RESUMO
Many proposed mechanisms for influenza A viral RNA synthesis include an interaction of the nucleoprotein (NP) with the viral polymerase. To identify an NP sequence required for this interaction, we used the cryoelectron microscopic structure of an influenza virus miniribonucleoprotein as a guide for choosing promising surface-exposed sequences. We show that three amino acids (R204, W207, and R208) located in a loop at the top of the head domain of NP are required for functional interaction with the viral polymerase. Quantitative reverse transcription-PCR (RT-PCR) measurements of RNAs synthesized in minigenome assays established that each of these NP amino acids is required for viral RNA synthesis. The mutation of these three amino acids does not affect nuclear localization or RNA-binding and oligomerization activities of NP. In vitro binding experiments with purified virus polymerase and NPs established that these three amino acids are required for NP binding to the viral polymerase.
Assuntos
Vírus da Influenza A/fisiologia , Domínios e Motivos de Interação entre Proteínas , RNA Viral/biossíntese , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas do Core Viral/metabolismo , Replicação Viral , Linhagem Celular , Microscopia Crioeletrônica , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/ultraestrutura , Substâncias Macromoleculares/ultraestrutura , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Proteínas do Nucleocapsídeo , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/ultraestrutura , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real , Proteínas do Core Viral/genética , Proteínas do Core Viral/ultraestruturaRESUMO
The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10(-1) transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.
