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      ITGB1b-Deficient Rare Minnows Delay Grass Carp Reovirus (GCRV) Entry and Attenuate GCRV-Triggered Apoptosis

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          Abstract

          Integrin β-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b −/−) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b −/− rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b −/− rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection.

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          Most cited references40

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          ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool

          ZiFiT (Zinc Finger Targeter) is a simple and intuitive web-based tool that provides an interface to identify potential binding sites for engineered zinc finger proteins (ZFPs) in user-supplied DNA sequences. In this updated version, ZiFiT identifies potential sites for ZFPs made by both the modular assembly and OPEN engineering methods. In addition, ZiFiT now integrates additional tools and resources including scoring schemes for modular assembly, an interface with the Zinc Finger Database (ZiFDB) of engineered ZFPs, and direct querying of NCBI BLAST servers for identifying potential off-target sites within a host genome. Taken together, these features facilitate design of ZFPs using reagents made available to the academic research community by the Zinc Finger Consortium. ZiFiT is freely available on the web without registration at http://bindr.gdcb.iastate.edu/ZiFiT/.
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            The draft genome of the grass carp (Ctenopharyngodon idellus) provides insights into its evolution and vegetarian adaptation.

            The grass carp is an important farmed fish, accounting for ∼16% of global freshwater aquaculture, and has a vegetarian diet. Here we report a 0.9-Gb draft genome of a gynogenetic female adult and a 1.07-Gb genome of a wild male adult. Genome annotation identified 27,263 protein-coding gene models in the female genome. A total of 114 scaffolds consisting of 573 Mb are anchored on 24 linkage groups. Divergence between grass carp and zebrafish is estimated to have occurred 49-54 million years ago. We identify a chromosome fusion in grass carp relative to zebrafish and report frequent crossovers between the grass carp X and Y chromosomes. We find that transcriptional activation of the mevalonate pathway and steroid biosynthesis in liver is associated with the grass carp's adaptation from a carnivorous to an herbivorous diet. We believe that the grass carp genome could serve as an initial platform for breeding better-quality fish using a genomic approach.
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              AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection.

              Understanding the primary steps of viral entry can have important implications for strategies to prevent infection of known viral pathogens as well as determining parameters for efficient gene delivery using viral vectors. Recently, a two-step process for viral infection involving attachment of virus to a primary receptor (coxsackievirus adenovirus receptor and heparan sulfate proteoglycan) and subsequent mediation of virus entry by a co-receptor (alphaV integrins and HVEM) has been determined for both adenovirus and HSV, respectively. Heparan sulfate proteoglycan serves as a primary attachment receptor for adeno-associated virus type 2 (AAV-2)(ref. 5). Here we determined that alphaVbeta5 integrin plays a part in efficient AAV infection. Experiments using the chelating agent EDTA to disrupt integrin function resulted in a corresponding decrease in AAV infection, consistent with the possibility that integrin mediates infection. Viral overlay experiments on purified plasma membrane proteins as well as immunoprecipitated integrin beta5 subunit demonstrated that AAV directly associates with the beta5 subunit of alphaVbeta5 integrin. Genetically defined cells expressing alphaVbeta5 integrin showed increased susceptibility to AAV infection, demonstrating a biological role of this integrin in AAV infection. Finally, viral binding and internalization studies indicate that alphaVbeta5 integrin is not a primary attachment receptor for AAV-2, but is instead involved in facilitating virus internalization. This study supports the idea that alphaVbeta5 integrin serves as a co-receptor for AAV-2 virions, and should have a substantial effect on the use of AAV vectors in human gene therapy.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                15 October 2018
                October 2018
                : 19
                : 10
                : 3175
                Affiliations
                [1 ]State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430074, China; chengeng@ 123456ihb.ac.cn (G.C.); 18963973548@ 123456163.com (L.X.); wymlemon@ 123456whu.edu.cn (Y.W.); helibowudi@ 123456ihb.ac.cn (L.H.); huangrong@ 123456ihb.ac.cn (R.H.); liaolj@ 123456ihb.ac.cn (L.L.); zyzhu@ 123456ihb.ac.cn (Z.Z.)
                [2 ]University of Chinese Academy of Sciences, Beijing 101408, China
                [3 ]College of Life Sciences, Wuhan University, Wuhan 430072, China
                Author notes
                [* ]Correspondence: wangyp@ 123456ihb.ac.cn ; Tel.: +86-27-68780081
                Author information
                https://orcid.org/0000-0003-3619-3951
                https://orcid.org/0000-0003-0200-346X
                Article
                ijms-19-03175
                10.3390/ijms19103175
                6214113
                30326628
                0f85194c-0e21-434e-b9f3-a820c38394ab
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 21 September 2018
                : 14 October 2018
                Categories
                Article

                Molecular biology
                grass carp,grass carp reovirus,integrin,endocytosis,clathrin,apoptosis
                Molecular biology
                grass carp, grass carp reovirus, integrin, endocytosis, clathrin, apoptosis

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