RESUMO
Herpes simplex virus type 1 (HSV-1) infection induces severe alterations of the translational apparatus, including the phosphorylation of a few ribosomal proteins, and the progressive association of several nonribosomal proteins to ribosomes. Therefore, we hypothesized that ribosomes themselves could contribute to the HSV-1-induced translational control of host and viral gene expression. As a prerequisite to test this hypothesis, we undertook the identification of the nonribosomal proteins associated to the ribosomes during the course of HSV-1 infection. After separation by two-dimensional polyacrylamide gel electrophoresis of basic proteins extracted from the ribosomal fraction, the identification of unknown protein spots was carried out by N-terminal sequencing and peptide mass determination by mass spectrometry. This allowed us to identify HSV-1 VP19C and VP26 that associated to ribosomes with different kinetics. Another nonribosomal protein turned out to be the poly(A)-binding protein 1 (PAB1P). Newly synthesized PAB1P continued to associate to ribosomes all along infection.
Assuntos
Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidade , Proteínas Ribossômicas/metabolismo , Proteínas Virais/metabolismo , Regulação Viral da Expressão Gênica , Células HeLa , Herpes Simples/genética , Herpesvirus Humano 1/genética , Humanos , Cinética , Biossíntese de Proteínas , Proteoma , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Proteínas Virais/genéticaRESUMO
Nucleolin is one of the major nonribosomal proteins of the nucleolus. Through its four RNA-binding domains, nucleolin interacts specifically with pre-rRNA as soon as synthesis begins, but it is not found in mature cytoplasmic ribosomes. Nucleolin is able to shuttle between the cytoplasm and the nucleus. These data suggest that nucleolin might be involved in the nucleolar import of cytoplasmic components and in the assembly of pre-ribosomal particles. Here we show, using two-dimensional blots in a ligand blotting assay, that nucleolin interacts with 18 ribosomal proteins from rat (14 and 4 from the large and small subunit, respectively). The C-terminal domain of nucleolin (p50) interacts with 10 of these identified ribosomal proteins. In vitro binding assays show that the glycine-arginine rich domain of nucleolin (RGG domain) is sufficient for the interaction with one of these proteins. Interestingly, most of the proteins that interact with p50 belong to the core ribosomal proteins, which are resistant to extraction with high salt concentration. These findings suggest that nucleolin might be involved in the nucleolar targeting of some ribosomal proteins and in their assembly within pre-ribosomal particles.
Assuntos
Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Sítios de Ligação , Western Blotting , Células CHO , Cricetinae , Eletroforese em Gel Bidimensional , Humanos , Ligação Proteica , RNA Ribossômico/metabolismo , Ratos , NucleolinaRESUMO
Herpes simplex virus type 1 (HSV- 1) US11 protein is an RNA-binding protein which is able to mediate post-transcriptional transactivation of human T-lymphotropic virus type I (HTLV-I) envelope glycoprotein gene expression by interacting with the Rex responsive element (XRE) located at the 3' end of the env mRNA. In view of this functional activity, and because US11 protein is capable of substituting for HTLV-I Rex protein, it was hypothesized that US11 protein should exhibit at least two functional domains, an RNA-binding domain for specific interaction with the target RNA, and an effector domain involved in transport and translation of this mRNA. Recombinant US11 wild-type and deleted proteins were tested for their ability (i) to bind to the XRE and to HSV-1 UL34 RNA, the natural target of US11 protein, and (ii) to transactivate HTLV-I env gene expression. The C-terminal half of US11 protein, consisting of 20-24 XPR repeats, was necessary and sufficient to mediate RNA-binding with a high affinity and specificity. Structure prediction analyses showed the likely conformation of this domain to be that of a polyproline type II helix. Localized within the first 40 amino acids of the N-terminal region of US11 protein was the effector domain, deletion of which created US11(delta1-40), a trans-dominant negative mutant. These results demonstrate structural differences between US11 protein and proteins like Rex and Rev, despite their functional similarities.
