RESUMEN

An open reading frame (ORF) with homology to interleukin-10 (IL-10) has been identified in rhesus cytomegalovirus (RhCMV). The IL-10-like protein is generated from a multispliced, polyadenylated early gene transcript encompassing part of the corresponding UL111A ORF of human CMV (HCMV). Immunological analyses confirm expression of the IL-10-like protein both in tissue culture and in RhCMV-infected rhesus macaques. Conserved ORFs were subsequently identified in human, baboon, and African green monkey CMV, and a fully processed transcript has been mapped in fibroblasts infected with the Towne strain of HCMV. The conservation of this previously unrecognized ORF suggests that the protein may play an essential role in primate CMV persistence and pathogenesis.

Asunto(s)

Citomegalovirus/genética , Interleucina-10/biosíntesis , Interleucina-10/genética , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/metabolismo , ADN Complementario/aislamiento & purificación , ADN Viral/genética , ADN Viral/aislamiento & purificación , Exones/genética , Regulación Viral de la Expresión Génica , Genes Virales/genética , Humanos , Interleucina-10/química , Intrones/genética , Macaca mulatta , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Papio , Conformación Proteica , ARN Viral/análisis , ARN Viral/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Virales/biosíntesis , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Estructurales Virales/genética

RESUMEN

Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef deletion (SIVmac239Delta(152)nef) or interleukin-2 in place of nef developed high virus loads and progressed to simian AIDS. Viruses recovered from both juvenile and neonatal macaques with disease produced a novel truncated Nef protein, tNef. Viruses recovered from juvenile macaques infected with serially passaged virus expressing tNef exhibited a pathogenic phenotype. These findings demonstrated strong selective pressure to restore expression of a truncated Nef protein, and this reversion was linked to increased pathogenic potential in live attenuated SIV vaccines.

Asunto(s)

Productos del Gen nef/genética , Vacunas contra el SIDAS/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Secuencia de Aminoácidos , Animales , Vectores Genéticos , Interleucina-2/genética , Macaca mulatta , Datos de Secuencia Molecular , Mutagénesis , Vacunas contra el SIDAS/efectos adversos , Análisis de Secuencia de ADN , Síndrome de Inmunodeficiencia Adquirida del Simio/etiología , Vacunas Atenuadas/genética

RESUMEN

The simian immunodeficiency virus (SIV) nef gene is an important determinant of viral load and acquired immunodeficiency syndrome (AIDS) in macaques. A role(s) for the HIV-1 nef gene in infection and pathogenesis was investigated by constructing recombinant viruses in which the nef gene of the pathogenic molecular clone SIVmac239 nef was replaced with either HIV-1sf2nef or HIV-1sf33nef. These chimeras, designated SHIV-2nef and SHIV-33nef, expressed HIV-1 Nef protein and replicated efficiently in cultures of rhesus macaque lymphoid cells. In two SHIV-2nef-infected juvenile rhesus macaques and in one of two SHIV-33nef-infected juvenile macaques, virus loads remained at low levels in both peripheral blood and lymph nodes in acute and chronic phases of infection (for >83 weeks). In striking contrast, the second SHIV-33nef-infected macaque showed high virus loads during the chronic stage of infection (after 24 weeks). CD4+ T-cell numbers declined dramatically in this latter animal, which developed simian AIDS (SAIDS) at 47-53 weeks after inoculation; virus was recovered at necropsy at 53 weeks and designated SHIV-33Anef. Sequence analysis of the HIV-1sf33 nef gene in SHIV-33Anef revealed four consistent amino acid changes acquired during passage in vivo. Interestingly, one of these consensus mutations generated a tyr-x-x-leu (Y-X-X-L) motif in the HIV-1sf33 Nef protein. This motif is characteristic of certain endocytic targeting sequences and also resembles a src-homology region-2 (SH-2) motif found in many cellular signaling proteins. Four additional macaques infected with SHIV-33Anef contained high virus loads, and three of these animals progressed to fatal SAIDS. Several of the consensus amino acid changes in Nef, including Y-X-X-L motif, were retained in these recipient animals exhibiting high virus load and disease. In summary, these findings indicate that the SHIV-33Anef chimera is pathogenic in rhesus macaques and that this approach, i.e., construction of chimeric viruses, will be important for analyzing the function(s) of HIV-1 nef genes in immunodeficiency in vivo, testing antiviral therapies aimed at inhibiting AIDS, and investigating adaptation of this HIV-1 accessory gene to the macaque host.

