RESUMEN
The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes tat and rev with guide RNAs (gRNA) selected from each gene based on CRISPR specificity and sequence conservation across six major HIV-1 subtypes. Each gRNA was cloned into lentiCRISPRv2 before co-transfection to create a lentiviral vector and transduction into target cells. CRISPR/Cas9 transduction into 293 T and HeLa cells stably expressing Tat and Rev proteins successfully abolished the expression of each protein relative to that in non-transduced and gRNA-absent vector-transduced cells. Tat functional assays showed significantly reduced HIV-1 promoter-driven luciferase expression after tat-CRISPR transduction, while Rev functional assays revealed abolished gp120 expression after rev-CRISPR transduction. The target gene was mutated at the Cas9 cleavage site with high frequency and various indel mutations. Conversely, no mutations were detected at off-target sites and Cas9 expression had no effect on cell viability. CRISPR/Cas9 was further tested in persistently and latently HIV-1-infected T-cell lines, in which p24 levels were significantly suppressed even after cytokine reactivation, and multiplexing all six gRNAs further increased efficiency. Thus, the CRISPR/Cas9 system targeting HIV-1 regulatory genes may serve as a favorable means to achieve functional cures.
Asunto(s)
Sistemas CRISPR-Cas , VIH-1/genética , Replicación Viral/genética , Línea Celular , Genes rev , Genes tat , Terapia Genética , Humanos , Linfocitos T/virologíaRESUMEN
Viruses are completely dependent upon cellular machinery to support replication and have therefore developed strategies to co-opt cellular processes to optimize infection and counter host immune defenses. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode a relatively small number of genes. Viruses with limited genetic content often encode multifunctional proteins that function at multiple stages of the viral replication cycle. In this review, we discuss the functions of HIV-1 regulatory (Tat and Rev) and accessory (Vif, Vpr, Vpu, and Nef) proteins. Each of these proteins has a highly conserved primary activity; however, numerous additional activities have been attributed to these viral proteins. We explore the possibility that HIV-1 proteins leverage their multifunctional nature to alter host transcriptional networks to elicit a diverse set of cellular responses. Although these transcriptional effects appear to benefit the virus, it is not yet clear whether they are strongly selected for during viral evolution or are a ripple effect from the primary function. As our detailed knowledge of these viral proteins improves, we will undoubtedly uncover how the multifunctional nature of these HIV-1 regulatory and accessory proteins, and in particular their transcriptional functions, work to drive viral pathogenesis.
Asunto(s)
Genes rev , Genes tat , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Transcripción Genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , VIH-1/química , VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Proteínas Reguladoras y Accesorias Virales/genética , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
A 70-year-old man presented with lower back pain and cyanotic changes in his left lower extremity. He was diagnosed with infected aortic aneurysm and infectious spondylitis. He had received intravesical Bacillus Calmette-Guerin (BCG) therapy up to 1 month before the onset of symptoms. The aneurysm was excised and an aorto-biiliac interposition graft was performed. Mycobacterium tuberculosis complex was cultured in the surgical specimens. Real-time polymerase chain reaction (PCR) targeting the senX3-regX3 region, and multiplex PCR using dual-priming oligonucleotide primers targeting the RD1 gene, revealed that the organism isolated was Mycobacterium bovis BCG. The patient took anti-tuberculosis medication for 1 year, and there was no evidence of recurrence at 18 months follow-up.
Asunto(s)
Anciano , Humanos , Administración Intravesical , Aneurisma , Aneurisma Infectado , Aneurisma de la Aorta , Bacillus , Cartilla de ADN , Estudios de Seguimiento , Genes rev , Dolor de la Región Lumbar , Extremidad Inferior , Reacción en Cadena de la Polimerasa Multiplex , Mycobacterium bovis , Mycobacterium tuberculosis , Mycobacterium , Reacción en Cadena en Tiempo Real de la Polimerasa , Recurrencia , Espondilitis , Trasplantes , Neoplasias de la Vejiga Urinaria , Vejiga UrinariaRESUMEN
To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2ß, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 in vitro, validating this novel approach to treatment of this infection.
