A high-throughput screening system targeting the nuclear export pathway via the third nuclear export signal of influenza A virus nucleoprotein

2016 ◽  
Vol 217 ◽  
pp. 23-31 ◽  
Author(s):  
Michinori Kakisaka ◽  
Takafumi Mano ◽  
Yoko Aida
Keyword(s):  
Influenza A Virus ◽  
High Throughput ◽  
Nuclear Export ◽  
High Throughput Screening ◽  
Influenza A ◽  
Nuclear Export Signal ◽  
Screening System ◽  
The Third ◽  
Export Pathway ◽  
Export Signal

scholarly journals Reply to “Nuclear Export Signal and Immunodominant CD8+T Cell Epitope in Influenza A Virus Matrix Protein 1”

2012 ◽  
Vol 86 (18) ◽  
pp. 10259-10260
Author(s):  
Shuai Cao ◽  
Yi Shi ◽  
Shuguang Tan ◽  
Hao Song ◽  
George F. Gao ◽  
...  
Keyword(s):  
T Cell ◽  
Influenza A Virus ◽  
Nuclear Export ◽  
Influenza A ◽  
Matrix Protein ◽  
Nuclear Export Signal ◽  
Cell Epitope ◽  
Cd8 T Cell ◽  
T Cell Epitope ◽  
Export Signal

scholarly journals Generation of Influenza A Virus NS2 (NEP) Mutants with an Altered Nuclear Export Signal Sequence

2004 ◽  
Vol 78 (18) ◽  
pp. 10149-10155 ◽  
Author(s):  
Kiyoko Iwatsuki-Horimoto ◽  
Taisuke Horimoto ◽  
Yutaka Fujii ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza A Virus ◽  
Nuclear Export ◽  
Influenza A ◽  
Signal Sequence ◽  
Nuclear Export Signal ◽  
Wild Type Virus ◽  
Wild Type ◽  
Amino Terminal ◽  
Ribonucleoprotein Complexes ◽  
Export Signal

ABSTRACT The NS2 (NEP) protein of influenza A virus contains a highly conserved nuclear export signal (NES) motif in its amino-terminal region (12ILMRMSKMQL21, A/WSN/33), which is thought to be required for nuclear export of viral ribonucleoprotein complexes (vRNPs) mediated by a cellular export factor, CRM1. However, simultaneous replacement of three hydrophobic residues in the NES with alanine does not affect NS2 (NEP) binding to CRM1, although the virus with these mutations is not viable. To determine the extent of sequence conservation required by the NS2 (NEP) NES for its export function during viral replication, we randomly introduced mutations by degenerative mutagenesis into the region of NS cDNA encoding the NS2 (NEP) NES and then attempted to generate mutant viruses containing these alterations by reverse genetics. Sequence analysis of the recovered viruses showed that although some of the mutants possessed amino acids other than those conserved in the NES, hydrophobicity within this motif was maintained. Nuclear export of vRNPs representing all of the mutant viruses was completely inhibited in the presence of a CRM1 inhibitor, leptomycin B, as was the transport of wild-type virus, indicating that the CRM1-mediated pathway is responsible for the nuclear export of both wild-type and mutant vRNPs. The vRNPs of some of the mutant viruses were exported in a delayed manner, resulting in limited viral growth in cell culture and in mice. These results suggest that the NES motif may be an attractive target for the introduction of attenuating mutations in the production of live vaccine viruses.

scholarly journals High-Throughput Screening for Anti–Influenza A Virus Drugs and Study of the Mechanism of Procyanidin on Influenza A Virus–Induced Autophagy

2012 ◽  
Vol 17 (5) ◽  
pp. 605-617 ◽  
Author(s):  
Jianping Dai ◽  
Gefei Wang ◽  
Weizhong Li ◽  
Lin Zhang ◽  
Jiacai Yang ◽  
...  
Keyword(s):  
Western Blot ◽  
Influenza A Virus ◽  
High Throughput ◽  
High Throughput Screening ◽  
Influenza A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bimolecular Fluorescence Complementation ◽  
Vitis Vinifera L ◽  
Cinnamomum Cassia Presl

In this research, we have established a high-throughput screening (HTS) platform based on the influenza A virus (IAV) vRNA promoter. Using this HTS platform, we selected 35 medicinal plants out of 83 examples of traditional Chinese medicine and found that 7 examples had not been reported. After examining many previous reports, we found that Vaccinium angustifolium Ait., Vitis vinifera L, and Cinnamomum cassia Presl had a common active compound, procyanidin, and then determined the anti-IAV effect of procyanidin and explored its mechanism of action. With a plaque inhibition assay and a time-of-addition experiment, we found that procyanidin could inhibit the IAV replication at several stages of the life cycle. In the Western blot and EGFP-LC3 localization assays, we found that procyanidin could inhibit the accumulation of LC3II and the dot-like aggregation of EGFP-LC3. In the RT-PCR and Western blot assays, we found procyanidin could inhibit the expression of Atg7, Atg5, and Atg12. Finally, by the bimolecular fluorescence complementation–fluorescence resonance energy transfer and co-immunoprecipitation assays, we found that procyanidin could inhibit the formation of the Atg5-Atg12/Atg16 heterotrimer and the dissociation of the beclin1/bcl2 heterodimer. In conclusion, we have established an HTS platform and identified procyanidin as a novel and promising anti-IAV agent.

scholarly journals Adenovirus Late Gene Expression Does Not Require a Rev-Like Nuclear RNA Export Pathway

2000 ◽  
Vol 74 (14) ◽  
pp. 6684-6688 ◽  
Author(s):  
Claudia Rabino ◽  
Anders Aspegren ◽  
Kara Corbin-Lickfett ◽  
Eileen Bridge
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Late Gene Expression ◽  
Export Pathway ◽  
Early Proteins ◽  
Immunodeficiency Virus ◽  
Rna Export ◽  
Nuclear Rna Export ◽  
Export Signal

ABSTRACT Adenovirus late mRNA export is facilitated by viral early proteins of 55 and 34 kDa. The 34-kDa protein contains a leucine-rich nuclear export signal (NES) similar to that of the human immunodeficiency virus Rev protein. It was proposed that the 34-kDa protein might facilitate the export of adenovirus late mRNA through a Rev-like NES-mediated export pathway. We have tested the role of NES-mediated RNA export during adenovirus infection, and we find that it is not essential for the expression of adenovirus late genes.

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