RESUMO
Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the â¼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3'-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3'-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3'-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.
Assuntos
Regiões 3' não Traduzidas/genética , Infecções por Citomegalovirus/virologia , MicroRNAs/metabolismo , Muromegalovirus/fisiologia , RNA Viral/metabolismo , Replicação Viral/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação , Linhagem Celular , Regulação para Baixo/genética , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , MicroRNAs/genética , Muromegalovirus/genética , Mutação , Processamento Pós-Transcricional do RNA , Estabilidade de RNA/genética , RNA Viral/genética , Análise de Sequência de RNARESUMO
Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication.