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1.
Nat Struct Mol Biol ; 12(8): 671-7, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16041387

RESUMEN

Formation of infectious HIV-1 involves assembly of Gag polyproteins into immature particles and subsequent assembly of mature capsids after proteolytic disassembly of the Gag shell. We report a 12-mer peptide, capsid assembly inhibitor (CAI), that binds the capsid (CA) domain of Gag and inhibits assembly of immature- and mature-like capsid particles in vitro. CAI was identified by phage display screening among a group of peptides with similar sequences that bind to a single reactive site in CA. Its binding site was mapped to CA residues 169-191, with an additional contribution from the last helix of CA. This result was confirmed by a separate X-ray structure analysis showing that CAI inserts into a conserved hydrophobic groove and alters the CA dimer interface. The CAI binding site is a new target for antiviral development, and CAI is the first known inhibitor directed against assembly of immature HIV-1.


Asunto(s)
Antivirales/metabolismo , Proteínas de la Cápside/metabolismo , Cápside/fisiología , Productos del Gen gag/metabolismo , VIH-1/fisiología , Péptidos/metabolismo , Ensamble de Virus/fisiología , Secuencia de Aminoácidos , Antivirales/genética , Sitios de Unión , Cápside/ultraestructura , Proteínas de la Cápside/genética , Ensayo de Inmunoadsorción Enzimática , Productos del Gen gag/genética , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Biblioteca de Péptidos , Péptidos/genética
2.
J Virol ; 79(15): 9608-17, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16014923

RESUMEN

Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor alpha (TNF-alpha), alpha/beta interferon (IFN-alpha/beta), and IFN-like interleukin-28A/B (IFN-lambda2/3) and IL-29 (IFN-lambda1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-alpha, IFN-alpha/beta, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-alpha, IFN-alpha, IFN-beta, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-alpha. IFN-alpha priming led to increased expression of Toll-like receptor 3 (TLR3), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF3. Influenza A virus also enhanced the binding of NF-kappaB to the respective NF-kappaB elements of the promoters of IFN-beta and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-alpha or IFN-beta. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response.


Asunto(s)
Virus de la Influenza A/fisiología , Interferón-alfa/biosíntesis , Interferón beta/biosíntesis , Interleucinas/biosíntesis , Virus Sendai/fisiología , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/biosíntesis , Proteínas Adaptadoras del Transporte Vesicular/genética , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Antivirales/genética , Antivirales/farmacología , Diferenciación Celular , Células Cultivadas , Citocinas , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Células Dendríticas/citología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Expresión Génica/efectos de los fármacos , Humanos , Factor 3 Regulador del Interferón , Interferón-alfa/farmacología , Interferón beta/farmacología , Interferones , Interleucinas/farmacología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Factor 88 de Diferenciación Mieloide , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptor Toll-Like 3 , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Receptores Toll-Like , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología
3.
Biochemistry ; 44(6): 1909-18, 2005 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-15697216

RESUMEN

The biologically active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase (RT) found in infectious virions are heterodimers. We have previously shown that the dimeric nature of reverse transcriptase represents an important target for the design of a new class of antiviral agents and have designed a short peptide (Pep-7) derived from the tryptophan-rich motif of the connection subdomain that blocks dimerization of reverse transcriptase in vitro and abolishes viral infection. In the present work, we have investigated the mechanism through which this peptide inhibits RT dimerization and consequently viral propagation. We demonstrate that Pep-7 interacts preferentially with the p51 subunit within the heterodimeric reverse transcriptase, which destabilizes reverse transcriptase dimer conformation, thereby triggering dissociation. We have identified two residues Trp(24) and Phe(61), located on the fingers subdomain of p51, required for Pep-7 binding. Selective mutation of these residues on p51 to a glycine dramatically alters the stability of the RT-heterodimer suggesting that the fingers subdomain of p51 is also involved in stabilization of reverse transcriptase. We propose that the binding site of Pep-7 is located in a cleft between the fingers and the connection subdomains of p51 that contains the two highly conserved residues Phe(61) and Trp(24).


