Related Experiment Videos
A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag
Jeffrey M Slack1, Edward M Dougherty1, Susan D Lawrence1
1United State Department of Agriculture, Agriculture Research Service, Plant Sciences Institute, Insect Biocontrol Laboratory, Bldg 011A, Rm 214, BARC-West, Beltsville, MD 20705-2350, USA1.
Abstract:
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74-GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74-GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74-GFP chimeras in recombinant baculoviruses. When C-terminal region S580-F645 was deleted from p74, p74-GFP chimera localization became non-specific and chimeras became soluble. p74 region S580-F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.
Insights
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p74 protein is crucial for viral infectivity. Its C-terminal region anchors the protein, ensuring proper localization within infected cells for virus function.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a significant insect pathogen.
- The p74 protein is essential for the oral infectivity of occlusion-derived virus (ODV).
- p74's role in viral entry, specifically midgut attachment and fusion, has been hypothesized.
Purpose of the Study:
- To investigate the localization and function of the AcMNPV p74 protein within infected cells.
- To identify the specific region of p74 responsible for its localization and membrane association.
- To understand the role of the C-terminal hydrophobic region in p74 function.
Main Methods:
- Expression of p74 fused to green fluorescent protein (GFP) in baculovirus-infected Spodoptera frugiperda (Sf-9) cells.
- Visualization of p74-GFP chimera localization using fluorescence microscopy.
- Biochemical fractionation and co-precipitation assays to determine p74's association with cellular components.
- Analysis of p74 truncations to map functional domains.
Main Results:
- p74-GFP chimeras localized to the intranuclear ring zone of Sf-9 cells.
- p74-GFP co-precipitated with the microvesicle fraction, suggesting membrane association.
- Deletion of the C-terminal region (S580-F645) resulted in non-specific localization and soluble chimeras.
- The S580-F645 region alone directed GFP to the intranuclear ring zone, mimicking full-length p74.
Conclusions:
- The hydrophobic C-terminal region of AcMNPV p74 is critical for its specific nuclear localization.
- This C-terminal domain likely mediates transmembrane anchoring or insertion, essential for p74 function.
- Understanding p74's localization mechanism provides insights into baculovirus entry and infection processes.