RESUMEN
The transcription control region of the archetype strain of the human polyomavirus JC virus (JCV(Cy)), unlike its neurotropic counterpart (JCV(Mad-1)), contains only one copy of the 98-bp enhancer/promoter repeat with the 23-bp and the 66-bp insertion blocks. Early studies by us and others have indicated that the structural organization of JCV(Mad-1) is critical for glial cell-specific transcription of the viral genome. In addition, the kappa B regulatory motif found in the JCV(Mad-1) genome, which also exists in JCV(Cy), confers inducibility to the JCV(Mad-1) early and late promoters in response to extracellular stimuli. In this study, we have investigated the regulatory role of the 23- and the 66-bp blocks and their functional relationship to the kappa B motif in stimulating transcription of the Cy early and late promoters in glial cells. We demonstrate that mutations in the kappa B motif reduce the basal activity of the Cy early promoter and decrease the levels of its induction by phorbol myristate acetate or factors derived from activated T cells. Under similar circumstances, mutation in the kappa B motif completely abrogated the basal and the induced levels of transcription of the viral late promoter. Using deletion and hybrid promoter constructs, we have demonstrated that the 23-bp block of the Cy promoter plays a critical role in the observed inactivation of Cy late promoter transcription in glial cells. Results from DNA binding studies have indicated the formation of a common nucleoprotein complex with the 23-bp sequence, mutant kappa B (kappa B(mut)), and wild-type kappa B (kappa B(wt)). Analysis of this complex by UV cross-linking has identified a 40-kDa protein which binds to the 23-bp sequence and the kappa B motif. The importance of these findings for the activation of JCV(Cy) under various physiological conditions is discussed.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus JC/genética , FN-kappa B , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Sitios de Unión , Chlorocebus aethiops , ADN Viral , Genoma Viral , Humanos , Riñón/citología , Datos de Secuencia Molecular , Mutagénesis , Neuroglía , Proteínas Nucleares/metabolismo , Células Tumorales CultivadasRESUMEN
Two forms of JC virus (JCV) have been isolated from its human host, an archetype found in kidney tissue and urine of nonimmunocompromised individuals and a rearranged type detected in lymphocytes and brain tissue of patients with and without progressive multifocal leukoencephalopathy. To investigate the hypothesis that alterations to the archetype transcriptional control region yield rearranged forms of the virus exhibiting new tissue tropic and pathogenic potentials, attempts were made to propagate archetype JCV in human renal and glial cell cultures. Although rearranged forms of JCV multiplied in these cells, archetype JCV failed to do so. Through the use of chimeric and mutant viral genomes, and a cell line that constitutively expresses viral T protein, we demonstrated that archetype's inactivity relative to that of rearranged forms was due to differences in the promoter-enhancer and not in the protein coding regions or origin of DNA replication. Additional analyses revealed that the absence of a large tandem duplication and the presence of a 23- and a 66-base pair sequence in the archetype transcriptional control region were responsible for this restricted lytic behavior. We discuss the possibility that deletion and duplication events within the archetype promoter-enhancer might yield more active viral variants via the loss of a negative, or the creation of a positive, transcriptional control signal(s).
Asunto(s)
ADN Viral/biosíntesis , Virus JC/genética , Regiones Promotoras Genéticas , Adulto , Línea Celular , Línea Celular Transformada , Cloranfenicol O-Acetiltransferasa/genética , Replicación del ADN/genética , ADN Viral/genética , Regulación Viral de la Expresión Génica , Genoma Viral , Humanos , Virus JC/patogenicidad , Túbulos Renales Proximales/citología , Mutación , Neuroglía/citología , Recombinación Genética , Células Tumorales Cultivadas , Virión/genética , Virión/patogenicidad , Replicación Viral/genéticaRESUMEN
Brain tissue of a patient with multiple myeloma suffering from neurological disorders similar to those seen in progressive multifocal leukoencephalopathy (PML) patients was evaluated for the presence of the papovavirus, JCV. Results from polymerase chain reaction (PCR) revealed the presence of JCV with structural organization at the control region which is distinct from well-characterized isolates, ie Mad-1 and Mad-4. The control region of the new isolate, named JCVPhila-1' contains a 22 nucleotide insertion which separates the TATA box from the NF-1 regulatory motif. Only 18 nucleotides of the insert are duplicated in the second copy of the enhancer/promoter of the new isolate, which is 84 nucleotides in size. Results from a transcription assay indicate a modest elevated level of JCVPhila-1 early promoter activity compared to that of JCVMad-4 in glial cell lines. The basal and T-antigen-induced transcriptional activities of the JCVPhila-1 late promoter was lower with respect to Mad-4 late gene activity in glial cells. Of particular interest was the observation that in the cells producing the early protein, T-antigen, JCVPhila-1 DNA replicated more efficiently then the Mad-4 DNA. These results suggest that the alterations seen in the JCVPhila-1 control region may differentially influence early and late gene expression and facilitate amplification of the viral genome in cells derived from the CNS.