Assuntos
Produtos do Gene env/genética , Produtos do Gene rex/metabolismo , Glicoproteínas/genética , Herpesvirus Humano 1/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/genética , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Ativação Transcricional , Proteínas Virais/metabolismo , Sítios de Ligação , Simulação por Computador , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Modelos Moleculares , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/genéticaRESUMO
One of the herpes simplex virus type 1 (HSV-1) true late gene products, Us11 protein, is brought into the cell by the infecting virion and may play a role in the virally-induced post-transcriptional control of gene expression. Us11 protein forms large oligomers, exhibits RNA binding features, concentrates into the nucleolus and is able to replace Rex protein in post-transcriptional control of human T-cell leukemia/lymphoma virus type I (HTLV-I) expression. As heat shock drastically alters protein synthesis, and because HSV-1 infection stimulates heat shock protein (Hsp) expression, we analyzed the consequence of heat shock in HeLa cells expressing Us11 alone, either transiently or constitutively. No detectable modification of the overall pattern of protein synthesis was observed in cells growing at normal temperatures, including no induction of Hsp expression or accumulation. However, Us11 protein expression induced an enhanced recovery of protein synthesis after heat shock. Moreover, the level of Us11 protein-mediated protection of protein synthesis was similar to that observed for cells made thermotolerant, but only when submitted to a mild heat shock. Finally, Us11 protein expression induced in cells an enhanced survival to heat shock.
Assuntos
Herpes Simples/metabolismo , Herpesvirus Humano 1/genética , Proteínas de Ligação a RNA/genética , Estresse Fisiológico/virologia , Proteínas Virais/genética , Sobrevivência Celular/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Células HeLa , Herpesvirus Humano 1/metabolismo , Temperatura Alta , Humanos , Immunoblotting , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico/metabolismo , Fatores de Tempo , Proteínas Virais/biossíntese , Proteínas Virais/metabolismoRESUMO
Nuclear distribution and migration of herpes simplex virus type 1 Us11 transcripts were studied in transient expression at the ultrastructural level and compared to that of RNA polymerase II protein. Transcription was monitored by autoradiography following a short pulse with tritiated uridine. Us11 transcripts accumulated mainly over the foci of intermingled RNP fibrils as demonstrated by the presence of silver grains localizing incorporated radioactive uridine superimposed to these structures in which the presence of Us11 RNA and poly(A) tails was previously demonstrated. Silver grains were also scattered over the remaining nucleoplasm but not in the clusters of interchromatin granules, and over the dense fibrillar component of the nucleolus as in control, nontransfected HeLa cells. Pulse-chase experiments revealed the transient presence of migrating RNA in the clusters of interchromatin granules. RNA polymerase II was revealed by immunogold labeling following the use of two monoclonal antibodies: mAb H5, which recognizes the hyperphosphorylated form of the carboxy-terminal domain (CTD) of the molecule, and mAb 7C2, which recognizes both its hyperphosphorylated and unphosphorylated forms. The two mAbs bind to the newly formed Us11 transcription factories and the clusters of interchromatin granules of transfected cells. In control cells, however, clusters of interchromatin granules were labeled with mAb H5 but not with mAB 7C2. Taken together, our data demonstrate the involvement of the clusters of interchromatin granules in the intranuclear migration of Us11 RNA in transient expression. They also suggest the occurrence of changes in the accessibility of the RNA polymerase II CTD upon expression of the Us11 gene after transfection by exposing some epitopes, otherwise masked in nontransfected cells.
Assuntos
Herpesvirus Humano 1/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Proteínas Virais/genética , Núcleo Celular , Genes Virais , Células HeLa , Humanos , RNA Polimerase II/metabolismo , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/genética , TrítioRESUMO
The distribution of Us11 RNA and of its encoded protein have been investigated at the ultrastructural level in HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1. In these transfected cells, Us11 protein accumulates at sites identical to those of lytically infected cells, i.e., in nucleoli and in regions of the cytoplasm that contain ribosomes. Us11 RNA and polyadenylated RNA are scattered over the ribosome-rich areas of the cytoplasm. They also accumulate in the nucleoplasm on clustered ribonucleoprotein (RNP) fibrils but also in clusters of interchromatin granules, some of them contiguous to nucleoli. However they are never found in nucleoli. These data reveal the involvement of interchromatin granules in some steps of Us11 mRNA maturation and/or transport.
