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Characterization of two Autographa californica nucleopolyhedrovirus proteins, Ac145 and Ac150, which affect oral infectivity in a host-dependent manner.

ABSTRACT: The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent.

SUBMITTER: Lapointe R

PROVIDER: S-EPMC416519 | biostudies-literature | 2004 Jun

REPOSITORIES: biostudies-literature

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Characterization of two Autographa californica nucleopolyhedrovirus proteins, Ac145 and Ac150, which affect oral infectivity in a host-dependent manner.

Lapointe Renee R Popham Holly J R HJ Straschil Ursula U Goulding David D O'Reilly David R DR Olszewski Julie A JA

Journal of virology 20040601 12

The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this prote ...[more]

PMID: 15163737

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Project description:Productive 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.Baculoviruses 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 encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10-kDa auxiliary protein produced in the very late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeleton-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated perinuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate the horizontal transmission of the virus between insect hosts. Here, using mass spectrometric analysis, it is demonstrated that the C terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed aberrant formation of the perinuclear tubules. Thus, the phosphorylation of serine 93 may induce the aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects the structural conformation of P10.IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for the survival of its progeny, and therefore, P10 presents an enigma. As P10 polymerizes to form organized cytoskeletal structures that colocalize with host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus may potentially be responsible for its dissemination and survival.

Dataset Information

Characterization of two Autographa californica nucleopolyhedrovirus proteins, Ac145 and Ac150, which affect oral infectivity in a host-dependent manner.

Publications

Characterization of two Autographa californica nucleopolyhedrovirus proteins, Ac145 and Ac150, which affect oral infectivity in a host-dependent manner.

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