RESUMEN
In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date. In contrast, here we report high levels of deaminase activity at TC motifs when whole PBMCs or isolated primary monocyte-derived cells were treated with interferon-alpha (IFNalpha) or IFNalpha-inducing toll-like receptor ligands. Induction of TC-specific deaminase activity required new transcription and translation and correlated with the appearance of two APOBEC3A (A3A) isoforms. Knockdown of A3A in monocytes with siRNA abolished TC-specific deaminase activity, confirming that A3A isoforms are responsible for all TC-specific deaminase activity observed. Both A3A isoforms appear to be enzymatically active; moreover, our mutational studies raise the possibility that the smaller isoform results from internal translational initiation. In contrast to the high levels of TC-specific activity observed in IFNalpha-treated monocytes, CC-specific activity remained low in PBMCs, suggesting that A3G deaminase activity is relatively inhibited, unlike that of A3A. Together, these findings suggest that deaminase activity of A3A isoforms in monocytes and macrophages may play an important role in host defense against viruses.
Asunto(s)
Citidina Desaminasa/metabolismo , Regulación Enzimológica de la Expresión Génica/inmunología , Inmunidad Innata/genética , Monocitos/enzimología , Monocitos/inmunología , Proteínas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Transducción de Señal/genética , Desaminasa APOBEC-3G , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Citidina Desaminasa/química , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , Técnicas de Silenciamiento del Gen , Humanos , Interferón-alfa/metabolismo , Macrófagos/citología , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/metabolismo , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/metabolismo , Biosíntesis de Proteínas/inmunología , Proteínas/química , Proteínas/genética , ARN Interferente Pequeño/genética , Especificidad por Sustrato , Receptores Toll-Like/metabolismo , Transcripción Genética/inmunologíaRESUMEN
Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architectures comprised of an electrode-immobilized, redox-tagged probe oligonucleotide. Recent studies suggest that E-DNA signaling is predicated on hybridization-linked changes in probe flexibility, which will alter the efficiency with which the terminal redox tag strikes the electrode. This, in turn, suggests that probe length, probe geometry, and redox-tag placement will affect E-DNA signaling. To test this we have characterized E-DNA sensors comprised of linear or stem-loop probes of various lengths and with redox tags placed either distal to the electrode or internally within the probe sequence (proximal). We find that linear probes produce larger signal changes upon target binding than equivalent stem-loop probes. Likewise, long probes exhibit greater signal changes than short probes provided that the redox tag is placed proximal to the electrode surface. In contrast to their improved signaling, the specificity of long probes is poorer than that of short probes, suggesting that sensor optimization represents a trade off between sensitivity and specificity. Finally, we find that sensor response time and selectivity are only minimally affected by probe geometry or length. The results of this comparative study will help guide future designs and applications of these sensors.