Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology submitted time 2016-05-12
Chang, Shenghai
Sun, Dapeng
Liang, Huanhuan
Li, Jun
Guo, Lu
Wang, Xiangli
Guan, Chengcheng
Boruah, Bhargavi M.
Yuan, Lingmin
Feng, Feng
Yang, Mingrui
Liu, Yingfang
Wang, Jia
Wang, Jiawei
Wang, Hong-Wei
Wojdyla, Justyna
Wang, Meitian
Li, Lanjuan
Liu, Yingfang
Wang, Lulan
Abstract： Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the underlying mechanism of the RdRP complex is still unclear. Here we report the biochemical characterization of influenza RdRP subcomplex comprising PA, PB1, and N terminus of PB2, which exist as dimer in solution and can assemble into a tetramer state, regulated by vRNA promoter. Using single-particle cryo-electron microscopy, we have reconstructed the RdRP tetramer complex at 4.3 angstrom, highlighting the assembly and interfaces between monomers within the tetrameric structure. The individual RdRP subcomplex contains all the characterized motifs and appears as a cage-like structure. High-throughput mutagenesis profiling revealed that residues involved in the oligomer state formation are critical for viral life cycle. Our results lay a solid base for understanding the mechanism of replication of influenza and other negative-stranded RNA viruses.
Peer Review Status:
Awaiting Review
Subjects: Biology >> Biophysics submitted time 2016-05-11
Phiwpan, Krung
Guo, Jie
Zhang, Wei
Hu, Tanyu
Zhang, Jianhua
Zhou, Xuyu
Phiwpan, Krung
Hu, Tanyu
Boruah, Bhargavi M.
Abstract： MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.
Peer Review Status:Awaiting Review
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