Identification and Characterization of a Major Early-Transcribed Gene of Trichoplusia ni Single Nucleocapsid Nucleopolyhedrovirus Using the Baculovirus Expression System

Wang, Weizhou; Davison, Sean; Krell, Peter J.

doi:10.1023/b:viru.0000032786.45323.6b

Cited by 6 publications

(5 citation statements)

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“…Pr70 (BV/ODV-C42) has been proven to mediate the nuclear entry of P78/83. , As a conserved ODV envelope protein, ODV-E18 has been shown to mediate budded virus production, whereas ODV-E28 has been characterized as a structural ODV-associated envelope protein . LEF3 also participates in DNA replication and (or) the very late expression of genes. ,, PEP1 and PEP2 are a type of polyhedrin envelope proteins that are located on the surface of OBs and play an important role in polyhedrin formation. , ME-53 is an early expression protein with zinc finger-like motifs at its C-terminus . An me53 knockout could block viral DNA replication, nucleocapsid formation, and consequently BV and ODV production .…”

Section: Resultsmentioning

confidence: 99%

“…15,22 ME-53 is an early expression protein with zinc finger-like motifs at its C-terminus. 71 An me53 knockout could block viral DNA replication, nucleocapsid formation, and consequently BV and ODV production. 80 However, new data suggest that ME-53 does not affect DNA replication but is required for efficient BV production.…”

Section: Comparison Of Odv Proteins Identified In This Study and Prot...mentioning

confidence: 99%

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ODV-Associated Proteins of the Pieris rapae Granulovirus

Zhang

Cui

et al. 2011

J. Proteome Res.

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Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPV) and Betabaculovirus (granuloviruses, GV) are two main genera of the family Baculoviridae. The virion proteomes of Alphabaculovirus have been well studied; however, the Betabaculovirus virion compositions remain unclear. Pieris rapae granulovirus (PrGV) can kill larvae of P. rapae, a worldwide and important pest of mustard family crops. In this study, the occlusion-derived virus (ODV)-associated proteins of PrGV were identified using three mass spectrometry (MS) approaches. The MS analyses demonstrated that 47 proteins were present in PrGV-ODV. Of the 47 PrGV-ODV proteins, 33 have homologues identified previously in other baculovirus ODV/BVs, whereas 14 (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were newly identified ODV proteins. Seven of the 14 newly identified ODV proteins are specific to Betabaculovirus, including Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114. Furthermore, the data derived from these MS approaches were validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV-ODV proteins (including 5 Betabaculovirus-unique proteins). Comparison analyses revealed the similar and different compositions between Betabaculovirus and Alphabaculovirus virions, which deepen our understanding of the baculovirus virion structure and provide helpful information on Betabaculovirus–host interaction studies.

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Section: Resultsmentioning

confidence: 99%

Section: Comparison Of Odv Proteins Identified In This Study and Prot...mentioning

confidence: 99%

ODV-Associated Proteins of the Pieris rapae Granulovirus

Zhang

Cui

et al. 2011

J. Proteome Res.

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“…With the early and late specific motifs in the putative promoter regions, me53 gene was reported to transcribe at the early and late phases of viral infection [21]. It is unclear what is the relationship between DNA replication and late gene expression.…”

Section: Discussionmentioning

confidence: 99%

Characterization of AcMNPV with a deletion of me53 gene

Wang

Deng

et al. 2006

Virus Genes

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The Autographa Californica multiple nucleopolyhedrovirus (AcMNPV) me53 gene, which was previously reported as one of the major early-transcribed genes, was deleted through homologous recombination from an AcMNPV genome propagated as a bacmid DNA in E. coli, generating a me53 gene knockout bacmid. Green fluorescent protein (GFP) expression analysis and supernatant passage assay revealed that the me53 knockout bacmid was unable to replicate in cell culture, while me53 repair bacmid, which was generated by reinsertion of the me53 gene into the mutant at a different locus (the gentamicin locus) with ET-recombination technique, resumed viral replication ability at wild-type levels, indicating that the defective phenotype of the me53 knockout virus was directly due to the deletion of the me53-ORF. Subsequent electron microscopy revealed that the me53 knockout bacmid failed to form nucleocapsid in the nuclei of the transfected cells, though viral infection seemed to be initiated. Meanwhile, real-time PCR analysis based on SYBR Green fluorescence indicated abolishment of the viral DNA replication by me53 gene inactivation. Thus, it is demonstrated for the first time that me53 knockout blocked viral DNA replication, nucleocapsid formation, and consequent BV and ODV production.

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“…A study by Wang et al (33) revealed that me53 from Trichoplusia ni single nucleocapsid nucleopolyhedrosis virus could be successfully expressed from its native promoters in a recombinant AcMNPV in the Sf9 and BTI-Tn-5b1 (Hi5) insect cell lines. Their study confirmed the transcriptional profile of AcMNPV me53 and indicated that the T. ni single nucleocapsid nucleopolyhedrosis virus me53 was similarly transcribed from unique sites at both early and late times p.i.…”

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confidence: 99%

Autographa californica Multiple Nucleopolyhedrovirus me53 ( ac140 ) Is a Nonessential Gene Required for Efficient Budded-Virus Production

Jong

Arif

Theilmann

et al. 2009

J Virol

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me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an me53-null bacmid (Ac⌬me53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-5b1 cells transfected with Ac⌬me53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10-to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160-to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53, although not essential for viral replication, is required for efficient BV production.Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) is the type species for the genus Alphabaculovirus in the family Baculoviridae. Members of the Baculoviridae are characterized by having enveloped, rod-shaped virions with large circular double-stranded DNA genomes (13). Infection is restricted to arthropods, with the vast majority of known permissive species falling within the class Insecta. A hallmark of baculovirus infection is the production of two morphologically distinct virion phenotypes. At late times of infection, nucleocapsids bud from infected cells to form the budded-virus (BV) phenotype, which is responsible for the transmission of infection from cell-to-cell within a host. However, at very late times in infection, nucleocapsids are enveloped and occlusion-derived virions (ODVs) are packaged into intranuclear occlusion bodies (OBs) (3). OBs generally range from 1 to 5 m in diameter and are responsible for horizontal transmission of the virus from insect to insect (13).Baculovirus infection is characterized by the temporal cascade of expression consisting of immediate-early, early, late, and very late gene expression, with the onset of DNA replication separating the early and late phases. Early genes are transcribed by host RNA polymerase II, and some early genes are transcribed from promoters containing a TATA-N(24-28)-CAKT at the transcriptional start site (12). Late genes are transcribed by an ␣-amanitin-resistant viral RNA polymerase, from ATAAG promote...

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Identification and Characterization of a Major Early-Transcribed Gene of Trichoplusia ni Single Nucleocapsid Nucleopolyhedrovirus Using the Baculovirus Expression System

Cited by 6 publications

References 45 publications

ODV-Associated Proteins of the Pieris rapae Granulovirus

ODV-Associated Proteins of the Pieris rapae Granulovirus

Characterization of AcMNPV with a deletion of me53 gene

Autographa californica Multiple Nucleopolyhedrovirus me53 ( ac140 ) Is a Nonessential Gene Required for Efficient Budded-Virus Production

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