RESUMEN
Filoviruses, which include Ebola virus (EBOV) and Marburg virus, are negative-sense RNA viruses associated with sporadic outbreaks of severe viral hemorrhagic fever characterized by uncontrolled virus replication. The extreme virulence and emerging nature of these zoonotic pathogens make them a significant threat to human health. Replication of the filovirus genome and production of viral RNAs require the function of a complex of four viral proteins, the nucleoprotein (NP), viral protein 35 (VP35), viral protein 30 (VP30) and large protein (L). The latter performs the enzymatic activities required for production of viral RNAs and capping of viral mRNAs. Although it has been recognized that interactions between the virus-encoded components of the EBOV RNA polymerase complex are required for viral RNA synthesis reactions, specific molecular details have, until recently, been lacking. New efforts have combined structural biology and molecular virology to reveal in great detail the molecular basis for critical protein-protein interactions (PPIs) necessary for viral RNA synthesis. These efforts include recent studies that have identified a range of interacting host factors and in some instances demonstrated unique mechanisms by which they act. For a select number of these interactions, combined use of mutagenesis, over-expressing of peptides corresponding to PPI interfaces and identification of small molecules that disrupt PPIs have demonstrated the functional significance of virus-virus and virus-host PPIs and suggest several as potential targets for therapeutic intervention.
Asunto(s)
Filoviridae/fisiología , Interacciones Microbiota-Huesped , ARN Viral/biosíntesis , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacos , Animales , Ebolavirus/efectos de los fármacos , Ebolavirus/fisiología , Filoviridae/efectos de los fármacos , Humanos , Marburgvirus/efectos de los fármacos , Marburgvirus/fisiología , Unión Proteica , Proteínas Virales/antagonistas & inhibidores , Virosis/tratamiento farmacológicoRESUMEN
This study was designed to discover filovirus entry inhibitors in a drug library of commercial medicines. One thousand and six hundred drugs were screened using the ZEBOV-GP/HIV model, a pseudovirus formed by an HIV-core packed with the Zaire Ebola virus glycoprotein. We identified 12 gonadal hormone drugs with inhibitory activities in ZEBOV-GP/HIV entry at final concentration of 10 µmol x L(-1). Among them, three drugs exhibited strong activities with IC50 < 1 µmol x L(-1), such as toremifene citrate (IC50: 0.19 ± 0.02 µmol x L(-1)), tamoxifen citrate (IC50: 0.32 ± 0.01 µmol x L(-1)) and clomiphene citrate (IC50: 0.53 ± 0.02 µmol x L(-1)); seven drugs had moderate activities with IC50 between 1 and 10 µmol x L(-1), such as estradiol benzoate (IC50: 1.83 ± 5.69 µmol x L(-1)), raloxifene hydrochloride (IC50: 3.48 ± 0.07 µmol x L(-1)), equilin (IC50: 4.00 ± 9.94 µmol x L(-1)), estradiol (IC50: 5.26 ± 9.92 µmol x L(-1)), quinestrol (IC50: 6.36?5.37 gmol-L1), estrone (IC50: 6.87 ± 0.03 µmol-L1) and finasteride (IC50: 9.94 ± 0.45 µmol x L(-1)); two drugs, hexestrol (IC50: 14.20 ± 0.55 µmol x L(-1)) and chlormadinone acetate (IC50: 24.60 ± 0.36 µmol x L(-1)), had weak activities against ZEBOV. Further, toremifene citrate, tamoxifen citrate, clomiphene citrate, raloxifene hydrochloride and quinestrol could block both pseudovirus type Sudan ebola virus (SEBOV-GP/HIV) and Marburg virus (MARV-GP/HIV) entries.
Asunto(s)
Antivirales/farmacología , Ebolavirus/efectos de los fármacos , Marburgvirus/efectos de los fármacos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Internalización del Virus/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Ebolavirus/fisiología , Fiebre Hemorrágica Ebola , Humanos , Marburgvirus/fisiologíaRESUMEN
Marburg virus (MARV) and Ebola virus (EBOV), members of the family Filoviridae, represent a significant challenge to global public health. Currently, no licensed therapies exist to treat filovirus infections, which cause up to 90% mortality in human cases. To facilitate development of antivirals against these viruses, we established two distinct screening platforms based on MARV and EBOV reverse genetics systems that express secreted Gaussia luciferase (gLuc). The first platform is a mini-genome replicon to screen viral replication inhibitors using gLuc quantification in a BSL-2 setting. The second platform is complementary to the first and expresses gLuc as a reporter gene product encoded in recombinant infectious MARV and EBOV, thereby allowing for rapid quantification of viral growth during treatment with antiviral compounds. We characterized these viruses by comparing luciferase activity to virus production, and validated luciferase activity as an authentic real-time measure of viral growth. As proof of concept, we adapt both mini-genome and infectious virus platforms to high-throughput formats, and demonstrate efficacy of several antiviral compounds. We anticipate that both approaches will prove highly useful in the development of anti-filovirus therapies, as well as in basic research on the filovirus life cycle.
Asunto(s)
Antivirales/aislamiento & purificación , Evaluación Preclínica de Medicamentos/métodos , Ebolavirus/efectos de los fármacos , Marburgvirus/efectos de los fármacos , Genética Inversa/métodos , Animales , Antivirales/farmacología , Línea Celular , Ebolavirus/genética , Ebolavirus/fisiología , Genes Reporteros , Luciferasas/análisis , Luciferasas/genética , Marburgvirus/genética , Marburgvirus/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain.