RESUMEN
A subset of viral genes is required for the long-term latent infection of hematopoietic cells by human cytomegalovirus (HCMV). Here, we show that a latency-associated gene product (LUNA) promotes the disruption of cellular PML bodies during latency. Mutation and inhibitor studies reveal that LUNA encodes a deSUMOylase activity responsible for this disruption. Specifically, LUNA encodes a conserved Asp-Cys-Gly motif common to all deSUMOylases. Importantly, mutation of the putative catalytic cysteine is sufficient to reverse LUNA-mediated PML dispersal and markedly reduces the efficiency of viral reactivation. The depletion of PML from cells is sufficient to rescue the reactivation of the LUNA-deficient viruses, arguing that targeting PML is an important biological role of LUNA. Finally, we demonstrate that reactivation of naturally latent HCMV is blocked by deSUMOylase inhibitors. Thus, latent HCMV primes the cellular environment for efficient reactivation via the activity of a virally encoded deSUMOylase.
Asunto(s)
Citomegalovirus/fisiología , Proteínas Virales/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiología , Secuencia de Aminoácidos , Antígenos CD34/metabolismo , Liasas de Carbono-Nitrógeno/química , Liasas de Carbono-Nitrógeno/genética , Dominio Catalítico , Células Dendríticas/metabolismo , Células Dendríticas/virología , Humanos , Cuerpos de Inclusión/metabolismo , Mutación/genética , Células THP-1RESUMEN
The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Dominio T Box/metabolismo , Células TH1/metabolismo , Elongación de la Transcripción Genética , Animales , Linaje de la Célula/genética , Elementos de Facilitación Genéticos/genética , Humanos , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica/genética , ARN/genética , ARN/metabolismo , Células Th2/metabolismo , Factor de Transcripción ReIA/metabolismo , Factores de Transcripción/metabolismo , Uveítis/genéticaRESUMEN
Latent infection of primary CD34(+) progenitor cells by human cytomegalovirus (HCMV) results in their increased survival in the face of pro-apoptotic signals. For instance, we have shown previously that primary myeloid cells are refractory to FAS-mediated killing and that cellular IL-10 (cIL-10) is an important survival factor for this effect. However, how cIL-10 mediates this protection is unclear. Here, we have shown that cIL-10 signalling leading to upregulation of the cellular factor PEA-15 mediates latency-associated protection of CD34(+) progenitor cells from the extrinsic death pathway.