RESUMO
Functions of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) were tested in lymphoblastoid cell lines containing EBER mutants of EBV. Binding of EBER1 to ribosomal protein L22 (RPL22) was confirmed. Deletion of EBER1 or EBER2 correlated with increased levels of cytoplasmic EBV LMP2 RNA and with small effects on specific cellular microRNA (miRNA) levels, but protein levels of LMP1 and LMP2A were not affected. Wild-type EBV and EBER deletion EBV had approximately equal abilities to infect immunodeficient mice reconstituted with a human hematopoietic system.
Assuntos
Herpesvirus Humano 4/genética , RNA Viral/genética , Proteínas da Matriz Viral/metabolismo , Animais , Linhagem Celular , Cisplatino/farmacologia , Humanos , Camundongos , Camundongos Knockout , Camundongos SCID , MicroRNAs/genética , RNA Viral/biossíntese , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas da Matriz Viral/genéticaRESUMO
Novel Epstein-Barr Virus (EBV) strains with deletion of either EBER1 or EBER2 and corresponding revertant viruses were constructed and used to infect B lymphocytes to make lymphoblastoid cell lines (LCLs). The LCLs were used in microarray expression profiling to identify genes whose expression correlates with the presence of EBER1 or EBER2. Functions of regulated genes identified in the microarray analysis include membrane signaling, regulation of apoptosis, and the interferon/antiviral response. Although most emphasis has previously been given to EBER1 because it is more abundant than EBER2, the differences in cell gene expression were greater with EBER2 deletion. In this system, deletion of EBER1 or EBER2 had little effect on the EBV transformation frequency of primary B cells or the growth of the resulting LCLs. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed, but in LCLs almost all of the cells had a predominantly cytoplasmic expression of this ribosomal protein, which was not detectably changed by EBER1. The changes in LCL gene expression identified here will provide a basis for identifying the mechanisms of action of EBER RNAs.
Assuntos
Linfócitos B/virologia , Perfilação da Expressão Gênica , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno , Ativação Linfocitária , RNA Viral/metabolismo , Linhagem Celular , Deleção de Genes , Humanos , Análise em Microsséries , RNA Viral/genéticaRESUMO
The essential splicing factors SF1 and U2AF play an important role in the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. The structure of the C-terminal RRM (RRM3) of human U2AF(65) complexed to an N-terminal peptide of SF1 reveals an extended negatively charged helix A and an additional helix C. Helix C shields the potential RNA binding surface. SF1 binds to the opposite, helical face of RRM3. It inserts a conserved tryptophan into a hydrophobic pocket between helices A and B in a way that strikingly resembles part of the molecular interface in the U2AF heterodimer. This molecular recognition establishes a paradigm for protein binding by a subfamily of noncanonical RRMs.