snyder-Theilen, have been shown to encode polyproteins of around 115,000 molecular weight. Both polyproteins contain FeLV structural components (p15, p12) at their amino terminus in addition to nonstructural carboxyl terminal components encoded by acquired sequences within the FeSV genome. We have previously shown that Gardner FeSV P115 contains multiple sites of phosphorylation within its nonstructural component and possesses an associated protein kinase activity. In the present study we describe the expression in cells derived from a number of mammalian species, of a highly conserved celklular phosphoprotein with binding affinity for Gardner FeSV P115. This protein, designated P150, exhibits an associated protein kinase activity and is immunologically and structurally distinct from polyproteins encoded by the Gardner or Snyder-Theilen strains of FeSV.

Animals↗
PubMed2002

Enhancement of the immune response generated against hepatitis C virus envelope proteins after DNA vaccination with polyprotein-encoding plasmids.

Plasmids expressing variants of the hepatitis C virus (HCV) core, E1 and E2 proteins individually or as polyproteins were administered to BALB/c mice. All plasmids induced a detectable and specific antibody response. Antibody titres against core, E1 and E2 proteins, 19 weeks after primary immunization, ranged from 1:50 to 1:4500 depending on the inoculated plasmid and the HCV antigen evaluated. Constructs expressing HCV envelope proteins as polyprotein variants including the core amino acid region induced statistically stronger antibody responses than plasmids encoding individual E1 and E2 proteins. Particularly, the pIDKE2 plasmid, expressing the first 650 amino acids in the viral polyprotein, induced a potent and multispecific antibody and lymphoproliferative response against HCV core, E1 and E2 proteins. Anti-E2 antibodies generated by pIDKE2 immunization were cross-reactive to hypervariable region-1 peptides from different genotypes. Immunization with the pIDKE2 also generated a positive cellular immune response against the core antigen, determined by interferon-gamma enzyme-linked immunospot (ELISPOT) assay, and induced detectable levels of interferon-gamma but not interleukin-4 in vaccinated mice. The detection of both antibody and cytotoxic T-lymphocyte responses, potentially targeted to circulating or cell-infecting virions respectively, in mice vaccinated with the pIDKE2 plasmid is very attractive for the effective eradication of HCV infection.

Animals↗
PubMed2002

Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein.

The retroviral Gag polyprotein directs budding from the plasma membrane of infected cells. Until now, it was believed that Gag proteins of type C retroviruses, including the prototypic oncoretrovirus Rous sarcoma virus, were synthesized on cytosolic ribosomes and targeted directly to the plasma membrane. Here we reveal a previously unknown step in the subcellular trafficking of the Gag protein, that of transient nuclear localization. We have identified a targeting signal within the N-terminal matrix domain that facilitates active nuclear import of the Gag polyprotein. We also found that Gag is transported out of the nucleus through the CRM1 nuclear export pathway, based on observations that treatment of virus-expressing cells with leptomycin B resulted in the redistribution of Gag proteins from the cytoplasm to the nucleus. Internal deletion of the C-terminal portion of the Gag p10 region resulted in the nuclear sequestration of Gag and markedly diminished budding, suggesting that the nuclear export signal might reside within p10. Finally, we observed that a previously described matrix mutant, Myr1E, was insensitive to the effects of leptomycin B, apparently bypassing the nuclear compartment during virus assembly. Myr1E has a defect in genomic RNA packaging, implying that nuclear localization of Gag might be involved in viral RNA interactions. Taken together, these findings provide evidence that nuclear entry and egress of the Gag polyprotein are intrinsic components of the Rous sarcoma virus assembly pathway.

Active Transport, Cell Nucleus↗
PubMed1978

Chemical determination of the m1 Moloney sarcoma virus pP60gag gene order: evidence for unique peptides in the carboxy terminus of the polyprotein.

The gene order of the ml Moloney sarcoma virus (mlMSV) specific pP60gag (P60) was determined by direct chemical analysis of the polyprotein. P60 was cleaved with cyanogen bromide (CNBr) into eight partial and complete fragments ranging in mass from 10,000 daltons to 58,000 daltons. Peptide maps of these fragments were compared to maps of p15, p12, and three CNBr fragments of p30. The polarity of p15 and p12 in a CNBr fragment of P60 was determined by carboxypeptidase A digestion; likewise the CNBr fragments of p30 were ordered by aminopeptidase digestion. The linear arrangement of P60 CNBr fragments gave the gene order of NH2-p15-p12-p30-COOH. The m3 isolate of MSV expresses a P70 gag polyprotein. Peptide maps of 48,000-dalton CNBr fragments of m3 P70 and ml P60 were similar and suggested that both polyproteins were similar through the NH2-terminal two-thirds of p30. However, the presence of peptides unique to the 10,500-dalton COOH-terminal fragment of m1MSV p30 and not present in the p30 of either m3MSV or Moloney leukemia virus suggested that the gag gene deletion in the m1 isolate begins in the p30 reading frame.

Cyanogen Bromide↗