RESUMO
Oncogenic viruses promote cell proliferation through the dramatic reorganization of host transcriptomes. In addition to regulating mRNA abundance, changes in mRNA isoform usage can have a profound impact on the protein output of the transcriptome. Using Epstein-Barr virus (EBV) transformation of primary B cells, we have studied the ability of an oncogenic virus to alter the mRNA isoform profile of its host. Using the algorithm called SplicerEX with two complementary Affymetrix microarray platforms, we uncovered 433 mRNA isoform changes regulated by EBV during B-cell transformation. These changes were largely orthogonal with the 2,163 mRNA abundance changes observed during transformation, such that less than one-third of mRNAs changing at the level of isoform also changed in overall abundance. While we observed no preference for a mechanistic class of mRNA isoform change, we detected a significant shortening of 3' untranslated regions and exclusion of cassette exons in EBV-transformed cells relative to uninfected B cells. Gene ontology analysis of the mRNA isoform changes revealed significant enrichment in nucleic acid binding proteins. We validated several of these isoform changes and were intrigued by those in two mRNAs encoding the proteins XBP1 and TCF4, which have both been shown to bind and activate the promoter of the major EBV lytic trans-activator BZLF1. Our studies indicate that EBV latent infection promotes the usage of mRNA isoforms of XBP1 and TCF4 that restrict BZLF1 activation. Therefore, characterization of global changes in mRNA isoform usage during EBV infection identifies a new mechanism for the maintenance of latent infection.
Assuntos
Transformação Celular Viral/genética , Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , RNA Viral/genética , Latência Viral/genética , Replicação Viral , Linfócitos B/metabolismo , Linfócitos B/virologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Biomarcadores/metabolismo , Western Blotting , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Perfilação da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Isoformas de RNA , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição de Fator Regulador X , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo , Fator de Transcrição 4 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
The recombination directionality factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein-DNA architectures. Although the attachment site DNA of mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head to tail. The structure implies a DNA bend of ~120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that, in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation.