The influenza virus PB1 protein functions as a catalytic subunit of the viral RNA-dependent RNA polymerase and contains the highly conserved motifs of RNA-dependent RNA polymerases together with putative nucleotide-binding sites. PB1 also binds to viral genomic RNAs and its replicative intermediates through the promoter regions. The detail function and interplay between functional domains are not clarified although a part of structures and functions of PB1 have been clarified. In this study, we analyzed the function of PB1 subunit in the sense of nucleotide recognition using ribavirin, which is a nucleoside analog and inhibits viral RNA synthesis of many RNA viruses including influenza virus. We screened ribavirin-resistant PB1 mutants from randomly mutated PB1 cDNA library using a mini-replicon assay, and we identified a single mutation at the amino acid position 27 of PB1 as an important residue for the nucleotide recognition.
The influenza virus genome forms viral ribonucleoprotein (vRNP) complexes with nucleoprotein and viral RNA-dependent RNA polymerases (RdRp), PB1, PB2, and PA subunits. The vRNP complex catalyzes both genome replication and transcription reactions. PB1 contains the motifs highly conserved among RdRps and functions as a catalytic subunit of RdRp. The N-terminal region of PB1 between amino acid (a.a.) positions 1–83 contains both putative vRNA and cRNA promoter binding sites and a PA binding site. However, except for the PA binding site, the crystal structure and the function of the N-terminal region of PB1 are poorly understood. Here, we have examined the functional structure of the N-terminal region of PB1. The regions between a.a. positions 1–50 are highly conserved between influenza A and B viruses, but amino acids at positions 16, 27, and 44 are different between two viruses. To elucidate the functional importance of these amino acids in replication and transcription of the viral genome, we generated viruses containing mutations at these positions by reverse genetics and examined replication and transcription activities of these mutants. We found that a.a. positions 27 and 44 are responsible for the viral replication activity but not transcription activity.
Selenium nanoparticles (SeNPs) with diameters from 64.8 to 110.1 nm were successfully synthesized by γ-irradiation of solutions containing Se4+ and water-soluble yeast β-glucan. The size and size distribution of SeNPs were analyzed by dynamic light scattering (DLS). Analytical X-ray diffraction (XRD) pattern results confirmed the crystal structure of the Se nanoparticles and Fourier transform infrared (FTIR) spectroscopy revealed that β-glucan could interact with SeNPs through steric (Se…O) linkages leading to a homogeneous and translucent solution state for 60 days without any precipitates. In vivo tests in cytoxan-induced immunosuppressed mice revealed that the daily supplementation of SeNPs/β-glucan at concentrations of 6 mg per kg body weight of tested mice significantly stimulated the generation of cellular immune factors (white blood cells, neutrophil, lymphocyte, B cells, CD4+ cells, CD34+ cells and natural killer cells) and humoral immune indexes (IgM, IgG, TNF-α, IFN-γ and IL-2) in peripheral blood, bone marrow and spleen of the immunosuppressed mice. The obtained results indicated that radiation-synthesized SeNPs/β-glucan may be a candidate for further evaluation as an agent for the prevention of immunosuppression in chemotherapy.
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