Asunto(s)
Fármacos Anti-VIH/química , Antivirales/síntesis química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , Oligopéptidos/síntesis química , Proteínas Virales/química , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Fármacos Anti-VIH/metabolismo , Antivirales/genética , Antivirales/metabolismo , Dimerización , Estabilidad de Enzimas/genética , Transcriptasa Inversa del VIH/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fenilalanina/genética , Unión Proteica/genética , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Triptófano/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo
4.
J Immunol ; 174(3): 1574-9, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15661918

RESUMEN

CD8(+) cells from HIV-infected individuals showing the CD8(+) cell noncytotoxic antiviral response unexpectedly revealed mRNA for VCAM-1, a cell surface molecule found on endothelial cells. Uninfected subjects had undetectable levels of VCAM-1 mRNA in their CD8(+) cells. Flow cytometry analysis showed that up to 12% of the CD8(+) cells from HIV-positive individuals expressed VCAM-1 compared with 0.8% of the CD8(+) cells of HIV-negative individuals. Enrichment of the CD8(+)VCAM-1(+) cell population and subsequent coculture with CD4(+) cells acutely infected with HIV-1 showed that the VCAM-1(+)CD8(+) cells were able to suppress viral replication with 50% less input cells than the unseparated CD8(+) cell population. This study demonstrates, for the first time, the expression of VCAM-1 on CD8(+) cells. Moreover, the CD8(+)VCAM-1(+) cells show enhanced CD8(+) cell noncytotoxic antiviral response activity that could have clinical importance in HIV infection.


Asunto(s)
Adyuvantes Inmunológicos/biosíntesis , Fármacos Anti-VIH/metabolismo , Antivirales/biosíntesis , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Regulación hacia Arriba/inmunología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Enfermedad Aguda , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Antivirales/genética , Antivirales/fisiología , Linfocitos T CD8-positivos/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/virología , Células Cultivadas , Técnicas de Cocultivo , Seronegatividad para VIH/inmunología , Seropositividad para VIH/genética , Seropositividad para VIH/inmunología , Seropositividad para VIH/metabolismo , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Humanos , Interleucina-15/farmacología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , ARN Mensajero/biosíntesis , Linfocitos T/inmunología , Linfocitos T/virología , Regulación hacia Arriba/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/fisiología
5.
J Immunol ; 174(3): 1587-93, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15661920

RESUMEN

Human CMV is often associated with transplant rejection and opportunistic infections such as pneumonia in immunosuppressed patients. Current anti-CMV therapies, although effective, show relatively high toxicity, which seriously limits their long-term use. In this study, we provide evidence that leukotriene B(4) (LTB(4)) plays an important role in the fight against murine CMV (MCMV) infection in vivo. Intravenous administration of 50 and 500 ng/kg/day of LTB(4) to mice infected with a lethal dose of MCMV significantly increases their survival (50 and 70%, respectively), compared with the placebo-treated group (10% of survival). In mice infected with a sublethal dose of MCMV and treated daily with 50 ng/kg/day of LTB(4), the salivary gland viral loads were found to be reduced by 66% compared with the control group. Furthermore, using an allogeneic bone marrow transplantation mouse model, the frequency of MCMV reactivation from latently infected mice was much lower (38%) in LTB(4) (500 ng/kg)-treated mice than in the placebo-treated group (78%). Finally, in experiments using 5-lipoxygenase-deficient mice, MCMV viral loads in salivary glands were found to be higher in animals unable to produce leukotrienes than in the control groups, supporting a role of endogenous 5-lipoxygenase products, possibly LTB(4), in host defense against CMV infection.


Asunto(s)
Antivirales/uso terapéutico , Trasplante de Médula Ósea/efectos adversos , Infecciones por Citomegalovirus/prevención & control , Leucotrieno B4/uso terapéutico , Muromegalovirus/fisiología , Activación Viral , Latencia del Virus , Animales , Antivirales/deficiencia , Antivirales/genética , Antivirales/fisiología , Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/fisiología , Trasplante de Médula Ósea/inmunología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/mortalidad , Infecciones por Citomegalovirus/virología , Femenino , Rechazo de Injerto/genética , Rechazo de Injerto/prevención & control , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/prevención & control , Inyecciones Intravenosas , Leucotrieno B4/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Glándulas Salivales/efectos de los fármacos , Glándulas Salivales/virología , Bazo/citología , Bazo/trasplante , Bazo/virología , Carga Viral , Activación Viral/genética , Activación Viral/inmunología , Latencia del Virus/genética , Latencia del Virus/inmunología
6.
Curr Biol ; 15(1): 73-8, 2005 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-15649369

