RESUMEN
The S fragment of the FMDV 5' UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A24 FMDV-S4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A24 FMDV, indicating that the A24 FMDV-S4 virus is highly attenuated in vivo. Additionally, mice exposed to high doses of live A24 FMDV-S4 virus are fully protected when challenged with parental A24 FMDV virus.
Asunto(s)
Regiones no Traducidas 5'/genética , Virus de la Fiebre Aftosa/fisiología , Inmunidad Innata/fisiología , Replicación Viral/fisiología , Animales , Bovinos , Línea Celular , Cricetinae , Virus de la Fiebre Aftosa/genética , ARN Viral/genética , Eliminación de Secuencia , Replicación Viral/genéticaRESUMEN
Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.
Asunto(s)
Antígenos Virales/genética , Antígenos Virales/metabolismo , Virus de la Fiebre Aftosa/enzimología , Virus de la Fiebre Aftosa/patogenicidad , Ingeniería de Proteínas , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Sustitución de Aminoácidos , Animales , Antivirales , Células Cultivadas , Cricetinae , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Farmacorresistencia Viral , Fluorouracilo/farmacología , Fiebre Aftosa/patología , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/crecimiento & desarrollo , Ratones , Mutagénesis Sitio-Dirigida , Mutación Puntual , Ribavirina/farmacología , Porcinos , Triptófano/genética , Triptófano/metabolismo , Ensayo de Placa ViralRESUMEN
Foot-and-mouth disease (FMD) is a highly contagious disease of livestock caused by a highly variable RNA virus (FMDV) that has seven serotypes and more than sixty subtypes. Both prophylactic and post-infection means of controlling the disease outbreak, including universally applicable vaccines and emergency response measures such as therapeutic treatments, are on high demand. In this study, we analyzed the long-term exposure outcome to a previously identified inhibitor of 3D polymerase (FMDV 3Dpol) for controlling FMDV infection and for the selection of resistance mutants. The results showed that no escape mutant viruses were isolated from FMDV A24 Cruzeiro infections in cell culture treated with gradually increasing concentrations of the antiviral compound 5D9 (4-chloro-N'-thieno, [2,3-d]pyrimidin-4-ylbenzenesulfonohydrazide) over ten passages. Biochemical and plaque assays revealed that when 5D9 was used at concentrations within a non-toxic range in cells, it drove the virus to undetectable levels at passage eight to ten. This is in contrast with observations made on parallel control (untreated) passages exhibiting fully viable and stable virus progenies. Collectively, the results demonstrated that under the experimental conditions, treatment with 5D9 does not confer a resistant phenotype and the virus is unable to evade the antiviral effect of the inhibitor. Further efforts using quantitative structure-property relationship (QSPR) based modifications of the 5D9 compound may result in the successful development of an effective in vivo antiviral drug targeting FMDV.
Asunto(s)
Antivirales/farmacología , Virus de la Fiebre Aftosa/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Antígenos Virales , Línea Celular , Cricetinae , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Farmacorresistencia Viral , Virus de la Fiebre Aftosa/enzimología , Virus de la Fiebre Aftosa/fisiología , Compuestos Heterocíclicos con 2 Anillos/farmacología , Concentración 50 Inhibidora , Viabilidad Microbiana/efectos de los fármacos , Ribavirina/farmacología , Pase Seriado , Sulfonamidas/farmacología , Factores de Tiempo , Ensayo de Placa ViralRESUMEN
Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C(pro) induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5' non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C(pro).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cisteína Endopeptidasas/metabolismo , Citoplasma/metabolismo , Virus de la Fiebre Aftosa/patogenicidad , Riñón/virología , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Virus de la Fiebre Aftosa/enzimología , Riñón/citología , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribosomas/virologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) plays a crucial role in viral replication and pathogenesis by inducing cell cycle arrest, apoptosis, translocation of preintegration complex, potentiation of glucocorticoid action, impairment of dendritic cell (DC) maturation, and T-cell activation. Recent studies involving the direct effects of Vpr on DCs and T cells indicated that HIV-1 containing Vpr selectively impairs phenotypic maturation, cytokine network, and antigen presentation in DCs and dysregulates costimulatory molecules and cytokine production in T cells. Here, we have further investigated the indirect effect of HIV-1 Vpr(+) virus-infected DCs on the bystander CD8(+) T-cell population. Our results indicate that HIV-1 Vpr(+) virus-infected DCs dysregulate CD8(+) T-cell proliferation and induce apoptosis. Vpr-containing virus-infected DC-mediated CD8(+) T-cell killing occurred in part through enhanced tumor necrosis factor alpha production by infected DCs and subsequent induction of death receptor signaling and activation of the caspase 8-dependent pathway in CD8(+) T cells. Collectively, these results provide evidence that Vpr could be one of the important contributors to the host immune escape by HIV-1 through its ability to dysregulate both directly and indirectly the DC biology and T-cell functions.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , VIH-1/fisiología , Linfocitos T/citología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Células Cultivadas , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The HIV-1 viral protein R, Vpr, increases virus replication in T cells and is necessary for the optimal infection of primary monocytes/macrophages and other non-dividing cells. Vpr interacts with the cellular glucocorticoid receptor (GR) and transactivates the HIV-1 LTR through glucocorticoid response element (GRE), an event that can be blocked by the GR antagonist, mifepristone. Results demonstrated that Vpr-induced transactivation of the HIV-1 LTR was inhibited by mifepristone in a dose-dependent manner by >60% at a 10 microM concentration. Infectivity assays using X4 and R5 viruses demonstrated antiviral effects on a dose-dependent regimen of mifepristone. The effects of mifepristone were also tested in latently infected cells that could be activated with extracellular Vpr protein and results indicated specific inhibition of virus reactivation in the presence of this antagonist.
