RESUMO
Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design.
Assuntos
Genes Reporter , Engenharia Genética/métodos , Infecções por Henipavirus/genética , Vírus Nipah/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Feminino , Regulação da Expressão Gênica , Infecções por Henipavirus/mortalidade , Infecções por Henipavirus/patologia , Infecções por Henipavirus/virologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mesocricetus , Dados de Sequência Molecular , Mutagênese Insercional , Vírus Nipah/patogenicidade , Faloidina/genética , Faloidina/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Análise de Sobrevida , Transcrição Gênica , Replicação Viral/genética , Proteína Vermelha FluorescenteRESUMO
Mechanisms coordinating cell-cell interaction have appeared early in evolution. Allatotropin (AT), a neuropeptide isolated based on its ability to stimulate the synthesis of juvenile hormones (JHs) in insects has also been found in other invertebrate phyla. Despite this function, AT has proved to be myotropic. In the present study we analyze its expression in two groups of Turbellaria (Catenulida, Macrostomida), and its probable relationship with muscle tissue. The results show the presence of an AT-like peptide in the free living turbellaria analyzed. The analysis of the expression of the peptide together with phalloidin, suggests a functional relationship between the peptide and muscle tissue, showing that it could be acting as a myoregulator. The finding of immunoreactive fibers associated with sensory organs like ciliated pits in Catenulida and eyes in Macrostomida makes probable that AT could play a role in the physiological mechanisms controlling circadian activities. Furthermore, the existence of AT in several phyla of Protostomata suggests that this peptide could be a synapomorphic feature of this group. Indeed, the presence in organisms that do not undergo metamorphosis, could be signaling that it was first involved in myotropic activities, being the stimulation of the synthesis of JHs a secondary function acquired by the phylum Arthropoda.
Assuntos
Hormônios de Inseto/metabolismo , Músculos/metabolismo , Neuropeptídeos/metabolismo , Faloidina/metabolismo , Turbelários/metabolismo , Animais , Regulação da Expressão Gênica , Hormônios de Inseto/genética , Hormônios Juvenis/metabolismo , Músculos/fisiologia , Neuropeptídeos/genética , Faloidina/genética , Turbelários/citologia , Turbelários/genéticaRESUMO
The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, alpha-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide and was inhibited by the specific POP inhibitor benzyloxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.
