RESUMEN

The hamster polyomavirus major capsid protein VP1 was modified in its carboxy-terminal region by consecutive truncations and single amino acid exchanges. The ability of yeast-expressed VP1 variants to form virus-like particles (VLPs) strongly depended on the size and position of the truncation. VP1 variants lacking 21, 69, and 79 amino acid (aa) residues in their carboxy-terminal region efficiently formed VLPs similar to those formed by the unmodified VP1 (diameter 40-45 nm). In contrast, VP1 derivatives with carboxy-terminal truncations of 35 to 56 aa residues failed to form VLPs. VP1 mutants with a single A336G aa exchange or internal deletions of aa 335 to aa 346 and aa 335 to aa 363 resulted in the formation of VLPs of a smaller size (diameter 20 nm). These data indicate that certain parts of the carboxy-terminal region of VP1 are not essential for pentamer-pentamer interactions in the capsid, at least in the yeast expression system used.

Asunto(s)

Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Poliomavirus/genética , Poliomavirus/fisiología , Virosomas/metabolismo , Ensamble de Virus , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Codón sin Sentido , Electroforesis en Gel de Poliacrilamida , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Virosomas/ultraestructura

RESUMEN

Foreign protein sequences presented on hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic. The current study was aimed to evaluate VP1-derived chimeric VLPs as tools for hybridoma technology to generate monoclonal antibodies (mAbs) of desired specificity. Chimeric VLPs containing inserts of different size and origin were used as immunogens. Chimeric VLPs carrying a 9 amino acid (aa)-long cytotoxic T-cell epitope (STAPPVHNV) of human mucin 1 (MUC1) elicited a strong epitope-specific humoral immune response in mice and promoted the production of MUC1-specific mAbs. From a total of seven mAbs of IgG isotype generated against the chimeric VLPs, two mAbs were directed against the MUC1 epitope and five mAbs against the VP1-carrier. Two out of five anti-VP1 mAbs recognized epitopes located at the previously defined insertion site #2 (aa 223/224), which confirms its surface-exposed localization. Chimeric VLPs carrying a 120-aa long sequence of Puumala hantavirus (PUUV) nucleocapsid protein (NP) promoted the generation of five mAbs of IgG isotype specific to PUUV NP. All mAbs recognized the full-length NP of different PUUV strains. In contrast, no VP1-specific mAbs were obtained. The ability of chimeric VLPs to activate antigen-presenting cells was evaluated by studying the uptake of chimeric VLPs by murine spleen cell-derived dendritic cells (DCs). Efficient uptake of VLPs and activation of murine DCs were demonstrated, which may represent the basis of the strong immunogenicity of chimeric VLPs. In conclusion, chimeric VLPs effectively stimulated the production of IgG antibodies specific for foreign epitopes presented at surface-exposed regions. Thus, chimeric HaPyV VP1-derived VLPs represent efficient immunogens for hybridoma technology and provide a promising alternative to chemical coupling of synthetic peptides to carrier proteins.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Proteínas de la Cápside/genética , Poliomavirus/genética , Animales , Especificidad de Anticuerpos , Antígenos de Neoplasias , Western Blotting , Proteínas de la Cápside/inmunología , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos/inmunología , Hibridomas/inmunología , Inmunoglobulina G/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Mucina-1 , Mucinas/inmunología , Proteínas de la Nucleocápside/genética , Virus Puumala/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

The Saccharomyces cerevisiae [PSI] factor, a cytoplasmic omnipotent nonsense suppressor, is a conformationally changed (prion) form of translation termination factor eRF3 (Sup35p). Induction and maintenance of the [PSI] factor depend on the prionizing peptide located in the N domain of Sup35p. The N domain of Sup35p was fused with phosphoribosylaminoimidazole carboxylase (Ade2p), a purine biosynthesis enzyme, and the hybrid protein (NM-Sup35p::Ade2p) was tested for induction of the [PSI] factor. Transformation with a centromeric plasmid carrying the gene for NM-Sup35p::Ade2p induced a [PSI]-like factor in yeast cells, which was evident from efficient nonsense suppression. The suppressory effect depended on the presence of the prionizing peptide both in the hybrid protein and in Sup35p synthesized from the chromosomal gene, as well as on the presence of the prion-like [PIN] factor in the cell.

Asunto(s)

Carboxiliasas/genética , Proteínas Fúngicas/genética , Priones/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Carboxiliasas/metabolismo , Proteínas Fúngicas/metabolismo , Genes Supresores , Factores de Terminación de Péptidos , Priones/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformación Genética

RESUMEN

OBJECTIVES: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. METHODS: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. RESULTS: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. CONCLUSION: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.

Asunto(s)

Proteínas de la Cápside/biosíntesis , Poliomavirus/fisiología , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/genética , Virión/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Humanos , Datos de Secuencia Molecular

RESUMEN

We generated highly immunogenic virus-like particles that are based on the capsid protein VP1 of the hamster polyomavirus (HaPV-VP1) and harbor inserted foreign epitopes. The HaPV-VP1 regions spanning amino acids 81-88 (position 1), 222/223 (2), 244-246 (3), and 289-294 (4) were predicted to be surface exposed. An epitope of the pre-S1 region of the hepatitis B virus (designated S1; amino acid sequence DPAFR) was introduced into the predicted positions of VP1. All VP1/S1 fusion proteins were expressed in yeast and generated virus-like particles. Immunoassays using the S1-specific monoclonal antibody MA18/7 and immunization of C57Bl6 mice with different VP1/S1 constructs showed a pronounced reactivity and a strong S1-specific antibody response for particles carrying the insert in position 1, 2, 1+2, and 1+3. Our results suggest that HaPV-VP1 represents a highly flexible carrier moiety for the insertion of foreign sequences offering a broad range of potential uses, especially in vaccine development.

