Data from: Coordination of the host Vps4-Vta1 complex and the viral core protein Ac93 facilitates entry of Autographa californica multiple nucleopolyhedrovirus budded virions
Data files
Mar 13, 2025 version files 3.22 KB
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GUS_activities.csv
142 B
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LacZ-activities.csv
140 B
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README.md
2.43 KB
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Virus-titers-vAc93-HA-Cm.csv
279 B
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Virus-titers-wild_type_virus.csv
230 B
Abstract
The endosomal sorting complex required for transport (ESCRT) is a protein machine mediating membrane scission. In intraluminal vesicles (ILVs) formation, ESCRT-0 targets cargoes and recruits ESCRT-I/-II to create membrane invagination, whereas ESCRT-III coordinates with the AAA ATPase Vps4 and its cofactor Vta1 to catalyze the membrane fission. Recently, ESCRT-I/-III and Vps4 were found to involve in entry of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, the necessity of other ESCRT components and the interplay of viral proteins and ESCRTs in regulating the virus entry remain elusive. Here, we identified ESCRT-0 (Hse1 and Vps27), ESCRT-II (Vps22, Vps25, and Vps36), and Vta1 of S. frugiperda. RNAi depletion of Vta1 but not the components of ESCRT-0 or ESCRT-II in Sf9 cells significantly reduced budded virus (BV) production. Quantitative PCR together with confocal microscopy analyses indicated that Vta1 was required for internalization and endosomal trafficking of BV. In the late phase of infection, although Vps4 and Vta1 were both distributed to the nucleus and at the plasma membrane, depletion of Vta1 did not affect BV release. Further analysis revealed that 7 of 14 BV envelope proteins (Ac75, Ac93, E25, F-like, P33, P48, and vUbiquitin) interacted with Vps4 and Vta1. Intriguingly, Ac93 adopted a similar mode as ESCRT-III proteins to interact with the MIT domains of Vps4 and Vta1 via its C-terminal MIM1 motif, and the interactions were necessary for BV internalization. Together, our studies highlight the coordination of Vps4-Vta1 and Ac93, and probably other BV envelope proteins, in facilitating entry of AcMNPV.
https://doi.org/10.5061/dryad.g1jwstr2k
Description of the data and file structure
The data files contain the LacZ ((β-galactosidase) and GUS (β-glucuronidase) activities and the virus titers. The extended data file contains the supplemental figures.
For measurement of LacZ and GUS activities, a brief description of the methods as below (detailed Materials and Methods see the associated paper):
Sf9 cells in 12-well plates (200000 cells/well) were transfected with the dsRNA of gfp, Vps4, or Vta1 (5 g/well for each gene) using the standard CaPO4 precipitation method. At 48 h post-transfection (p.t.), the cells were transfected again with the bacmid DNA of AcMNPV-LacZGUS (4 g/well). After transfecting the viral DNA for 24 h, the cells were lysed using 1% Triton X-100 containing 2 mg/ml Chlorophenol red--D-galactopyranoside (Roche Diagnostics GmbH) or 1mM 4-Nitrophenyl -D-glucuronide (Merck) to measure the activity of -Gal or -Gluc.
For measurement of the titers of wild-type virus (AcMNPV) and vAc93-HA-Cm, a brief description of the methods as below (detailed Materials and Methods see the associated paper):
Sf9 cells were infected with each of the virus (MOI=5) and the virus titers were measured by a 50% tissue culture infection dose (TCID50) assay at indicated time points.
Files and variables
The data files listed as:
- File 1: LacZ activities
- File 2: GUS activities
- File 3: Virus titers of vAc93-HA-Cm
- File 4: Virus titers of wild-type AcMNPV
The extended data file contains 6 figures:
- Extended Figure S1: Amino acid sequence alignment of the homologs of ESCRT-0 components Hse1 and Vps27.
- Extended Figure S2: Amino acid sequence of Hse1 and Vps27 of S. frugiperda.
- Extended Figure S3: The activities of -galactosidase and -glucuronidase.
- Extended Figure S4: Yeast-two hybrid analysis of the interaction between Vps4 or Vta1 of S. frugiperda and BV-envelope proteins of AcMNPV.
- Extended Figure S5: Western blot analysis of the expression of Nm- and Cm-fused Vps4 and Vta1 of S. frugiperda, and BV envelope proteins of AcMNPV.
- Extended Figure S6: One-step growth curves of wild-type (wt) AcMNPV or Ac93-HA-Cm-repaired ac93-knockout virus.