RESUMEN
Influenza A virus encodes two segments (7 and 8) that produce mRNAs that can be spliced. We have investigated if naturally occurring sequence polymorphisms in the influenza A virus family affects splicing of these viral mRNAs, as that could potentially alter the NS1/NS2- and/or M1/M2-protein ratios. We compared splicing efficiency of segment 7 and 8 mRNAs of A/Brevig Mission/1918/1 (H1N1) and A/Netherlands/178/95 (H3N2), as well as various H5N1 avian strains. Results revealed that both segment 7 and 8 mRNAs of A/Brevig Mission/1918/1 (H1N1) were inefficiently spliced compared to other influenza virus segment 7 and 8 mRNAs. This resulted in production of higher levels of functional NS1 protein, which could potentially contribute to the pathogenic properties of the A/Brevig Mission/1918/1 (H1N1). We also show that A/Brevig Mission/1918/1 (H1N1) segment 8 mRNAs responded differently to overexpression of SR proteins than A/Netherlands/178/95 (H3N2).
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Empalme del ARN , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Genes Virales , Células HEK293 , Humanos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Plásmidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
We have previously shown that SRp55 inhibits splicing from HIV-1 exon 3, thereby generating partially spliced mRNAs encoding HIV-1 vpr. Here we show that SRp55 also inhibits splicing from HIV-1 exon 5 to generate HIV-1 vpu/env mRNA, albeit with lower efficiency. We also show that inhibition of HIV-1 splicing by SRp55 causes the appearance of partially spliced vpu, env and vpr mRNAs in the cytoplasm. SRp55 could also induce production of extracellular p24gag from a rev-defective HIV-1 provirus. These results indicate that SRp55 aids in the generation of partially spliced and unspliced HIV-1 mRNAs.
Asunto(s)
VIH-1/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Empalme del ARN , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/virología , Exones , Genoma Viral , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Plásmidos , Provirus/genética , ARN Mensajero , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Empalme Serina-Arginina , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was seven fold decreased whereas K M was unaffected in the presence of 10 microM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible approximately 40% decrease in tryptophan fluorescence, consistent with the ion inducing a conformational change in the protein. The apparent K D was 7.4 +/- 1.5 microM and thus correlated well with the apparent K I. In the presence of physiological Ca2+ concentration the K I and K D values were three to four fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding. The magnitude of the apparent KI in relation to the blood and platelet concentrations of Zn2+ supports a physiological role for this ion in the regulation of anticoagulant activity of APC. These findings broaden the understanding of this versatile serine protease and enable the future development of potentially more efficient anticoagulant APC variants for treatments of thrombotic diseases.
