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Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.

ABSTRACT: The insect baculovirus AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells, we show that entry is favoured by low pH and by increasing the available cell-surface area through a transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268-281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell-to-cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies, suggesting that additional factors also govern entry.

SUBMITTER: O'Flynn NM

PROVIDER: S-EPMC3522474 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.

O'Flynn Neil M J NM Patel Avnish A Kadlec Jan J Jones Ian M IM

Bioscience reports 20121130 1

The insect baculovirus AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells, we show that entry is favoured by low pH and by increasing the available cell-surface area through a transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, ...[more]

PMID: 23035899

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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.

Project description:UnlabelledProductive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) leads to expression of ~156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an ~60-Gb RNA sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i. but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathway members that were upregulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure.ImportanceBaculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell transduction and protein expression for vaccine production. While there is considerable information regarding viral gene expression in infected cells, little is known regarding responses of the host cell to baculovirus infection. In these studies, we assembled a cell transcriptome from the host Trichoplusia ni and used that transcriptome to analyze changes in host cell gene expression throughout the infection cycle. The study was performed in parallel with a prior study of changes in viral gene expression. Combined, these studies provide an unprecedented new level of detail and an overview of events in the infection cycle, and they will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.

Project description:Baculoviruses are large double-stranded DNA viruses that are virulent pathogens of certain insect species. In a natural host, Trichoplusia ni, infection by the model baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) begins when the occluded form of the virus disassembles in the midgut and virions infect midgut epithelial cells to establish the primary phase of the infection. To better understand the primary phase of the AcMNPV infection cycle, newly molted 5th-instar T. ni larvae were orally infected with AcMNPV occlusion bodies and the transcriptional responses of the T. ni midgut were analyzed at various times from 0 to 72 h postinfection, using transcriptome sequencing analysis and a T. ni reference genome. The numbers of differentially expressed host genes increased as the infection progressed, and we identified a total of 3,372 differentially expressed T. ni transcripts in the AcMNPV-infected midgut. Genes encoding orthologs of HMG176, atlastin, and CPH43 were among the most dramatically upregulated in response to AcMNPV infection. A number of cytochrome P450 genes were downregulated in response to infection. We also identified the effects of AcMNPV infection on a large variety of genes associated with innate immunity. This analysis provides an abundance of new and detailed information on host responses to baculovirus infection during the primary phase of the infection in the midgut and will be important for understanding how baculoviruses establish productive infections in the organism.IMPORTANCE Baculoviruses are virulent pathogens of a number of important insect pest species. In the host Trichoplusia ni, infection begins in the midgut when infectious virions of the occlusion-derived virus (ODV) phenotype enter and subsequently replicate in cells of the midgut epithelium. A second virion phenotype (budded virus [BV]) is produced there, and BV mediates systemic infection of the animal. Most prior detailed studies of baculovirus infections have focused on BV infections of cultured cells. In this study, we examined the transcriptional responses of the T. ni midgut to infection by ODV of the baculovirus AcMNPV and identified a variety of host genes that respond dramatically to viral infection. Understanding the transcriptional responses of the host midgut to viral infection is critically important for understanding the biphasic infection in the animal as a whole.

Dataset Information

Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.

Publications

Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.

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