RESUMEN
Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging-uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.
Asunto(s)
Vectores Genéticos/genética , Lentivirus/genética , Lisina/análogos & derivados , Transducción Genética , Azidas/efectos de la radiación , Línea Celular , Terapia Genética/métodos , Vectores Genéticos/efectos de la radiación , VIH-1/genética , Humanos , Lentivirus/efectos de la radiación , Lisina/genética , Lisina/efectos de la radiación , Rayos Ultravioleta , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/efectos de la radiaciónRESUMEN
Ultraviolet (254 nm) irradiation of the bacteriophage MS2 results in the decrease of the number of antigenic determinants exposed on the virion surface. The cross-section of the decrease, as measured by the number of anti-MS2 IgG molecules bound per virion, is 10(-16) mm2 per photon. The decrease of the phage-antibody binding proceeds after irradiation with a rate constant of about 5 x 10(-3) min-1. Since the antigenic determinants of the phage MS2 coat protein does not contain photoreactive amino acid residues, the irradiation-induced decrease of the phage antibody binding is determined, most probably, by the shielding of the antigenic determinants. Such shielding could be caused by rearrangement of coat protein molecules and/or of the capsid induced by photomodification of non-antigenic fragments of coat protein and/or of intraphage RNA.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Bacteriófagos/inmunología , Inmunoglobulina G/inmunología , Rayos Ultravioleta , Animales , Antígenos Virales/efectos de la radiación , Bacteriófagos/genética , Bacteriófagos/efectos de la radiación , Cromatografía en Gel , Epítopos/inmunología , Epítopos/efectos de la radiación , Genes Virales , Conejos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/efectos de la radiación , Virión/inmunología , Virión/efectos de la radiaciónRESUMEN
Recent studies suggested a role for the human endogenous retrovirus (HERV) group HERV-K(HML-2) in melanoma because of upregulated transcription and expression of HERV-K(HML-2)-encoded proteins. Very little is known about which HML-2 loci are transcribed in melanoma. We assigned >1,400 HML-2 cDNA sequences generated from various melanoma and related samples to genomic HML-2 loci, identifying a total of 23 loci as transcribed. Transcription profiles of loci differed significantly between samples. One locus was found transcribed only in melanoma-derived samples but not in melanocytes and might represent a marker for melanoma. Several of the transcribed loci harbor ORFs for retroviral Gag and/or Env proteins. Env-encoding loci were transcribed only in melanoma. Specific investigation of rec and np9 transcripts indicated transcription of protein encoding loci in melanoma and melanocytes hinting at the relevance of Rec and Np9 in melanoma. UVB irradiation changed transcription profiles of loci and overall transcript levels decreased in melanoma and melanocytes. We further identified transcribed HML-2 loci formed by reverse transcription of spliced HML-2 transcripts by L1 machinery or in a retroviral fashion, with loci potentially encoding HML-2-like proteins. We reveal complex, sample-specific transcription of HML-2 loci in melanoma and related samples. Identified HML-2 loci and proteins encoded by those loci are particularly relevant for further studying the role of HML-2 in melanoma. Transcription of HERVs appears as a complex mechanism requiring specific studies to elucidate which HERV loci are transcribed and how transcribed HERVs may be involved in disease.
Asunto(s)
Retrovirus Endógenos/genética , Melanoma/genética , Activación Transcripcional/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Bases , Línea Celular Tumoral , Retrovirus Endógenos/metabolismo , Retrovirus Endógenos/efectos de la radiación , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Genoma Humano , Humanos , Melanocitos/metabolismo , Melanoma/metabolismo , Rayos Ultravioleta , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/efectos de la radiaciónRESUMEN
The steady-state fluorescence anisotropy measurements on influenza and parainfluenza viruses, showed no changes in the microviscosity of the viral membranes after exposure to UV-irradiation, when a fluorescent probe was used, but the intrinsic fluorescence of viral proteins presented, under the same experimental conditions, a significant difference of anisotropy behaviour in the two viruses used.