RESUMEN

Retroviral restriction factors are cellular proteins that interfere with retrovirus replication at a postpenetration, preintegration stage in the viral life cycle. The first restriction activity described was the mouse Fv1 gene. Three different alleles of Fv1, capable of restricting different murine leukaemia viruses (MLV), have been characterized at the molecular level. Two further activities, Ref1, which acts on MLV, and Lv1, which acts on lentiviruses, have been identified in other mammalian species. Recently, it has become clear that Ref1 and Lv1 are encoded by the same gene, Trim5alpha, which inhibits retrovirus replication in a species-specific manner. A series of chimeras between the human and rhesus monkey Trim5 genes were created to map and identify these specificity determinants. The Trim5alpha SPRY domain was found to be responsible for targeting HIV-1 restriction. By contrast, N-MLV restriction appears dependent on both the coiled-coil domain and the SPRY domain. A single amino acid substitution (R332P) in the human Trim5alpha can confer the ability to restrict HIV-1, suggesting that small changes during evolution may have profound effects on our susceptibility to cross-species infection.


Asunto(s)
Sustitución de Aminoácidos/genética , Antivirales/genética , Proteínas Portadoras/genética , VIH-1 , Proteínas Recombinantes de Fusión/genética , Replicación Viral/genética , Secuencia de Aminoácidos , Animales , Factores de Restricción Antivirales , Citometría de Flujo , Proteínas Fluorescentes Verdes , Haplorrinos , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Transducción Genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas
7.
Protein Pept Lett ; 11(6): 551-61, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15579125

RESUMEN

Plants antiviral proteins are being used as anticancer agents and inhibit other viral diseases in humans. We modified the purification protocol of the two N-terminally blocked antiviral glycoproteins, CCP-25 and CCP-27, purified from the leaves of Celosia cristata. This not only gave rise to single pure samples with few steps of purification but also resulted in N-terminally free proteins. The extra purity of the samples was analyzed by reverse phase HPLC. Deglycosylation studies of CCP-25 with PNGase F enzyme revealed that its asparagine or asparagine-linked glycon contents are negligible. Partial N-terminal sequence of the CCP-25 showed the sequence (ANDIS), which seems to be conserved among plant antiviral proteins.


Asunto(s)
Antivirales/aislamiento & purificación , Celosia/genética , Proteínas de Plantas/aislamiento & purificación , Secuencia de Aminoácidos , Antivirales/genética , Antivirales/farmacología , Bioensayo , Celosia/virología , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Proteínas de Plantas/genética , Proteínas de Plantas/farmacología , Virus de Plantas/efectos de los fármacos , Alineación de Secuencia , Análisis de Secuencia de Proteína
8.
J Immunol ; 173(10): 6303-11, 2004 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-15528369

RESUMEN

A large proportion of the CD8(+) T cell pool in persons chronically infected with HIV consists of cells that show features of replicative senescence, an end stage characterized by irreversible cell cycle arrest, multiple genetic and functional changes, and shortened telomeres. The objective of our research was to determine whether constitutive expression of the gene for the human telomerase (hTERT) can prevent senescence-induced impairments in human virus-specific CD8(+) T cells, particularly in the context of HIV-1 disease. Our results indicate that hTERT-expressing HIV-specific CD8(+) lymphocytes show both an enhanced and sustained capacity to inhibit HIV-1 replication in in vitro coculture experiments, as well as prolonged ability to produce IFN-gamma and TNF-alpha in response to stimulation with HIV-1-derived peptides, as compared with vector-transduced controls. Loss of CD28 expression, the signature change of replicative senescence in cell culture, was retarded in those CD8(+) T cell cultures that had high levels of CD28 at the time of hTERT transduction. These findings suggest that telomere shortening may be the primary driving force behind several aspects of CD8(+) T cell dysfunction associated with replicative senescence. We also demonstrate reduced accumulation of the p16(INK4a) and p21(WAF1) cell cycle inhibitors in hTERT-transduced lymphocytes, providing a possible mechanism by which stable hTERT expression is able to circumvent the senescence barrier in CD8(+) T cells. Given the key role of CD8(+) T cell function in controlling a variety of acute and latent viral infections, approaches to retard the functional decrements associated with replicative senescence may lead to novel types of immunotherapy.