Asunto(s)
Antivirales/farmacología , Digoxina/farmacología , Inhibidores Enzimáticos/farmacología , VIH-1/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antígenos CD4/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes rev/efectos de los fármacos , VIH-1/genética , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Proteínas del Tejido Nervioso/metabolismo , Empalme del ARN/efectos de los fármacos , ARN Viral/efectos de los fármacos , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina , Proteínas ViralesRESUMEN
We analyzed RNA splice site usage in three HIV-1 subtype B primary isolates through reverse transcriptase polymerase chain reaction (RT-PCR) amplification of spliced RNAs using a fluorescently labeled primer, with computerized size determination and quantification of PCR products, which were also identified by clone sequencing. In one isolate, P2149-3, unusual and unreported spliced transcripts were detected. This isolate preferentially used for rev RNA generation a 3' splice site (3'ss) located five nucleotides upstream of A4a, previously identified only in a T cell line-adapted virus and in a group O isolate, and designated A4d. P2149-3 also used an unreported 3'ss for rev RNA generation, designated A4h, located 20 nucleotides upstream of 3'ss A4c. Additionally, unusual nef RNAs using 3'ss A5a and A7a and with exon composition 1.3.7 were identified. The identification of several unusual and unreported spliced transcripts in an HIV-1 primary isolate suggests a greater diversity of splice site usage in HIV-1 than previously appreciated.
Asunto(s)
Genes nef , Genes rev , VIH-1/genética , Sitios de Empalme de ARN/genética , ARN Viral/genética , Secuencia de Bases , Variación Genética , VIH-1/clasificación , VIH-1/aislamiento & purificación , Humanos , Leucocitos Mononucleares/virología , Datos de Secuencia MolecularRESUMEN
The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. We previously reported the regulatory effect of a small upstream open reading frame (ORF) on Rev and Env translation. Here we study this mechanism in further detail by modulating the strength of the translation signals upstream of the open reading frames in subgenomic reporters. Furthermore, the effects of these mutations on SIV gene expression and viral replication are analyzed. An intricate regulatory mechanism is disclosed that allows the virus to express a balanced amount of these two proteins.
Asunto(s)
Codón Iniciador , Productos del Gen env/genética , Productos del Gen rev/genética , Sistemas de Lectura Abierta , Virus de la Inmunodeficiencia de los Simios/genética , Línea Celular , Regulación Viral de la Expresión Génica , Genes env , Genes rev , Células HEK293 , Humanos , Mutación , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismoRESUMEN
The HIV-1 Rev response element (RRE) is a ~350 nucleotide, highly structured, cis-acting RNA element essential for viral replication. It is located in the env coding region of the viral genome and is extremely well conserved across different HIV-1 isolates. It is present on all partially spliced and unspliced viral mRNA transcripts, and serves as an RNA framework onto which multiple molecules of the viral protein Rev assemble. The Rev-RRE oligomeric complex mediates the export of these messages from the nucleus to the cytoplasm, where they are translated to produce essential viral proteins and/or packaged as genomes for new virions.
Asunto(s)
Regulación Viral de la Expresión Génica , Genes rev , VIH-1/genética , Elementos de Respuesta , Ribonucleoproteínas/metabolismo , Ensamble de Virus , Secuencia de Bases , Sitios de Unión , Bases de Datos Genéticas , VIH-1/metabolismo , VIH-1/fisiología , Datos de Secuencia Molecular , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Conformación de Ácido Nucleico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteínas/genética , Alineación de SecuenciaRESUMEN
The ability of lentiviruses to continually evolve and escape immune control is the central impediment in developing an effective vaccine for HIV-1 and other lentiviruses. Equine infectious anemia virus (EIAV) is considered a useful model for immune control of lentivirus infection. Virus-specific cytotoxic T lymphocytes (CTL) and broadly neutralizing antibody effectively control EIAV replication during inapparent stages of disease, but after years of low-level replication, the virus is still able to produce evasion genotypes that lead to late re-emergence of disease. There is a high rate of genetic variation in the EIAV surface envelope glycoprotein (SU) and in the region of the transmembrane protein (TM) overlapped by the major exon of Rev. This review examines genetic and phenotypic variation in Rev during EIAV disease and a possible role for Rev in immune evasion and virus persistence.