Asunto(s)
Fármacos Anti-VIH/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Productos del Gen vpr/metabolismo , VIH-1/efectos de los fármacos , Mifepristona/farmacología , Receptores de Glucocorticoides/metabolismo , Replicación Viral/efectos de los fármacos , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Proteína p24 del Núcleo del VIH/biosíntesis , Duplicado del Terminal Largo de VIH/genética , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/fisiología , Células HeLa , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/virología , Regiones Promotoras Genéticas , Activación Transcripcional , Activación Viral , Latencia del Virus , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).
Asunto(s)
Ancirinas/química , Ancirinas/fisiología , Apoptosis/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Supervivencia Celular/fisiología , Empalme del ARN , Secuencia de Aminoácidos , Ancirinas/metabolismo , Secuencia de Bases , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Línea Celular , Cartilla de ADN , Técnica del Anticuerpo Fluorescente Indirecta , Productos del Gen vpr/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Antigen presentation and T-cell activation are dynamic processes involving signaling molecules present in both APCs and T cells. Effective APC function and T-cell activation can be compromised by viral immune evasion strategies, including those of human immunodeficiency virus type 1 (HIV-1). In this study, we determined the effects of HIV-1 Vpr on one of the initial target of the virus, dendritic cells (DC), by investigating DC maturation, cytokine profiling, and CD8-specific T-cell stimulation function followed by a second signal. Vpr impaired the expression of CD80, CD83, and CD86 at the transcriptional level without altering normal cellular transcription. Cytokine profiling indicated that the presence of Vpr inhibited production of interleukin 12 (IL-12) and upregulated IL-10, whereas IL-6 and IL-1beta were unaltered. Furthermore, DC infected with HIV-1 vpr+ significantly reduced the activation of antigen-specific memory and recall cytotoxic-T-lymphocyte responses. Taken together, these results indicate that HIV-1 Vpr may in part be responsible for HIV-1 immune evasion by inhibiting the maturation of costimulatory molecules and cytokines essential for immune activation.
Asunto(s)
Células Dendríticas/fisiología , Productos del Gen vpr/farmacología , VIH-1/fisiología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/inmunología , Antígenos CD/genética , Apoptosis , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Citocinas/genética , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , VIH-1/inmunología , Humanos , Linfocitos T/efectos de los fármacos , Transcripción Genética , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
HIV-1 Vpr has been shown to transactivate LTR-directed expression through its interaction with several proteins of cellular origin including the glucocorticoid receptor (GR). Upon activation, steroid receptors bind to proteins containing the signature motif LxxLL, translocate into the nucleus, bind to their response element, and activate transcription. The presence of such motifs in HIV-1 Vpr has prompted us to undertake the analysis of the role of specific leucine residue(s) involved in Vpr-GR interaction, subcellular localization and its effect on Vpr-GR-mediated transactivation. The individual leucine residues present in H I, II, and III were mutated in the Vpr molecule and evaluated for their ability to interact with GR, transactivate GRE and HIV-1 LTR promoters, and their colocalization with GR. While Vpr mutants L42 and L67 showed reduced activation, substitutions at L20, L23, L26, L39, L64, and L68 exhibited a similar and slightly higher level of activation compared to Vprwt. Interestingly, a substitution at residue L22 resulted in a significantly higher GRE and HIV-1 LTR transactivation (8- to 11-fold higher) in comparison to wild type. Confocal microscopy indicated that Vpr L22A exhibited a distinct condensed nuclear localization pattern different from the nuclear/perinuclear pattern noted with Vprwt. Further, electrophoretic mobility shift assay (EMSA) revealed that the VprL22A-GR complex had higher DNA-binding activity when compared to the wild type Vpr-GR complex. These results suggest a contrasting role for the leucine residues on HIV-1 LTR-directed transactivation dependent upon their location in Vpr.
Asunto(s)
Productos del Gen vpr/fisiología , Infecciones por VIH/virología , VIH-1/fisiología , Leucina/fisiología , Fragmentos de Péptidos/fisiología , Activación Transcripcional , Replicación Viral , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Productos del Gen vpr/química , Productos del Gen vpr/genética , Productos del Gen vpr/metabolismo , Duplicado del Terminal Largo de VIH , Humanos , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores del VIH/metabolismo , Factores de Tiempo , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
HIV-1 Vpr is a protein with multiple functions. It has been suggested that such pleiotropic effects by a viral protein may be mediated by its association with viral and cellular proteins or through modulation of expression of specific cellular genes. To address this, we used cDNA microarray techniques to analyze the regulation of a panel of host cellular genes by HIV-1 Vpr using isogenic HIV-1 either with or without Vpr expression. Results indicate that Vpr downregulated the expression of genes involved in cell cycle/proliferation regulation, DNA repair, tumor antigens, and immune activation factors, and upregulated many ribosomal and structural proteins. These results for the first time reveal the involvement of several potential cellular genes, which may be useful, both for understanding Vpr functions and for the development of therapeutics targeting the Vpr molecule.