Asunto(s)
Adyuvantes Inmunológicos/fisiología , Antivirales/fisiología , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , VIH-1/inmunología , Telomerasa/fisiología , Telómero/metabolismo , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Antivirales/biosíntesis , Antivirales/genética , Antivirales/metabolismo , Antígenos CD28/biosíntesis , Antígenos CD28/fisiología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/virología , Ciclo Celular/genética , Ciclo Celular/inmunología , Proliferación Celular , Células Cultivadas , Senescencia Celular/genética , Senescencia Celular/inmunología , Citotoxicidad Inmunológica/genética , Proteínas de Unión al ADN , Inhibidores de Crecimiento/antagonistas & inhibidores , Inhibidores de Crecimiento/biosíntesis , VIH-1/crecimiento & desarrollo , VIH-1/fisiología , Humanos , Interferón gamma/biosíntesis , Interferón gamma/fisiología , Telomerasa/biosíntesis , Telomerasa/genética , Telomerasa/metabolismo , Telómero/enzimología , Telómero/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba/inmunología , Replicación Viral/genética , Replicación Viral/inmunología
10.
Proc Natl Acad Sci U S A ; 101(29): 10774-9, 2004 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-15249685

RESUMEN

Mammalian cells express several factors that act in a cell-autonomous manner to inhibit retrovirus replication. Among these are the Friend virus susceptibility factor 1/lentivirus susceptibility factor 1/restriction factor 1 (Ref1) class of restriction factors, which block infection by targeting the capsids of diverse retroviruses. Here we show that lentivirus susceptibility factor 1 and Ref1 are species-specific variants of tripartite interaction motif 5alpha (TRIM5alpha), a cytoplasmic body component recently shown to block HIV-1 infection in rhesus macaque cells, and can indeed block infection by widely divergent retroviruses. Depletion of TRIM5alpha from human cells relieved restriction of N-tropic murine leukemia virus (N-MLV), and expression of human TRIM5alpha in otherwise nonrestricting cells conferred specific resistance to N-MLV infection, indicating that TRIM5alpha is Ref1 or an essential component of Ref1. TRIM5alpha variants from humans, rhesus monkeys, and African green monkeys displayed different but overlapping restriction specificities that were quite accurately predicted by the restriction properties of the cells from which they were derived. All TRIM5alpha variants could inhibit infection by at least two different retroviruses, and African green monkey TRIM5alpha was able to inhibit infection by no less than four divergent retroviruses of human, non-human primate, equine, and murine origin. However, each TRIM5alpha variant was unable to restrict retroviruses isolated from the same species. These data indicate that TRIM5alpha can confer broad innate immunity to retrovirus infection in primate cells and is likely to be an important natural barrier to cross-species retrovirus transmission.


Asunto(s)
Antivirales/metabolismo , Proteínas Portadoras/metabolismo , Retroviridae/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/genética , Factores de Restricción Antivirales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Infecciones por Retroviridae/metabolismo , Alineación de Secuencia , Especificidad de la Especie , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas
11.
Proc Natl Acad Sci U S A ; 101(29): 10780-5, 2004 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-15249687

RESUMEN

The rhesus macaque tripartite motif containing protein TRIM5alpha specifically restricts HIV-1 infection at an early post-entry step before reverse transcription [Stremlau, M., Owens, C. M., Perron, M. J., Kiessling, M., Autissier, P. & Sodroski, J. (2004) Nature 427, 848-853]. Here, we show that the human and African green monkey (AGM) TRIM5alpha genes encode Ref1 and Lv1 antiretroviral activities, respectively. Expression of TRIM5alpha in permissive cat cells renders them resistant to restriction-sensitive murine leukemia virus but not closely related insensitive virus. Disruption of TRIM5alpha expression in human and AGM cells with small interfering RNA rescues infectivity of restricted virus without affecting unrestricted virus. We also demonstrate that the activity of the murine restriction factor Fv1 depends on TRIM5alpha expression when Fv1 is expressed in human cells. Furthermore, a drug that modifies the behavior of the related promyelocytic leukemia protein PML specifically rescues infection by viruses restricted by human TRIM5alpha. Alignment of the TRIM5alpha proteins from rhesus macaque and AGM indicates an 18-aa insertion. We speculate that this insertion may contribute to the broader specificity of the AGM TRIM5alpha restriction as compared with the human and rhesus macaque proteins.


Asunto(s)
Antivirales/metabolismo , Proteínas Portadoras/metabolismo , Chlorocebus aethiops/genética , Factores R/metabolismo , Retroviridae/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/genética , Factores de Restricción Antivirales , Trióxido de Arsénico , Arsenicales/metabolismo , Proteínas Portadoras/genética , Gatos , Línea Celular , Inhibidores de Crecimiento/metabolismo , Humanos , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/metabolismo , Ratones , Datos de Secuencia Molecular , Óxidos/metabolismo , Factores R/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Infecciones por Retroviridae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas
12.
Hum Mol Genet ; 13(16): 1785-91, 2004 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-15198990

RESUMEN

Human cytidine deaminase APOBEC3G and the virion infectivity factor (vif) of the human immunodeficiency virus (HIV) are a pair of antagonistic molecules. In the absence of vif, APOBEC3G induces a high rate of dC to dU mutations in the nascent reverse transcripts of HIV that leads to the degradation of the HIV genome. HIV vif, on the other hand, can suppress the translation and trigger the degradation of human APOBEC3G. Here, we studied the rate of APOBEC3G gene evolution from five hominoids and two Old World monkeys. Averaged across the entire coding region, the rate of non-synonymous nucleotide substitutions is approximately 1.4 times the rate of synonymous substitutions, strongly suggesting that APOBEC3G has been under positive Darwinian selection. A comparison between the nucleotide polymorphisms within humans and the substitutions among the seven primates reveals a significant excess of non-synonymous substitutions. Furthermore, the rate of charge-altering non-synonymous substitution is approximately 1.8 times that of charge-conserving substitution, indicating that the selection is promoting the diversity of the protein charge profile. However, no difference in selective pressure on APOBEC3G is detected between hosts and non-hosts of HIV or simian immunodeficiency virus (SIV). These results, together with recent findings that the antiviral activity of APOBEC3G is not limited to HIV/SIV, suggest that the selective pressure on APOBEC3G is not solely from HIV/SIV and that APOBEC3G is a broad antiviral enzyme. The identification of pervasive positive selection for charge-altering amino acid substitutions supports the hypothesis of electrostatic interactions between APOBEC3G and vif or its functional equivalents.


Asunto(s)
Antivirales/genética , Evolución Molecular , Filogenia , Primates/genética , Proteínas/genética , Selección Genética , Desaminasa APOBEC-3G , Secuencia de Aminoácidos , Animales , Análisis por Conglomerados , Citidina Desaminasa , Productos del Gen vif/metabolismo , VIH/genética , Humanos , Datos de Secuencia Molecular , Mutación/genética , Nucleósido Desaminasas , Polimorfismo Genético , Proteínas/metabolismo , Proteínas Represoras , Alineación de Secuencia , Análisis de Secuencia de ADN , Productos del Gen vif del Virus de la Inmunodeficiencia Humana
13.
Anim Genet ; 35(3): 182-7, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15147388

RESUMEN

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.


Asunto(s)
Antivirales/genética , Bovinos/genética , Proteínas de Unión al GTP/genética , Filogenia , Polimorfismo Genético , Virus de la Estomatitis Vesicular Indiana/genética , Células 3T3 , Empalme Alternativo , Animales , Secuencia de Bases , Análisis por Conglomerados , Cartilla de ADN , ADN Complementario/genética , Proteínas de Unión al GTP/clasificación , Ratones , Datos de Secuencia Molecular , Mutación Missense/genética , Proteínas de Resistencia a Mixovirus , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transfección
14.
Biochemistry ; 43(6): 1410-7, 2004 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-14769016

RESUMEN

The human monoclonal antibody Fab X5 neutralizes a broad range of HIV-1 primary isolates. The crystal structure of X5 has been determined at 1.9 A resolution. There are two crystallographically independent Fab fragments in the asymmetric unit. The crystallographic R value for the final model is 0.22. The antibody-combining site features a long (22 amino acid residues) CDR H3 with a protruding hook-shaped motif. The X5 structure and site-directed mutagenesis data suggest that X5 amino acid residues W100 and Y100F in the CDR H3 motif may be critical for the binding of Fab X5 to gp120. X5 bound to a complex of a CD4 mimetic and gp120 with approximately the same kinetics and affinity as to a CD4-gp120 complex, suggesting that specific interactions between CD4 and X5 are unlikely to contribute to the binding of X5 to gp120-CD4 complexes. Binding of X5 to alanine scanning mutants of gp120JR-CSF complexed with CD4 suggested a critical role of the highly conserved amino acid residues at positions 423 and 432. The X5 structure and fine mapping of its epitope may assist in the elucidation of the mechanisms of viral entry and neutralization, and the development of HIV-1 inhibitors and vaccines.


Asunto(s)
Anticuerpos Monoclonales/química , Epítopos/química , Epítopos/inmunología , VIH-1/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Mapeo Peptídico , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Antivirales/química , Antivirales/genética , Antivirales/metabolismo , Sitios de Unión/genética , Antígenos CD4/química , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Reacciones Cruzadas/genética , Cristalización , Cristalografía por Rayos X , Epítopos/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/patogenicidad , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Mapeo Peptídico/métodos , Estructura Terciaria de Proteína/genética , Resonancia por Plasmón de Superficie
15.
J Biol Chem ; 278(45): 44412-6, 2003 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-12970355

RESUMEN

Human immunodeficiency virus, type 1 (HIV-1) Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion. Vif functions to counteract an anti-HIV-1 cellular factor in non-permissive cells, CEM15/Apobec-3G, which shares a cytidine deaminase motif. CEM15/Apobec-3G deaminates dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. In this study, we have done the mutagenesis analysis on two cytidine deaminase motifs in CEM15/Apobec-3G and examined their antiviral functions as well as the DNA editing activity. Point mutations in the C-terminal active site such as E259Q and C291A almost completely abrogated the antiviral function, while those in the N-terminal active site such as E67Q and C100A retained this activity to a lesser extent as compared with that of the wild type. The DNA editing activities of E67Q and E259Q mutants were both retained but impaired to the same extent. This indicates that the enzymatic activity of this protein is essential but not a sole determinant of the antiviral activity. Furthermore, all the deletion mutants tested in this study lost the antiviral activity because of the loss of the activity for dimerization, suggesting that the entire protein structure is necessary for the antiviral function.


Asunto(s)
Antivirales/fisiología , VIH-1/patogenicidad , Proteínas/fisiología , Desaminasa APOBEC-3G , Antivirales/química , Antivirales/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , Citidina Desaminasa/metabolismo , ADN/química , Dimerización , Eliminación de Gen , Expresión Génica , Humanos , Técnicas de Inmunoadsorción , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nucleósido Desaminasas , Mutación Puntual , Proteínas/química , Proteínas/genética , Edición de ARN , Proteínas Represoras , Relación Estructura-Actividad , Transfección
16.
J Neuroimmunol ; 141(1-2): 40-6, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12965252

RESUMEN

The induction of an antiviral state by type I interferons (IFN) was evaluated in primary trigeminal ganglion cell cultures using herpes simplex virus type 1 (HSV-1). Cells treated with mouse IFN-beta consistently showed the greatest resistance to HSV-1 infection in comparison to cells treated with IFN-alpha1, IFN-alpha4, IFN-alpha5, IFN-alpha6, or IFN-alpha9. The antiviral efficacy was dose-dependent and correlated with the induction of the IFN-inducible, antiviral genes, 2'-5' oligoadenylate synthetase (OAS) and double-stranded RNA-dependent protein kinase. In trigeminal ganglion cells deficient in the downstream effector molecule of the OAS pathway, RNase L, the antiviral state induced by IFN-beta was lost.


Asunto(s)
Antivirales/fisiología , Endorribonucleasas/fisiología , Herpesvirus Humano 1/inmunología , Interferón beta/fisiología , Transducción de Señal/inmunología , Ganglio del Trigémino/inmunología , Ganglio del Trigémino/virología , Replicación Viral/inmunología , Animales , Antivirales/deficiencia , Antivirales/genética , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Endorribonucleasas/deficiencia , Endorribonucleasas/genética , Femenino , Regulación Viral de la Expresión Génica/inmunología , Herpesvirus Humano 1/genética , Inmunidad Innata/genética , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Transducción de Señal/genética , Transfección , Ganglio del Trigémino/citología , Ganglio del Trigémino/enzimología , Células Vero , Replicación Viral/genética
17.
Virology ; 313(1): 170-83, 2003 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-12951031

RESUMEN

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.


Asunto(s)
Antivirales/genética , Péptidos/genética , Rhinovirus/efectos de los fármacos , Secuencia de Aminoácidos , Antivirales/aislamiento & purificación , Antivirales/farmacología , Secuencia de Bases , Clonación Molecular/métodos , Efecto Citopatogénico Viral/efectos de los fármacos , ADN Complementario/biosíntesis , ADN Complementario/genética , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Biblioteca de Genes , Vectores Genéticos , Células HeLa , Humanos , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Péptidos/farmacología , Placenta/química , Retroviridae/genética , Rhinovirus/fisiología , Transfección , Replicación Viral/efectos de los fármacos
18.
J Immunol ; 171(5): 2694-702, 2003 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-12928423

RESUMEN

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.


Asunto(s)
Perfilación de la Expresión Génica , Interferón beta/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/genética , Esclerosis Múltiple Recurrente-Remitente/inmunología , Adulto , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/genética , Antivirales/biosíntesis , Antivirales/genética , Teorema de Bayes , Biomarcadores/análisis , Femenino , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/estadística & datos numéricos , Variación Genética/inmunología , Humanos , Inyecciones Intramusculares , Interferón beta/administración & dosificación , Interferón beta/farmacología , Janus Quinasa 1 , Lectinas Tipo C , Activación de Linfocitos/genética , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Procesamiento Proteico-Postraduccional/inmunología , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/genética , ARN Mensajero/biosíntesis , Transducción de Señal/genética , Transducción de Señal/inmunología
19.
BMC Immunol ; 4: 8, 2003 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-12887736

RESUMEN

BACKGROUND: Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported. RESULTS: This study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1) Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-kappaB. (2) Interferon (IFN)-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-alpha/beta receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN. CONCLUSION: This study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-alpha/beta receptor-mediated paracrine pathway.


Asunto(s)
ADN/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Familia de Multigenes/inmunología , Oligodesoxirribonucleótidos/inmunología , Receptores de Interferón/metabolismo , Anticuerpos Monoclonales/farmacología , Antivirales/genética , Células Cultivadas , Citocinas/genética , ADN/farmacología , Humanos , Interferón Tipo I/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Proteínas de la Membrana , Oligodesoxirribonucleótidos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Receptor de Interferón alfa y beta , Receptores de Interferón/efectos de los fármacos , Regulación hacia Arriba
20.
J Immunol ; 170(7): 3565-71, 2003 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-12646618

RESUMEN

We have recently described an IFN regulatory factor 3-mediated antiviral gene program that is induced by both Toll-like receptor (TLR)3 and TLR4 ligands. In our current study, we show that activation of IFN/viral response gene expression in primary macrophage cells is stronger and prolonged with TLR3 stimulation compared with that of TLR4. Our data also reveal that the cytoplasmic tails of both TLR3 and TLR4 can directly interact with myeloid differentiation factor 88 (MyD88). However, although Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like is able to associate with TLR4, we were unable to detect any interaction between Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like and TLR3. By using quantitative real-time PCR assays, we found that TLR3 expression is inducible by both TLR3 and TLR4 ligands, while TLR4 expression is not inducible by these same stimuli. Furthermore, using cells derived from mice deficient in the IFN-alphabetaR, we show that both TLR3 and TLR4 require IFN-beta autocrine/paracrine feedback to induce TLR3 expression and activate/enhance genes required for antiviral activity. More specifically, a subset of antiviral genes is initially induced independent of IFN-beta, yet the cytokine further enhances expression at later time points. This was in contrast to a second set of genes (including TLR3) that is induced only after IFN-beta production. Taken together, our data argue that, despite both TLR3 and TLR4 being able to use IFN-beta to activate/enhance antiviral gene expression, TLR3 uses multiple mechanisms to enhance and sustain the antiviral response more strongly than TLR4.


Asunto(s)
Antivirales/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Proteínas Adaptadoras Transductoras de Señales , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/metabolismo , Antivirales/genética , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Gammaherpesvirinae/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Interferón-alfa/metabolismo , Interferón beta/biosíntesis , Interferón beta/genética , Interferón beta/metabolismo , Interferón beta/fisiología , Ligandos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide , Receptor de Interferón alfa y beta , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/metabolismo , Receptores de Interferón/fisiología , Receptores de Interleucina-1/biosíntesis , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/fisiología , Factor de Transcripción STAT1 , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 3 , Receptor Toll-Like 4 , Receptores Toll-Like , Transactivadores/metabolismo
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