RESUMEN
The two biological evidences to endorse the antiviral activity of RNA interference (RNAi) are biogenesis of viral-siRNA (v-siRNA) by the host and encoding of RNAi-suppressor protein by viral genome. It has been recently established that mammals and mammalian cell lines mount antiviral RNAi to defend themselves against the invading viruses. The large part of viral pathogenicity is also due to the RNAi suppressor proteins. In this context it is only natural to ask what kinds of RNAi suppressors are encoded by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the central character of the present pandemic. The following mini review addresses this question.
Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/patología , Neumonía Viral/patología , Interferencia de ARN/fisiología , ARN Interferente Pequeño/genética , Proteínas Virales/genética , Animales , COVID-19 , Línea Celular , Chlorocebus aethiops , Interacciones Huésped-Patógeno/fisiología , Humanos , Inmunidad Innata/inmunología , Pandemias , SARS-CoV-2 , Células VeroRESUMEN
Gene silencing is one of the limiting factors for transgene expression in plants. But the plant viruses have learnt to suppress gene silencing by encoding the protein(s), called RNA silencing suppressor(s) (RSS). Hence, these proteins could be used to overcome the limitation for transgene expression. The RNAi suppressors, namely HC-Pro and P19, have been shown to enhance the transgene expression but other RSS proteins have not been screened for similar role. Moreover, none of RSSs from the DNA viruses are known for enhancing the expression of transgenes. The Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus Begomovirus within the family of Geminiviridae encodes an RSS called the AC2 protein. Here, we used AC2 to elevate the expression of the transgenes. Upon introduction of MYMIV-AC2 in the silenced GFP transgenic tobacco lines, by either genetic hybridisation or transgenesis, the GFP expression was enhanced several fold in F1 and T0 lines. The GFP-siRNA levels were much reduced in F1 and T0 lines compared with those of the initial parental silenced lines. The enhanced GFP expression was also observed at the cellular level. This approach was also successful in enhancing the expression of another transgene, namely topoisomeraseII.
Asunto(s)
Geminiviridae/genética , Ingeniería Genética/métodos , Interferencia de ARN , Transgenes/genética , Proteínas Virales/genética , ADN-Topoisomerasas de Tipo II/genética , Expresión Génica/genética , Proteínas Fluorescentes Verdes/deficiencia , Proteínas Fluorescentes Verdes/genética , Hibridación Genética , Protoplastos/metabolismo , Nicotiana/citología , Nicotiana/genéticaRESUMEN
RNA silencing suppressors (RSSs) are well studied for plant viruses but are not well defined to date for animal viruses. Here, we have identified an RSS from a medically important positive-sense mammalian virus, Severe acute respiratory syndrome coronavirus. The viral 7a accessory protein suppressed both transgene and virus-induced gene silencing by reducing the levels of small interfering RNA (siRNA). The suppression of silencing was analyzed by two independent assays, and the middle region (amino acids [aa] 32 to 89) of 7a was responsible for suppression. Finally, the RNA suppression property and the enhancement of heterologous replicon activity by the 7a protein were confirmed for animal cell lines.
Asunto(s)
Interferencia de ARN/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/fisiología , Línea Celular , Genes Virales , Humanos , Virus de Plantas/genética , Virus de Plantas/fisiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/virología , ARN Interferente Pequeño/genética , ARN Viral/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Supresión Genética , Proteínas de la Matriz Viral/químicaRESUMEN
RNA silencing, including RNA interference, is a novel method of gene regulation and one of the potent host-defense mechanisms against the viruses. In the course of evolution, the viruses have encoded proteins with the potential to suppress the host RNA silencing mechanism as a counterdefense strategy. The virus-encoded RNA silencing suppressors (RSSs) can serve as important biological tools to dissect the detailed RNA silencing pathways and also to evolve the antiviral strategies. Screening and identification of the RSSs are indeed of utmost significance in the field of plant biotechnology. We describe two Green Fluorescent Protein (GFP) reporter-based plant assay systems that rely on two different principles, namely reversal of silencing and enhancement of rolling circle replication (RCR) of geminiviral replicon. These proof-of-concept examples and assay systems could be used to screen various plant, animal, and insect viral ORFs for identification of the RSS activities.
Asunto(s)
Geminiviridae/genética , Silenciador del Gen , Genes Reporteros , Proteínas Fluorescentes Verdes , Interferencia de ARN , Geminiviridae/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Sistemas de Lectura Abierta , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , TransgenesRESUMEN
Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.