Asunto(s)

Proteínas de la Cápside , Cápside/genética , Epítopos/genética , Epítopos/inmunología , Mutagénesis Insercional/genética , Poliomavirus/genética , Poliomavirus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Cápside/química , Cápside/inmunología , Cápside/metabolismo , Cricetinae , Técnica de Inmunoensayo de Enzimas Multiplicadas , Epítopos/química , Epítopos/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Antígenos de Superficie de la Hepatitis B/química , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Poliomavirus/química , Poliomavirus/metabolismo , Conformación Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Vacunas Sintéticas/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

Yeast phosphatidylinositol transfer protein (Sec14p) is required for the production of secretory vesicles from the Golgi. This requirement can be relieved by inactivation of the cytosine 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis, indicating that Sec14p is an essential component of a regulatory pathway linking phospholipid metabolism with vesicle trafficking (the Sec14p pathway). Sac1p (refs 7 and 8) is an integral membrane protein related to inositol-5-phosphatases such as synaptojanin, a protein found in rat brain. Here we show that defects in Sac1p also relieve the requirement for Sec14p by altering phospholipid metabolism so as to expand the pool of diacylglycerol (DAG) in the Golgi. Moreover, although short-chain DAG improves secretory function in strains with a temperature-sensitive Sec14p, expression of diacylglycerol kinase from Escherichia coli further impairs it. The essential function of Sec14p may therefore be to maintain a sufficient pool of DAG in the Golgi to support the production of secretory vesicles.

Asunto(s)

Proteínas Portadoras/metabolismo , Diglicéridos/metabolismo , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Clonación Molecular , Diacilglicerol Quinasa , Escherichia coli , Proteínas Fúngicas/genética , Glicósido Hidrolasas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Mutagénesis , Fosfatidilinositoles/metabolismo , Proteínas de Transferencia de Fosfolípidos , Monoéster Fosfórico Hidrolasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingolípidos/metabolismo , beta-Fructofuranosidasa

RESUMEN

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis.

Asunto(s)

Proteínas Fúngicas/metabolismo , Aparato de Golgi/fisiología , Proteínas de la Membrana , Receptores de Esteroides/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/metabolismo , Citidina Difosfato Colina/metabolismo , Cartilla de ADN , Proteínas Fúngicas/química , Aparato de Golgi/ultraestructura , Humanos , Fosfatidilinositoles/metabolismo , Proteínas de Transferencia de Fosfolípidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

The mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PI-TPs) catalyze exchange of phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayers in vitro. We find that Ser-25, Thr-59, Pro-78, and Glu-248 make up a set of rat (r) PI-TP residues, substitution of which effected a dramatic reduction in the relative specific activity for PI transfer activity without significant effect on PC transfer activity. Thr-59 was of particular interest as it is a conserved residue in a highly conserved consensus protein kinase C phosphorylation motif in metazoan PI-TPs. Replacement of Thr-59 with Ser, Gln, Val, Ile, Asn, Asp, or Glu effectively abolished PI transfer capability but was essentially silent with respect to PC transfer activity. These findings identify rPI-TP residues that likely cooperate to form a PI head-group binding/recognition site or that lie adjacent to such a site. Finally, the selective sensitivity of the PI transfer activity of rPI-TP to alteration of Thr-59 suggests a mechanism for in vivo regulation of rPI-TP activity.

Asunto(s)

Proteína de Unión a Andrógenos , Proteínas Portadoras/metabolismo , Proteínas de la Membrana , Mutación , Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfolípidos/metabolismo , Proteínas de Saccharomyces cerevisiae , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Transferencia de Fosfolípidos , Prostateína , Ratas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Secretoglobinas , Relación Estructura-Actividad , Uteroglobina

RESUMEN

The ADE2 gene encodes AIR-carboxylase which catalyzes the sixth step of the purine biosynthetic pathway in Saccharomyces cerevisiae. We have analyzed the effect of deletions in the promoter region of this gene on the expression of the enzyme using a fusion of the ADE2 gene promoter to the bacterial lacZ gene. Adenine added to the growth medium repressed the expression of the fusion at the level of mRNA. The ADE2-lacZ fusion expression can be slightly activated in response to amino-acid starvation, but only in Gcn4+ strains and in an adenine-supplemented medium. In the absence of adenine in the medium ADE2 gene expression is derepressed, and neither starvation for histidine nor a gcd1 general control regulatory mutation leads to additional derepression. Our experiments indicate that the ADE2 gene of the purine biosynthetic pathway is under both specific adenine control and the general amino-acid control system. The cis-acting promoter elements mediating both modes of regulation overlap each other and are located around the proximal TGACTC sequence.

Asunto(s)

Adenina/farmacología , Carboxiliasas/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Aminoácidos/metabolismo , Secuencia de Bases , Carboxiliasas/biosíntesis , Inducción Enzimática/efectos de los fármacos , Proteínas Fúngicas/biosíntesis , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN de Hongos/biosíntesis , ARN de Hongos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Eliminación de Secuencia