Assuntos
Genes Virais/genética , Proteínas de Ligação a RNA/genética , Simplexvirus/genética , Proteínas Virais/genética , Expressão Gênica , Herpes Simples/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , Hibridização In Situ , Microscopia Imunoeletrônica , Poli A/genética , Conformação Proteica , RNA Mensageiro/análise , Proteínas de Ligação a RNA/química , Proteínas Virais/químicaRESUMO
Herpes simplex virus type 1 (HSV-1) Us11 protein, a true late gene product packaged within the virion, is delivered into cells after infection, exhibits a nucleocytoplasmic localization at early times, and later accumulates in the nucleoli. This RNA-binding basic phosphoprotein, capable of oligomerization, is supposed to be involved in post-transcriptional regulation of gene expression after HSV-1 infection. Expression of human T-cell leukaemia/lymphoma virus type-I (HTLV-I) and of human immunodeficiency virus type 1 (HIV-1) is post-transcriptionally regulated by Rex and Rev, respectively. These proteins are required for the cytoplasmic expression of unspliced gag-pol and singly spliced env transcripts. Here we show that HSV-1 Us11 protein is able to bind Rex- and Rev-responsive elements and to transactivate envelope retroviral glycoprotein expression.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/genética , Herpesvirus Humano 1/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Proteínas de Ligação a RNA/fisiologia , Transativadores/fisiologia , Proteínas do Envelope Viral/genética , Proteínas Virais/fisiologia , Citoplasma/metabolismo , Produtos do Gene rev/genética , Produtos do Gene rev/fisiologia , Produtos do Gene rex/genética , Produtos do Gene rex/fisiologia , Produtos do Gene tax/genética , Células Gigantes/virologia , Células HeLa , Humanos , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transfecção , Proteínas Virais/genética , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The Us11 protein is a true late gene product of herpes simplex virus type 1 (HSV-1), whose exact function is unknown but which exhibits RNA-binding properties and which is phosphorylated on serine residues. In order to determine whether the Us11 protein is phosphorylated by cellular kinase(s) or by virally encoded kinase(s), the Us11 gene has been cloned and transiently expressed in HeLa cells. In addition, HeLa-derived cell lines have been selected for their ability to express Us11 protein constitutively. 32P-Labeling and analysis by two-dimensional electrophoresis of transiently and constitutively expressed Us11 protein demonstrated that, indeed, multiple phosphorylation of the protein occurs in absence of HSV-1 genome expression, indicating that the protein behaves as a natural substrate for cellular kinase(s). In addition, a sequence heterogeneity of the Us11 protein, due to a difference in the number of SPREPR repeats, has been characterized between different strains of HSV-1.
Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Genoma Viral , Herpesvirus Humano 1/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Eletroforese em Gel Bidimensional , Vetores Genéticos , Células HeLa , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfotransferases/metabolismo , Sequências Repetitivas de Ácido Nucleico , Especificidade por SubstratoRESUMO
In addition to an irreversible stimulation of S6 ribosomal protein phosphorylation, there is a modification of a subset of ribosomal proteins by phosphorylation after herpes simplex virus type 1 (HSV-1) infection. Moreover, in the course of this infection, three additional phosphorylated proteins can be extracted from ribosomes and separated by two-dimensional electrophoresis (2-DE) of total ribosomal proteins. One of them exhibits an identical molecular mass to L30, while being more acidic. This protein is phosphorylated on serine residues. The kinetics of appearance of this protein in the ribosomal fraction correlated with a decrease in L30 staining, as shown by 2-DE. Determination of the N-terminal amino acid sequence of this extra phosphoprotein and of L30-derived peptides demonstrated the identity of these two proteins.
Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Dados de Sequência Molecular , Fosforilação , Ratos , Homologia de Sequência de Aminoácidos , Serina/metabolismoRESUMO
The sequence coding for the 5' untranslated region (UTR) of ICP22 mRNA of herpes simplex virus type 1 has been tested for its ability to regulate gene expression. This sequence was placed in frame with the chloramphenicol acetyltransferase (CAT) coding sequence and under the control of the simian virus 40 early promoter-enhancer. Under these conditions, the sequence coding for the 5'UTR led to an increase of about 13-fold in CAT activity, measured during transient expression. The use of mutants with progressive deletions within the sequence coding for the 5'UTR allowed localization of the sequence responsible for the enhancement of gene expression to the first exon of the ICP22 gene. Precise quantification of hybrid ICP22-CAT mRNA showed that the sequence coding for the 5'UTR induced an increase in the amounts of transcripts, which resulted in a parallel increase in CAT activity. This increase in the level of hybrid ICP22-CAT mRNA is not the result of an increase in mRNA stability, nor is it due to more efficient nucleo-cytoplasmic transport of the transcripts. Moreover, the distribution of hybrid mRNA in the different ribosomal populations indicates that the 5'UTR of ICP22 mRNA does not induce a preferential recruitment of the transcripts by the translational apparatus. Taken together, these results indicate that a cis-acting element located in the sequence coding for the 5'UTR of ICP22 mRNA can mediate a high level of gene expression independently of the viral promoter and of viral trans-acting factors.
