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Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for budded-virus production and occlusion-derived-virus envelopment.

ABSTRACT: ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5' rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment.Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

SUBMITTER: Tao XY

PROVIDER: S-EPMC4301119 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for budded-virus production and occlusion-derived-virus envelopment.

Tao Xue Ying XY Choi Jae Young JY Kim Woo Jin WJ An Saes Byeol SB Liu Qin Q Kim Song Eun SE Lee Seok Hee SH Kim Jong Hoon JH Woo Soo Dong SD Jin Byung Rae BR Je Yeon Ho YH

Journal of virology 20141015 1

<h4>Unlabelled</h4>ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5' rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produce ...[more]

PMID: 25320313

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Project description:In eukaryotic cells, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV.IMPORTANCE Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses.

Project description:The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

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Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for budded-virus production and occlusion-derived-virus envelopment.

Publications

Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for budded-virus production and occlusion-derived-virus envelopment.

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