Asunto(s)
Anemia Infecciosa Equina/genética , Genes env/genética , Genes rev/genética , Virus de la Anemia Infecciosa Equina , Animales , Anemia Infecciosa Equina/inmunología , Productos del Gen rev/inmunología , VIH-1/genética , VIH-1/inmunología , Caballos , Humanos , Evasión Inmune/genética , Evasión Inmune/inmunología , Virus de la Anemia Infecciosa Equina/genética , Virus de la Anemia Infecciosa Equina/inmunología , Modelos Biológicos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Transcripción Genética/inmunología , Replicación ViralRESUMEN
OBJECTIVES: The objectives of this study were to assess the prevalence of transmitted HIV-1 drug resistances (TDR) and HIV-1 subtypes in recently infected patients in Catalonia between 2003 and 2005 and to describe the characteristics of these patients according to the presence or absence of TDR and HIV-1 subtype. METHODS: After application of the Serological Testing Algorithm for Recent HIV Seroconversion (STARHS), residual aliquots of serum samples from recently infected antiretroviral-naïve individuals were genotyped. FASTA sequences were analyzed using the HIVDB Program. The World Health Organization 2009 List of Mutations for Surveillance of Transmitted HIV-1 Drug Resistant HIV Strains was used to estimate the prevalence of TDR. RESULTS: Of 182 recently infected patients, 14 (7.7%) presented TDR. Seven (3.8%) had genotypic evidence of TDR against non-nucleoside reverse transcriptase inhibitors, 6 (3.3%) against nucleoside reverse transcriptase inhibitors, 3 (1.6%) against protease inhibitors (PIs), and only 2 individuals (1.1%) presented TDR against more than one class of drugs. Thirty-five (19.2%) patients were infected with a non-B HIV-1 subtype. CONCLUSION: This is the first study to estimate the prevalence of TDR in recently infected patients in Catalonia. The results are similar to those of studies performed in other Spanish regions. Correct monitoring of these parameters requires systematic epidemiologic surveillance of transmitted resistance.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Adulto , Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral Múltiple/genética , Farmacorresistencia Viral/genética , Emigrantes e Inmigrantes , Femenino , Genes pol , Genes rev , Genotipo , Infecciones por VIH/epidemiología , Infecciones por VIH/transmisión , Infecciones por VIH/virología , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Masculino , Mutación , Vigilancia de la Población , ARN Viral/genética , Estudios Retrospectivos , Análisis de Secuencia de ARN , España/epidemiología , Manejo de EspecímenesRESUMEN
Human papillomavirus (HPV) can infect oral mucosa, causing asymptomatic infection or warty lesions. Several case-control studies have confirmed HPV as an independent risk factor for squamous cell carcinoma. HPV-related cancers seem to have better prognoses and different risk factors than do HPV-negative ones. HIV-infected patients are known to be at increased risk for persistent genital and anal high-risk HPV infections and intraepithelial neoplasm. Since the era of highly active antiretroviral therapy, the prevalence and persistence of warty lesions in oral mucosa have increased. Oral squamous cell carcinoma was recently added in the case definitions for common HIV-related oral mucosa lesions. The increased risk of HPV infection in HIV patients has been associated with impaired immune response to HPV, highly active antiretroviral therapy, aging of the HIV-infected patients, and direct interaction between the 2 viruses. HPV32 seems to be much more prevalent in asymptomatic HPV infections and warts among those infected with HIV than among those in the general population. Regarding HIV genes, there is evidence of an interaction between HPV and tat, rev, and vpr. HIV might play a role in HPV-associated pathogenesis by exhorting oncogenic stimuli via tat and rev or visa versa.