Assuntos
Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces , RNA Mensageiro/biossíntese , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Virais/genética , Sequência de Bases , Cloranfenicol O-Acetiltransferase/biossíntese , DNA Viral/genética , Genes Precoces/genética , Células HeLa , Humanos , Íntrons , Dados de Sequência Molecular , Polirribossomos/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência/genética , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Proteínas Virais Reguladoras e AcessóriasRESUMO
Microsequencing of a cyanogen bromide peptide obtained from a basic phosphoprotein co-sedimenting with purified ribosomes extracted from herpes simplex virus type 1-infected human epidermoid carcinoma 2 cells identified this protein as a product of the true late US11 gene. An antibody was raised against a recombinant fusion protein expressed in Escherichia coli from a plasmid carrying 75% of the US11 coding sequence including the carboxy terminus. This antibody was used to probe Western blots carried out under various conditions of one- and two-dimensional electrophoresis. The electrophoretic behaviour of the immunoreactive proteins offered further proof that they were indeed products of the US11 gene. This US11 protein, which has phosphates on multiple serine residues, is brought into the cell by the virion and found to be present within ribosome fractions early after infection. This association with ribosomes is non-specific and due to probable aggregation or oligomerization of this proline-rich basic protein allowing its co-sedimentation with ribosomes during the different subcellular fractionation steps used for the purification of ribosomal subunits.
Assuntos
Ribossomos/microbiologia , Simplexvirus/química , Proteínas Estruturais Virais/análise , Centrifugação com Gradiente de Concentração , Humanos , Fosforilação , Ribossomos/química , Células Tumorais Cultivadas , Proteínas Estruturais Virais/fisiologiaRESUMO
We report a case of acute regressive meningoradiculitis concomitant with HIV1 primoinfection. The clinical symptoms were mild and disappeared spontaneously. Electromyographic studies confirmed the regressive demyelinating-type process. The biological diagnosis of HIV1 infection was demonstrated by viral antigen detection, by the presence of the integrated proviral DNA after gene amplification by a polymerase chain reaction, and in particular, by the steady progression in the appearance of different HIV1-specific antibodies. This was shown using three different Western blot kits whose performances were compared.
Assuntos
Soropositividade para HIV/complicações , HIV-1 , Meningite/complicações , Radiculopatia/complicações , Doença Aguda , Adulto , Western Blotting , Eletromiografia , Feminino , Soropositividade para HIV/diagnóstico , Humanos , Meningite/diagnóstico , Reação em Cadeia da Polimerase , Radiculopatia/diagnóstico , Kit de Reagentes para DiagnósticoRESUMO
The study of 500 blood donors, of 23 prostitutes, 22 confirmed AIDS cases and 19 ARC patients in Abidjan showed that HIV-2 prevalence in blood donors (3.4%) was somewhat higher than that of HIV-1 (2.4%). Furthermore HIV-2 alone was found associated with 14% of AIDS cases and 21% of ARC, while HIV-1 was associated with 32% of the cases. Antibodies to HIV-1 and HIV-2 were found in 54% of AIDS and ARC cases, as well as in 48% of the prostitutes sera. These preliminary results suggest that HIV-2 is pathogenic, being causally involved in some AIDS cases and does not protect from HIV-1 pathogenic potential.
Assuntos
Complexo Relacionado com a AIDS/complicações , Síndrome da Imunodeficiência Adquirida/complicações , Infecções por Deltaretrovirus/complicações , Complexo Relacionado com a AIDS/imunologia , Complexo Relacionado com a AIDS/microbiologia , Síndrome da Imunodeficiência Adquirida/epidemiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/microbiologia , Anticorpos Antivirais/análise , Doadores de Sangue , Côte d'Ivoire , Infecções por Deltaretrovirus/epidemiologia , Anticorpos Anti-HIV , Humanos , Trabalho SexualRESUMO
We used two-dimensional polyacrylamide gel electrophoresis and immunoblotting techniques to study serum proteins from a patient with a monoclonal gammopathy. Two-dimensional electrophoresis was optimized for serum proteins with two main goals: (a) to allow the resolution of many serum proteins in both directions, with penetration of the maximum number of proteins in the first dimension; and (b) to obtain the best reproducibility from one experiment to another, within the limits of the current technique. These analyses, combined with immunoblotting, permitted us to characterize a gamma heavy chain disease protein of 34 000-Da molecular mass. Moreover, two-dimensional mapping of the patient's serum proteins allowed demonstration of the microheterogeneity of this monoclonal component.