Asunto(s)

Productos del Gen nef/fisiología , VIH-1 , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Secuencia de Aminoácidos , Animales , Línea Celular , Productos del Gen nef/genética , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Recombinación Genética , Homología de Secuencia de Aminoácido , Virus de la Inmunodeficiencia de los Simios/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

The role of the feline immunodeficiency virus (FIV) vif gene in establishing productive infection in feline peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) was examined in cell culture systems. A 375-bp deletion was introduced into the vif gene of the wild-type FIV-pPPR infectious molecular clone to produce Vif deletion mutant FIV-pPPRDeltavif. This mutant FIV proviral construct expressed FIV proteins p24gag and gp100env in transfected Crandell feline kidney cells as measured by immunoprecipitation and Western blot analysis as well as immunocytochemical analysis; these cultures produced very low levels of virus by cocultivation of transfected cells with PBMCs and K-258 cells, as measured by production of p24gag. Replication kinetics of wild-type and vif-deleted virus were compared in PBMCs and monocyte-derived macrophages (MDMs) by infection with cell-free virus preparations. Similar to findings with other lentiviruses, the vif gene was found to be essential for establishment of productive FIV infection in both PBMCs and MDMs. This study indicates that vif is essential for productive FIV infection of host target cells in vitro and that FIV-pPPRDeltavif may be an excellent candidate viral mutant for attenuated virus vaccine studies.

Asunto(s)

Productos del Gen vif/genética , Virus de la Inmunodeficiencia Felina/genética , Virus de la Inmunodeficiencia Felina/fisiología , Leucocitos Mononucleares/virología , Macrófagos/virología , Animales , Gatos , Línea Celular , Células Cultivadas , Productos del Gen gag/metabolismo , Macrófagos/citología , Monocitos/citología , Transfección , Proteínas del Envoltorio Viral/metabolismo , Replicación Viral

RESUMEN

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P104A/P107A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.

Asunto(s)

Productos del Gen nef/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Virus de la Inmunodeficiencia de los Simios/fisiología , Dominios Homologos src , Animales , Recuento de Linfocito CD4 , Productos del Gen nef/química , Humanos , Ligandos , Macaca mulatta , Mutación Puntual , Conejos , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Quinasas p21 Activadas

Asunto(s)

Productos del Gen nef/fisiología , VIH/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Línea Celular , Productos del Gen nef/química , Genes nef , VIH/genética , Infecciones por VIH/virología , Humanos , Transducción de Señal , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas

RESUMEN

BACKGROUND: The negative factor (Nef) of human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV) is required for high levels of viremia and progression to AIDS. Additionally, Nef leads to cellular activation, increased viral infectivity and decreased expression of CD4 on the cell surface. Previously, we and others demonstrated that Nef associates with a cellular serine kinase (NAK) activity. Recently, it was demonstrated that NAK bears structural and functional similarity to p21-activated kinases (PAKs). RESULTS: In this study, we demonstrate that Nef not only binds to but also activates NAK via the small GTPases CDC42 and Rac1. First, the dominant-negative PAK (PAKR), via its GTPase-binding domain, and dominant-negative GTPases (CDC42Hs-N17 and Rac1-N17) block the ability of Nef to associate with and activate NAK. Second, constitutively active small GTPases (CDC42Hs-V12 and Rac1-V12) potentiate the effects of Nef. Third, interactions between Nef and NAK result in several cellular effector functions, such as activation of the serum-response pathway. And finally, PAKR, CDC42Hs-N17 and Rac1-N17 decrease levels of HIV-1 production to those of virus from which the nef gene is deleted. CONCLUSIONS: By activating NAK via small GTPases and their downstream effectors, Nef interacts with regulatory pathways required for cell growth, cytoskeletal rearrangement and endocytosis. Thus, NAK could participate in the budding of new virions, the modification of viral proteins and the increased endocytosis of surface molecules such as CD4. Moreover, blocking the activity of these GTPases could lead to new therapeutic interventions against AIDS.