Asunto(s)
Carcinoma de Células Escamosas/complicaciones , Infecciones por VIH/complicaciones , VIH-1/genética , Neoplasias de la Boca/complicaciones , Papillomaviridae/genética , Infecciones por Papillomavirus/complicaciones , Factores de Edad , Terapia Antirretroviral Altamente Activa , Infecciones Asintomáticas , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virología , Genes rev , Genes tat , Genes prv , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/virología , Humanos , Neoplasias de la Boca/virología , Papillomaviridae/clasificación , Infecciones por Papillomavirus/clasificación , Infecciones por Papillomavirus/genética , Factores de Riesgo , Neoplasias Urogenitales/complicaciones , Neoplasias Urogenitales/virología , Integración ViralRESUMEN
A number of proteins which are needed for the building of new immunodeficiency virus type 1 virions can only be translated from unspliced virus-derived pre-mRNAs. These unspliced mRNAs are shuttled through the nuclear pores reaching the cytosol when bound to the viral protein Rev. However, as a cellular co-factor Rev requires a Rev-binding protein of the AGFG family (nucleoporin-related Arf-GAP domain and FG repeats-containing proteins). In this article we address the evolution of the AGFGs by analyzing the first section of the coding mRNAs. This contains a "core module" which can be traced from Drosophilae to fish, amphibia, birds, and mammals, including man. In the subfamily of AGFG1 molecules the estimated conservation from Drosophilae to primates is 67% (with limited gaps). In some Drosophilae the core module is preceded by a long stretch of more than 300 coding nucleotides, but this additional module is absent in other Drosophilae and in all AGFG1s of other species. The AGFG2 molecules emerged later in evolution, possibly deriving from a duplication of AGFG1s. AGFG2s, present in mammals only, exhibit an additional module of about 50 coding nucleotides ahead of the core module, which is significantly less conserved (54%, with more remarkable gaps). This additional module does not seem to have homologies with the additional module of Drosophilae nor with the precoding section of AGFG1s. Interestingly, in birds a highly re-edited form of the AGFG1 core module (Gallus gallus, Galliformes) coexists with a typical form of the AGFG1 core module (Taeniopygia guttata, Passeriformes).
Asunto(s)
Evolución Molecular , Genes rev , VIH-1/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Unión al ARN/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Regiones no Traducidas 5'/genética , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero/genética , ARN Viral/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.
Asunto(s)
Farmacorresistencia Viral/fisiología , Transcriptasa Inversa del VIH/química , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Zidovudina/farmacología , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , ADN Viral/biosíntesis , Desoxirribonucleótidos/metabolismo , Didesoxinucleótidos/metabolismo , Diseño de Fármacos , Farmacorresistencia Viral/genética , Genes rev , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Modelos Moleculares , Mutación Missense , Mutación Puntual , Conformación Proteica , Relación Estructura-Actividad , Nucleótidos de Timina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/metabolismoRESUMEN
Equine infectious anemia virus (EIAV) is one of the most divergent members of the lentivirus subfamily of retroviruses and is considered a useful comparative model for molecular studies of lentivirus replication. The Rev protein of EIAV is functionally homologous with other lentiviral Revs and facilitates export of incompletely spliced viral mRNAs through a Crm1-dependent pathway. The trans- and cis-acting elements that mediate EIAV Rev function are similar to, but distinct from, the well-characterized elements in human immunodeficiency virus (HIV-1), the prototypical Rev protein. In addition, the EIAV rev sequence is highly variable in vivo, and changes in Rev phenotype correlate with changes in clinical stages of EIAV infection. This review summarizes the molecular biology of EIAV Rev-RRE interactions and the consequences of Rev variation in vivo. A comparative perspective of Rev activity may enhance understanding of an essential lentiviral protein and stimulate new strategies for treatment and prevention of lentivirus infections in vivo.
