The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-␣. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication. IMPORTANCEIt is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.
Anthocyanins play an important role in the growth of plants, and are beneficial to human health. In plants, the MYB-bHLH-WD40 (MBW) complex activates the genes for anthocyanin biosynthesis. However, in rice, the WD40 regulators remain to be conclusively identified. Here, a crucial anthocyanin biosynthesis gene was fine mapped to a 43.4-kb genomic region on chromosome 2, and a WD40 gene OsTTG1 (Oryza sativa TRANSPARENT TESTA GLABRA1) was identified as ideal candidate gene. Subsequently, a homozygous mutant (osttg1) generated by CRISPR/Cas9 showed significantly decreased anthocyanin accumulation in various rice organs. OsTTG1 was highly expressed in various rice tissues after germination, and it was affected by light and temperature. OsTTG1 protein was localized to the nucleus, and can physically interact with Kala4, OsC1, OsDFR and Rc. Furthermore, a total of 59 hub transcription factor genes might affect rice anthocyanin biosynthesis, and LOC_Os01g28680 and LOC_Os02g32430 could have functional redundancy with OsTTG1. Phylogenetic analysis indicated that directional selection has driven the evolutionary divergence of the indica and japonica OsTTG1 alleles. Our results suggest that OsTTG1 is a vital regulator of anthocyanin biosynthesis, and an important gene resource for the genetic engineering of anthocyanin biosynthesis in rice and other plants.
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