Asunto(s)

Proteínas de Ciclo Celular/genética , Proteínas de Unión al GTP/genética , Productos del Gen nef/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Animales , Células COS , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Productos del Gen nef/metabolismo , VIH-1/genética , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Células Jurkat , Ratones , Proteínas Nucleares/genética , Ratas , Factor de Respuesta Sérica , Transducción de Señal , Replicación Viral , Proteína de Unión al GTP cdc42 , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas , Proteínas de Unión al GTP rac

RESUMEN

BACKGROUND: The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), encode a conserved accessory gene product, Nef. In vivo, Nef is important for the maintenance of high virus loads and progression to AIDS in SIV-infected adult rhesus macaques. In tissue culture cells expressing Nef, this viral protein interacts with a cellular serine kinase, designated Nef-associated kinase. RESULTS: This study identifies the Nef-associated kinase as a member of the p21-activated kinase (PAK) family of kinases and investigates the role of this Nef-associated kinase in vivo. Mutants of Nef that do not associate with the cellular kinase are unable to activate the PAK-related kinase in infected cells. To determine the role of cellular kinase association in viral pathogenesis, macaques were infected with SIV containing point-mutations in Nef that block PAK activation. Virus recovered at early time points after inoculation with mutant virus was found to have reverted to prototype Nef function and sequence. Reversion of the kinase-negative mutant to a kinase-positive genotype in macaques infected with the mutant virus preceded the induction of high virus loads and disease progression. CONCLUSIONS: Nef associates with and activates a PAK-related kinase in lymphocytes infected in vitro. Moreover, the Nef-mediated activation of a PAK-related kinase correlates with the induction of high virus loads and the development of AIDS in the infected host. These findings reveal that there is a strong selective pressure in vivo for the interaction between Nef and the PAK-related kinase.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/metabolismo , Productos del Gen nef/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Animales , Modelos Animales de Enfermedad , Activación Enzimática , Eliminación de Gen , Productos del Gen nef/genética , Humanos , Macaca mulatta , Mapeo Peptídico , Fosfopéptidos/química , Conejos , Células Tumorales Cultivadas , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas

Asunto(s)

Retrovirus de los Simios/inmunología , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas Virales/inmunología , Vacunas contra el SIDA , Animales , Productos del Gen env/inmunología , Productos del Gen nef/genética , Productos del Gen nef/inmunología , VIH-1/inmunología , Humanos , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

Among the primate lentiviruses (human immunodeficiency virus (HIV) -1, HIV-2, and simian immunodeficiency virus (SIV), the nef gene is highly conserved and encodes a myristylated protein of approximately 27 kDa (HIV-1) or approximately 34 kDa (HIV-2, SIV). Previously, we found Nef expressed either as a CD8-Nef fusion protein or as a native protein in virally infected T cell lines associates with a cellular serine kinase. This kinase activity phosphorylated two proteins of 62 and 72 kDa that coimmunoprecipitate with Nef in in vitro kinase assays. Using transient expression, various Nef alleles and mutants have been analyzed for association with the cellular kinase activity. The ability of Nef to associate with the kinase activity is conserved among several alleles of HIV-1 as well as SIVmac239 and is observed in non-lymphoid cell lines of simian and murine origins. Two separate regions of HIV-1SF2 Nef are critical for the associated kinase activity. One domain overlaps with a central highly conserved region found in all primate lentivirus nef genes and has been provisionally mapped to amino acids 45-127. Because membrane localization of Nef is important for the associated cellular kinase activity, the second domain represents a membrane targeting signal. Moreover, point mutations within the central region that abrogate the Nef-associated kinase activity in HIV-1SF2 Nef have the same effect when introduced into SIVmac239open Nef.

