RESUMEN
Semliki Forest virus (SFV), a neurotropic virus, has been used to deliver heterologous genes into cells in vitro and in vivo. In this study, we constructed a reporter SFV4-FL-EGFP and found that it can deliver EGFP into neurons located at the injection site without disseminating throughout the brain. Lacking of the capsid gene of SFV4-FL-EGFP does not block its life cycle, while forming replication-competent virus-like particles (VLPs). These VLPs hold subviral genome by using the packaging sequence (PS) located within the nsP2 gene, and can transfer their genome into cells. In addition, we found that the G protein of vesicular stomatitis virus (VSVG) can package SFV subviral genome, which is consistent with the previous reports. The G protein of rabies virus (RVG) could also package SFV subviral genome. These pseudo-typed SFV can deliver EGFP gene into neurons. Taken together, these findings may be used to construct various SFV-based delivery systems for virological studies, gene therapy, and neural circuit labeling.
Asunto(s)
Ingeniería Genética , Terapia Genética/métodos , Vectores Genéticos/metabolismo , Hipotálamo/virología , Neuronas/virología , Virus de los Bosques Semliki/genética , Animales , Línea Celular , Cricetulus , Células Epiteliales/ultraestructura , Células Epiteliales/virología , Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipotálamo/ultraestructura , Inyecciones Intraventriculares , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/ultraestructura , Cultivo Primario de Células , Virus de la Rabia/genética , Virus de la Rabia/metabolismo , Virus de los Bosques Semliki/metabolismo , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Virión/genética , Virión/metabolismo , Ensamble de Virus/genéticaRESUMEN
We have screened the NCI diversity set library for molecules that bind specifically to stem loop 3 (SL3) RNA of the packaging element Psi of HIV-1 using the docking programs DOCK and AutoDock, followed by MD simulations. The association of the predicted ligands with SL3 RNA was characterized using fluorescence, ITC, UV-melting, CD, and footprinting techniques. Nine ligands for SL3 RNA have been identified, four of which bind with higher affinity to SL3 RNA than to either single- or double-stranded RNA motifs. The most selective ligands, 9 (NSC252359) and 5 (NSC123111), bind SL3 RNA with dissociation constants of 11 microM and 98 microM, respectively. Compound 9 binds with 4-7-fold higher affinity to SL3 RNA than to the other tetraloops found in Psi-RNA, SL2 and SL4 RNAs. The results suggest that both 9 and 5 bind to the stem region of SL3 RNA without large distortions of the SL3 RNA.
Asunto(s)
VIH-1/genética , VIH-1/fisiología , Secuencias Invertidas Repetidas/genética , ARN Viral/genética , ARN Viral/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Ensamble de Virus , Acridinas/química , Acridinas/metabolismo , Secuencia de Bases , Sitios de Unión , Calorimetría , Evaluación Preclínica de Medicamentos , Ligandos , Modelos Moleculares , Conformación Molecular , Estabilidad del ARN , ARN Viral/química , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Especificidad por Sustrato , Volumetría , Ensamble de Virus/genéticaRESUMEN
We developed a yeast cell-free system suitable for in vitro translation of human papillomavirus 58 (HPV58) L1 mRNA. This system was systematically optimized resulting in enhanced translation efficiency. The optimal concentrations of potassium and magnesium ions observed were specific to the HPV58 L1 protein production. Supplementation with sucrose in the preparation of the yeast lysate greatly enhanced its stability. After optimization, protein production in this system was significantly superior to that produced by the rabbit reticulocyte (RRL) system. Finally, we demonstrated for the first time that virus-like particles (VLPs) were assembled from HPV58 L1 capsid protein in the yeast cell-free system. Thus, the system described here is a powerful tool for the HPV L1 protein production and will be useful for the study of VLP assembly and DNA encapsulation.
Asunto(s)
Proteínas de la Cápside/genética , Ingeniería Genética/métodos , Proteínas Oncogénicas Virales/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Virión/genética , Ensamble de Virus/genética , Sistema Libre de Células , ARN Viral/genéticaRESUMEN
Self-replicating, non-infectious flavivirus subgenomic replicons have been broadly used in the studies of trans-complementation, adaptive mutation, viral assembly and packaging in Kunjin, yellow fever and West Nile viruses. We describe here the construction of subgenomic EGFP- or Renilla luciferase-reporter based dengue replicons of the type 2 New Guinea C (NGC) strain and the establishment of stable BHK21 cell lines harboring the replicons. In replicon cells, viral proteins and RNAs are stably expressed at levels similar to cells transfected with the full length NGC infectious RNA. Furthermore, the replicon can be packaged by separately transfected C (core)-prM (pre-membrane)-E (envelope) polyprotein construct. The replicon cells were subjected to treatment with several antiviral compounds and inhibition of the replicon was observed in treatment with known nucleoside analog inhibitors of NS5 such as 2'-C-methyladenosine (EC(50)=2.42 +/- 0.59 microM), or ribavirin (EC(50)=6.77 +/- 1.33 microM), mycophenolic acid (EC(50)=1.31 +/- 0.27 microM) and siRNA against NS3. The BHK-replicon cells have been stably maintained for about 10 passages without significant loss in reporter intensity and are sufficiently robust for both research and drug discovery.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/genética , Evaluación Preclínica de Medicamentos/métodos , ARN Interferente Pequeño/genética , Replicón/genética , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Cricetinae , Virus del Dengue/efectos de los fármacos , Virus del Dengue/fisiología , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , ARN Viral/biosíntesis , Replicón/efectos de los fármacos , Proteínas Virales/biosíntesis , Ensamble de Virus/efectos de los fármacos , Ensamble de Virus/genética , Replicación Viral/genéticaRESUMEN
Soybean chlorotic mottle virus (SbCMV) is the type species of the genus 'Soybean chlorotic mottle-like viruses', within the family Caulimoviridae. The double-stranded DNA genome of SbCMV (8178 bp) contains eight major open reading frames (ORFs). Viral genes essential and non-essential for the replication and movement of SbCMV were investigated by mutational analysis of an infectious 1.3-mer DNA clone. The results indicated that ORFs Ia, II, III, IV and V were essential for systemic infection. The product of ORF Ib was non-essential, although the putative tRNA(Met) primer-binding site in ORF Ib was proved to be essential. Immunoselection PCR revealed that an ORF Ia deletion mutant was encapsidated in primarily infected cells, indicating that ORF Ia is required for virus movement but not for replication. ORF IV was confirmed to encode a capsid protein by peptide sequencing of the capsid. Analysis of the viral transcripts showed that the SbCMV DNA genome gives rise to a pregenomic RNA and an ORF VI mRNA, as shown in the case of Cauliflower mosaic virus.