Baculovirus as an efficient vector for gene delivery into mosquitoes

Naik, Nenavath Gopal; Lo, Yu‐Wen; Wu, Tzong‐Yuan; Lin, Cheng Wen; Kuo, Shin-Chang; Chao, Yu-Chan

doi:10.1038/s41598-018-35463-8

Cited by 28 publications

(29 citation statements)

References 50 publications

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“…For the insect experiment, the baculovirus enhancer sequence hr1 (homologous region 1) was obtained by PCR using 2ϫ Ultra Hi-Fi PCR Master Mix (TTC-PE15, TOOLS) as previously described (23,57) and inserted into the pAB-6ϫHis vector (Invitrogen). The PCR-amplified fragments of the Drosophila hsp70 promoter and baculovirus ie2 gene sequence were obtained as described previously (23,62) and subcloned into the pAB-6ϫHis vector containing the hr sequence to obtain the phr-hsp-IE2 plasmid. The plasmid was transfected into Sf21 cells by using Cellfectin.…”

Section: Methodsmentioning

confidence: 99%

Baculovirus IE2 Interacts with Viral DNA through Daxx To Generate an Organized Nuclear Body Structure for Gene Activation in Vero Cells

Wei

Tsai

Hsu

et al. 2019

J Virol

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Upon virus infection of a cell, the uncoated DNA is usually blocked by the host intrinsic immune system inside the nucleus. Although it is crucial for the virus to counteract the host intrinsic immune system and access its genome, little is known about how viruses can knock down host restriction and identify their blocked genomes for later viral gene activation and replication. We found that upon baculovirus transduction into Vero E6 cells, the invading viral DNA is trapped by the cellular death domain-associated protein (Daxx) and histone H3.3 in the nucleus, resulting in gene inactivation. IE2, a baculovirus transactivator, targets host Daxx through IE2 SUMO-interacting motifs (SIMs) to indirectly access viral DNA and forms unique nuclear body structures, which we term clathrate cage-like apparatus (CCLAs), at the early transduction stage. At the later transduction stage, CCLAs gradually enlarge, and IE2 continues to closely interact with viral DNA but no longer associates with Daxx. The association with Daxx is essential for IE2 CCLA formation, and the enlarged CCLAs are capable of transactivating viral but not chromosomal DNA of Vero E6 cells. Our study reveals that baculovirus IE2 counteracts the cellular intrinsic immune system by specifically targeting Daxx and H3.3 to associate with viral DNA indirectly and efficiently. IE2 then utilizes this association with viral DNA to establish a unique CCLA cellular nanomachinery, which is visible under light microscopy as an enclosed environment for proper viral gene expression. IMPORTANCE The major breakthrough of this work is that viral protein IE2 localizes and transactivates its own viral DNA through a most unlikely route, i.e., host proteins Daxx and H3.3, which are designed to efficiently restrict viral DNA from expression. By interacting with these host intrinsic immune factors, IE2 can thus target the viral DNA and then form a unique spherical nuclear body, which we name the CCLA, to enclose the viral DNA and necessary factors to assist in high-level transactivation. Our study represents one of the most complete investigations of nuclear body formation. In addition, so far only RNA or protein molecules have been reported as potential nucleators for initiating nuclear body formation; our study may represent the first example showing that DNA can be a nucleator for a new class of nuclear body formation.

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Section: Methodsmentioning

confidence: 99%

Baculovirus IE2 Interacts with Viral DNA through Daxx To Generate an Organized Nuclear Body Structure for Gene Activation in Vero Cells

Wei

Tsai

Hsu

et al. 2019

J Virol

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“…These 'BacMam'-type viruses (Boyce and Bucher, 1996) are a powerful tool for transgene delivery into mammalian systems due to the ability of the baculovirus BV to enter diverse cell types. Baculovirus can transduce human, rabbit, non-human primate, rodent, porcine, bovine, and ovine mammalian cells , as well as non-mammalian cells from fish (Kost et al, 2005), fly (Lee et al, 2000), and mosquito (Naik et al, 2018), albeit with variable transduction efficiencies. Baculovirus transduction has been studied in many cell types, including hepatic cells (Hofmann et al, 1995), A549 and HFL-1 (Chang et al, 2004), chondrocytes (Ho et al, 2004), kidney cells (Liang et al, 2004), osteosarcoma cells (Song and Boyce, 2001), mesenchymal stem cells (Ho et al, 2005), amongst many others.…”

Section: Cell Surface-display Strategiesmentioning

confidence: 99%

“…In addition to the mammalian systems, baculovirus are able to transduce the cells of several non-lepidopteran hosts. Naik et al (2018) established a 'BacMos' system using baculovirus as a vector to deliver genes of interest into mosquito cells, larvae, and adults. They introduced a neuraminidase (NA) from H5N3 influenza virus into the baculovirus vector, and then transduced mosquito C6/36 cells.…”

Section: Display Of Viral Protein For High-throughput Screeningmentioning

confidence: 99%

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Baculovirus as Versatile Vectors for Protein Display and Biotechnological Applications

Tsai¹,

Wei²,

Lo³

et al. 2020

Current Issues in Molecular Biology

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The baculovirus-insect cell system has long been deployed for a variety of applications including for use as biopesticides, for recombinant protein production, transient transgene expression, tissue therapy, and for vaccine production. Apart from the advantage of large-scale heterologous protein production with appropriate eukaryotic post-translational modification, foreign proteins can also be displayed on the viral envelope. This surface-display technology preserves the native multimeric structure of the protein, thereby expanding the clinical and pharmaceutical utility of the baculovirus system. Recombinant baculoviruses displaying major antigens for human or animal viruses can serve as appropriate vaccines. They can also serve as effective diagnostic platforms and various cell-based assay systems. In this review, we discuss progress in applying baculovirus surfacedisplay, including protein display on the envelope, capsid, and occlusion bodies of baculoviruses, as well as on cells. We will also describe strategies for improvement of this biotechnological approach.

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“…Trata-se de um vírus que infecta células de inseto, com 134 kb de material genético, de DNA de cadeia fechado, de fita dupla e apresenta capacidade de codificar 154 polipeptídios. Um tipo de baculovírus é o nucleopolyhedrovirus múltiplo de Autographa californica (AcMNPV) que é usado para expressão de genes de células de mamíferos (HOFMANN et al1995. NAIK et al 2018.…”

Section: Introductionunclassified

Expressão da glicoproteína do vírus Chikungunya em células de insetos visando desenvolvimento de insumo para diagnóstico e/ou vacina

Ferreira¹,

Lima²

2019

PIC

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INTRODUÇÃO Os mosquitos Aedes são vetores importantes para doenças emergentes causadas por arbovírus, como o Chikungunya. As principais espécies transmissoras desses vírus são Ae. aegypti e Ae. albopictus, que estão presentes em áreas climáticas tropicais e temperadas. O vírus Chikungunya é um patógeno artritogênico transmitido por mosquito, classificado como um Alfavírus da família Togaviridae, que possui um envelope e RNA de cadeia simples como ácidos nucléicos. De acordo com o Ministério da Saúde, a saúde pública no Brasil tem relatado um aumento na incidência de doenças tropicais “negligenciadas” emergentes e reemergentes causadas por arbovírus, como o vírus Chikungunya. Agências responsáveis no Brasil vêm demonstrando grande preocupação com todos os dados epidemiológicos recentes associados a esse vírus, uma vez que não há tratamento específico disponível ou vacina para seus programas públicos de imunização. Dessa forma, este projeto teve como objetivo expressar proteínas do vírus CHIKV em um sistema de expressão de proteínas heterólogas, utilizando o baculovírus, com intuito de obter antígenos específicos para testes imunológicos. METODOLOGIA: Após a síntese de oligonucleotídeos específicos para a região de interesse do vírus Chikungunya, analisamos a expressão dos epítopos específicos de proteínas do vírus, que foram fundidos à proteína poliedrina do baculovírus. Esse sistema de expressão de proteínas recombinante em células de inseto é um modelo de expressão bem estabelecido na literatura. Em seguida, foi construído um baculovírus recombinante contendo os epítopos do vírus Chikungunya, a confirmação se deu por sequenciamento e por fim, analisamos a expressão da proteína de interesse por western blot, assim como, realizamos testes preliminares para verificar possível reação cruzada entre outros arbovírus. RESULTADOS: O vírus recombinante construído foi utilizado para infectar células de inseto para a expressão da proteína recombinante. Este vírus recombinante possui os genes E2 e NSP3, que foi usado para infectar células de insetos (Tn5B) usando a estratégia bac-to-bac. A proteína expressa por este vírus recombinante foi então analisada por SDSPAGE e detectada por western blot, que confirmou a sua expressão, apresentando o tamanho esperado da proteína recombinante de 37 kDa. Um único ensaio de ELISA apontou que não houve reação cruzada com outros arbovírus, sendo necessário mais repetições de ensaios imunoenzimático. DISCUSSÃO/CONCLUSÃO: Sabe-se que as regiões do gene E2 e NSP3 do Chikungunya já foram expressas em estudos anteriores, no entanto, nenhuma delas utilizou as mesmas regiões antigênicas utilizadas como repetições descritas neste trabalho, as quais apresentaram uma significativa expressão em células de inseto e demonstrou ser reconhecido por antissoro contra as propriedades imunogênicas do vírus Chikungunya. Os resultados apontaram que a estratégia é promissora, utilizando regiões imunogênicas específicas do vírus Chikungunya, que poderiam ser utilizadas para produzir um kit de diagnóstico, bem como, para outras aplicações biotecnológicas, como auxiliar naprodução de uma potencial vacina subunitária

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Baculovirus as an efficient vector for gene delivery into mosquitoes

Cited by 28 publications

References 50 publications

Baculovirus IE2 Interacts with Viral DNA through Daxx To Generate an Organized Nuclear Body Structure for Gene Activation in Vero Cells

Baculovirus IE2 Interacts with Viral DNA through Daxx To Generate an Organized Nuclear Body Structure for Gene Activation in Vero Cells

Baculovirus as Versatile Vectors for Protein Display and Biotechnological Applications

Expressão da glicoproteína do vírus Chikungunya em células de insetos visando desenvolvimento de insumo para diagnóstico e/ou vacina

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