Asunto(s)

Productos del Gen nef/genética , Productos del Gen nef/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Secuencia Conservada , Fibroblastos/metabolismo , Productos del Gen nef/química , Genes nef , VIH-1/genética , VIH-2/genética , Humanos , Riñón , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Primates , Homología de Secuencia de Aminoácido , Virus de la Inmunodeficiencia de los Simios/genética , Transfección , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas

RESUMEN

Nef of primate lentiviruses is required for viremia and progression to AIDS in monkeys. Negative, positive, and no effects of Nef have also been reported on viral replication in cells. To reconcile these observations, we expressed a hybrid CD8-Nef protein in Jurkat cells. Two opposite phenotypes were found, which depended on the intracellular localization of Nef. Expressed in the cytoplasm or on the cell surface, the chimera inhibited or activated early signaling events from the T cell antigen receptor. Activated Jurkat cells died by apoptosis, and only cells with mutated nef genes expressing truncated Nefs survived, which rendered Nef nonfunctional. These mutations paralleled those in other viral strains passaged in vitro. Not only do these positional effects of Nef reconcile diverse phenotypes of Nef and suggest a role for its N-terminal myristylation, but they also explain effects of Nef in HIV infection and progression to AIDS.

Asunto(s)

Genes nef/inmunología , VIH-1/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linfocitos T CD8-positivos/química , Productos del Gen nef/análisis , Humanos , Células Híbridas/fisiología , Líquido Intracelular/química , Activación de Linfocitos , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Proteínas Recombinantes de Fusión/análisis , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/metabolismo , Proteínas Virales de Fusión/análisis , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

SV40 large T-antigen (T-ag) mutants were generated using a cassette mutagenesis strategy and naturally occurring restriction sites. T-ag mutant constructs included internal in-frame deletions, frame-shift deletions that resulted in amino-terminal fragments, and internal initiation mutants that produced carboxy-terminal fragments; no foreign amino acids were introduced. The deletion mutants were stably expressed in BALB/c 3T3E cells and were analyzed for ability to bind heat shock cognate protein 70 using an ATP release assay of T-ag immunoprecipitates. Complex formation between heat shock protein and T-ag was independent of p53 involvement. The heat shock protein binding domain was narrowed to the amino-terminal 97 amino acids of T-ag, with the first 29 residues influencing the interaction. The amino-terminal domain of T-ag is important in both viral replication and cell transformation. We propose that the functional interactions of this highly interactive region of T-ag may be modulated by heat shock cognate protein 70.

Asunto(s)

Antígenos Transformadores de Poliomavirus/química , Eliminación de Gen , Proteínas de Choque Térmico/metabolismo , Unión Proteica/genética , Células 3T3 , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Línea Celular , Regulación Viral de la Expresión Génica , Proteínas de Choque Térmico/genética , Ratones , Ratones Endogámicos BALB C , Mutación

RESUMEN

With T-cell lines constitutively expressing Nef from the SF2 strain of human immunodeficiency virus type 1 (HIV-1SF2) in the form of a hybrid CD8-Nef fusion protein or T-cell lines chronically infected with HIV-1SF2, a cellular serine kinase was found that specifically associates with Nef. Proteins of 62 kDa and 72 kDa, which coimmunoprecipitated with Nef, were phosphorylated in in vitro kinase assays. This Nef-associated serine kinase activity was not blocked by inhibitors of protein kinase C or protein kinase A and was lost when Nef was truncated at amino acid 94 or 99. These findings present evidence that a serine kinase activity is associated with Nef expressed in human T lymphocytes.

Asunto(s)

Productos del Gen nef/metabolismo , VIH-1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/microbiología , Antígenos CD8/genética , Antígenos CD8/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Análisis Mutacional de ADN , Productos del Gen nef/genética , Humanos , Sustancias Macromoleculares , Fosfoserina/análisis , Pruebas de Precipitina , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas

RESUMEN

Deletion mutants of SV40 T-antigen were stably expressed in BALB/c 3T3E cells and characterized in immunoprecipitation assays using T-antigen-specific monoclonal antibodies. The epitope recognized by antibody PAb602 was narrowed to T-antigen residues 230-362. Coprecipitation results were compatible with a p53-binding region between T-antigen amino acids 347 and 517. Amino terminal deletions (1-108 or 98-229) of T-antigen appeared to have pronounced effects on the conformation of distal regions of the molecule, based both on antibody binding and on association with p53.

Asunto(s)

Antígenos Transformadores de Poliomavirus/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/ultraestructura , Línea Celular , Análisis Mutacional de ADN , Epítopos , Ratones , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Relación Estructura-Actividad

RESUMEN

Many investigators have tried to prove a relationship between Crohn's disease and Mycobacteria. Recent evidence suggests that some autoimmune diseases may be initiated through "molecular mimicry" between mycobacterial stress protein antigens and their human homologs. We investigated whether antibody to stress proteins was more frequent in patients with Crohn's disease than controls. We used ATP binding to separate stress proteins (heat-shock-induced, de novo-synthesized, and constitutively expressed ATP-binding proteins) from crude extracts obtained from Mycobacteria and from an SV40-transformed human epithelial cell line that expresses a heat-shock protein, hsp73, as a complex with SV40 T antigen. We used immunoblots to compare sera from 34 patients with Crohn's disease, 14 with ulcerative colitis, and 14 with duodenal or gastric ulcers (noninflammatory bowel disease control patients). We found no statistically significant pattern or frequency of antibodies against single proteins or a combination of mycobacterial or human stress proteins. These observations do not support the hypothesis that a humoral immune response to stress proteins of Mycobacteria is important in the pathogenesis of Crohn's disease.

Asunto(s)

Enfermedades Autoinmunes/inmunología , Enfermedad de Crohn/inmunología , Proteínas de Choque Térmico/inmunología , Formación de Anticuerpos , Antígenos Bacterianos/inmunología , Humanos , Mycobacterium tuberculosis/inmunología

RESUMEN

The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000-60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the Mr 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the beta subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperature-dependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoelectron microscopy. Treatment of mKSA cells with 10 microM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control.

Asunto(s)

Antígenos Transformadores de Poliomavirus/fisiología , Transformación Celular Viral , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Aminoácidos , Animales , Antígenos Transformadores de Poliomavirus/aislamiento & purificación , Línea Celular Transformada , Colchicina/farmacología , Microtúbulos/química , Microtúbulos/efectos de los fármacos , Datos de Secuencia Molecular , Transducción de Señal , Virus 40 de los Simios/fisiología , Tubulina (Proteína)/metabolismo , Proteína p53 Supresora de Tumor/aislamiento & purificación

RESUMEN

The viral oncoprotein of simian virus 40, large T antigen (T-ag), is essential for viral replication and cellular transformation. To understand the mechanisms by which T-ag mediates its multifunctional properties, it is important to identify the cellular targets with which it interacts. A cellular protein of 73 kilodaltons (p73) which specifically associates with T-ag in simian virus 40-transformed BALB/c 3T3E cells has been identified. The binding of p73 to T-ag was demonstrated by coimmunoprecipitation analyses using polyclonal and monoclonal antibodies specific for T-ag. The interaction of p73 with T-ag was independent of T-ag complex formation with the cellular protein p53. Partial V8 protease cleavage maps for p73 and the cellular heat shock protein hsp70 were identical. Immunoblot analyses indicated that p73 complexed to T-ag was antigenically related to hsp70. T-ag deletion mutants were constructed that remove internal, amino-terminal, and carboxy-terminal sequences. These mutants mapped the p73 binding domain to the amino terminus of T-ag. The specific dissociation of p73 from the p73/T-ag complex was mediated by ATP; GTP, CTP, and UTP were also utilized as substrates. These characteristics suggest that p73 may be a member of the hsp70 family of heat shock proteins. The biologic significance of p73/T-ag complex formation has yet to be determined.

Asunto(s)

Antígenos Transformadores de Poliomavirus/fisiología , Proteínas de Choque Térmico/fisiología , Animales , Antígenos Transformadores de Poliomavirus/análisis , Antígenos Transformadores de Poliomavirus/inmunología , Sitios de Unión , Línea Celular Transformada , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/inmunología , Ratones , Mutación , Pruebas de Precipitina , Especificidad por Sustrato