Asunto(s)
Productos del Gen rev/fisiología , Genes env/fisiología , Virus de la Anemia Infecciosa Equina/genética , Empalme Alternativo/genética , Animales , Genes rev/genética , Caballos , Evasión Inmune/genética , Virus de la Anemia Infecciosa Equina/inmunología , Estructura Secundaria de Proteína , Replicación ViralRESUMEN
In the course of search for the robust analogs of 1'-acetoxychavicol acetate (ACA, 1), the Rev-export inhibitor from the medicinal plant Alpinia galanga, we clarified formation of the quinone methide intermediate ii to be essential for exerting the inhibitory activity of 1. Based on this mechanism of action, the rational design from the MO calculation of the conclusive activation energy to ii resulted in the four halogenated analogs with more potent activity than ACA (1). In particular, the difluoroanalog 20d exhibited approximately four-fold potent activity as compared with 1.
Asunto(s)
Alpinia/química , Fármacos Anti-VIH/farmacología , Alcoholes Bencílicos/farmacología , Genes rev/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Animales , Fármacos Anti-VIH/química , Alcoholes Bencílicos/química , Bovinos , VIH-1/efectos de los fármacos , Células HeLa , Humanos , Hidrocarburos Halogenados/química , Hidrocarburos Halogenados/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacologíaRESUMEN
The interaction of multiple HIV-1 regulator of virion (Rev) proteins with the viral RNA target, the Rev response element (RRE), is critical for nuclear export of incompletely spliced and unspliced viral RNA, and for the onset of the late phase in the viral replication cycle. The heterogeneity of the Rev-RRE complex has made it difficult to study using conventional structural methods. In the present study, atomic force microscopy is applied to directly visualize the tertiary structure of the RRE RNA alone and in complex with Rev proteins. The appearance of the RRE is compatible with the earlier proposed RRE secondary structure in dimensions and overall shape, including a stalk and a head interpreted as stem I, and stem-loops II-V in the secondary structure model, respectively. Atomic force microscopy imaging of the Rev-RRE complex revealed an increased height of the structure both in the stalk and head regions, which is in accordance with a binding model in which Rev binding to a high affinity site in stem IIB triggers oligomerization of Rev proteins through cooperative binding along stem I in RRE. The present study demonstrates that atomic force microscopy comprises a useful technique to study complex biological structures of nucleic acids at high resolution.
Asunto(s)
Genes rev , VIH-1/genética , ARN Viral/genética , Virión/genética , Secuencia de Bases , Calorimetría , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Viral/química , Termodinámica , Replicación Viral/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.
Asunto(s)
Núcleo Celular/metabolismo , Carioferinas/metabolismo , Luciferasas de Luciérnaga/genética , Proteínas de Complejo Poro Nuclear/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína de Unión al GTP ran/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular Tumoral , Ácidos Grasos Insaturados/farmacología , Genes rev , VIH-1/genética , Células HeLa , Humanos , Luciferasas de Luciérnaga/metabolismo , Señales de Exportación Nuclear/efectos de los fármacos , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Unión al ARN/genética , Transfección , Proteína Exportina 1RESUMEN
A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.
Asunto(s)
Equartevirus/genética , Genes rev , VIH-1/genética , Sitios de Unión , Equartevirus/metabolismo , VIH-1/metabolismo , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genéticaRESUMEN
We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.
Asunto(s)
Vectores Genéticos/biosíntesis , Lentivirus/crecimiento & desarrollo , Reactores Biológicos/virología , Técnicas de Cultivo de Célula , Línea Celular , Medio de Cultivo Libre de Suero/farmacología , Proteínas de Fusión gag-pol/genética , Genes rev/genética , Vectores Genéticos/genética , Humanos , Lentivirus/efectos de los fármacos , Lentivirus/genética , Modelos Genéticos , Plásmidos/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.
Asunto(s)
Genes env , VIH-1/genética , Línea Celular , Clonación Molecular , Productos del Gen env/metabolismo , Productos del Gen rev/metabolismo , Genes rev , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Plásmidos , Reacción en Cadena de la PolimerasaRESUMEN
The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS.