RESUMEN
BACKGROUND: Non-structural protein NS1 of influenza A viruses harbours several determinants of pathogenicity and host-range. However it is still unclear to what extent each of its two structured domains (i.e. RNA-binding domain, RBD, and effector domain, ED) contribute to its various activities. METHODS: To evaluate the respective contributions of the two domains, we genetically engineered two variants of an H7N1 low pathogenicity avian influenza virus harbouring amino-acid substitutions that impair the functionality of either domain. The RBD- and ED-mutant viruses were compared to their wt- counterpart in vivo and in vitro, notably in chicken infection and avian cell culture models. RESULTS: The double substitution R38A-K41A in the RBD dramatically reduced the pathogenicity and replication potential of the virus, whereas the substitution A149V that was considered to abrogate the IFN-antagonistic activity of the effector domain entailed much less effects. While all three viruses initiated the viral life cycle in avian cells, replication of the R38A-K41A virus was severely impaired. This defect was associated with a delayed synthesis of nucleoprotein NP and a reduced accumulation of NS1, which was found to reach a concentration of about 30 micromol.L- 1 in wt-infected cells at 8 h post-infection. When overexpressed in avian lung epithelial cells, both the wt-NS1 and 3841AA-NS1, but not the A149V-NS1, reduced the poly(I:C)-induced activation of the IFN-sensitive chicken Mx promoter. Unexpectedly, the R38A-K41A substitution in the recombinant RBD did not alter its in vitro affinity for a model dsRNA. When overexpressed in avian cells, both the wt- and A149V-NS1s, as well as the individually expressed wt-RBD to a lesser extent, enhanced the activity of the reconstituted viral RNA-polymerase in a minireplicon assay. CONCLUSIONS: Collectively, our data emphasized the critical importance and essential role of the RNA-binding domain in essential steps of the virus replication cycle, notably expression and translation of viral mRNAs.
Asunto(s)
Subtipo H7N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H7N1 del Virus de la Influenza A/patogenicidad , Gripe Aviar/virología , Motivos de Unión al ARN/fisiología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Sustitución de Aminoácidos , Animales , Línea Celular , Embrión de Pollo , Pollos , Modelos Animales de Enfermedad , Perros , Expresión Génica , Regulación Viral de la Expresión Génica , Subtipo H7N1 del Virus de la Influenza A/genética , Células de Riñón Canino Madin Darby , Motivos de Unión al ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas Virales/biosíntesis , Virulencia/genéticaRESUMEN
The nucleoid-associated protein HU is involved in numerous DNA transactions and thus is essential in DNA maintenance and bacterial survival. The high affinity of HU for SSBs (single-strand breaks) has suggested its involvement in DNA protection, repair and recombination. SSB-containing DNA are major intermediates transiently generated by bifunctional DNA N-glycosylases that initiate the BER (base excision repair) pathway. Enzyme kinetics and DNA-binding experiments demonstrate that HU enhances the 8-oxoguanine-DNA glycosylase activity of Fpg (formamidopyrimidine-DNA glycosylase) by facilitating the release of the enzyme from its final DNA product (one nucleoside gap). We propose that the displacement of Fpg from its end-DNA product by HU is an active mechanism in which HU recognizes the product when it is still bound by Fpg. Through DNA binding, the two proteins interplay to form a transient ternary complex Fpg/DNA/HU which results in the release of Fpg and the molecular entrapment of SSBs by HU. These results support the involvement of HU in BER in vivo.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN-Formamidopirimidina Glicosilasa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Guanina/análogos & derivados , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , ADN-Formamidopirimidina Glicosilasa/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanina/metabolismoRESUMEN
The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα2, EcHUß2, and EcHUαß) are in thermal equilibrium between two dimeric conformations (N2 â I2) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N2 to I2 thermal transition of the EcHUß2 homodimer. On the basis of these data, a realistic 3D model is proposed for the major I2 conformation of EcHUß2. This model is in agreement with previous experimental data.