Unique Requirement for ESCRT Factors in Flavivirus Particle Formation on the Endoplasmic Reticulum

Keisuke Tabata; Masaru Arimoto; Masashi Arakawa; Atsuki Nara; Kazunobu Saito; Hiroko Omori; Arisa Arai; Tomohiro Ishikawa; Eiji Konishi; Ryosuke Suzuki; Yoshiharu Matsuura; Eiji Morita

doi:10.1016/j.celrep.2016.07.068

Unique Requirement for ESCRT Factors in Flavivirus Particle Formation on the Endoplasmic Reticulum

Cell Rep. 2016 Aug 30;16(9):2339-47. doi: 10.1016/j.celrep.2016.07.068. Epub 2016 Aug 18.

Authors

Affiliations

¹ Laboratory of Viral Infection, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
² Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
³ Faculty of Bioscience, Nagahama Institute of Bio-science and Technology, Nagahama 526-0829, Japan.
⁴ Core Instrumentation Facility, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
⁵ Department of Microbiology, Dokkyo Medical University School of Medicine, Tochigi 321-0293 Japan.
⁶ BIKEN Endowed Department of Dengue Vaccine Development, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
⁷ Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.
⁸ Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
⁹ Laboratory of Viral Infection, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan. Electronic address: moritae@hirosaki-u.ac.jp.

PMID: 27545892
DOI: 10.1016/j.celrep.2016.07.068

Abstract

Flavivirus infection induces endoplasmic reticulum (ER) membrane rearrangements to generate a compartment for replication of the viral genome and assembly of viral particles. Using quantitative mass spectrometry, we identified several ESCRT (endosomal sorting complex required for transport) proteins that are recruited to sites of virus replication on the ER. Systematic small interfering RNA (siRNA) screening revealed that release of both dengue virus and Japanese encephalitis virus was dramatically decreased by single depletion of TSG101 or co-depletion of specific combinations of ESCRT-III proteins, resulting in ≥1,000-fold titer reductions. By contrast, release was unaffected by depletion of some core ESCRTs, including VPS4. Reintroduction of ESCRT proteins to siRNA-depleted cells revealed interactions among ESCRT proteins that are crucial for flavivirus budding. Electron-microscopy studies revealed that the CHMP2 and CHMP4 proteins function directly in membrane deformation at the ER. Thus, a unique and specific subset of ESCRT contributes to ER membrane biogenesis during flavivirus infection.

Keywords: CHMP2; CHMP4; ESCRT; TSG101; flavivirus.

MeSH terms

ATPases Associated with Diverse Cellular Activities
Animals
Cell Line
Chlorocebus aethiops
Cricetulus
DNA-Binding Proteins / antagonists & inhibitors
DNA-Binding Proteins / genetics*
DNA-Binding Proteins / metabolism
Dengue Virus / genetics*
Dengue Virus / growth & development
Dengue Virus / metabolism
Encephalitis Virus, Japanese / genetics*
Encephalitis Virus, Japanese / growth & development
Encephalitis Virus, Japanese / metabolism
Endoplasmic Reticulum / genetics*
Endoplasmic Reticulum / virology
Endosomal Sorting Complexes Required for Transport / antagonists & inhibitors
Endosomal Sorting Complexes Required for Transport / genetics*
Endosomal Sorting Complexes Required for Transport / metabolism
Epithelial Cells / metabolism*
Epithelial Cells / virology
Gene Expression Regulation
HEK293 Cells
Host-Pathogen Interactions
Humans
RNA, Small Interfering / genetics
RNA, Small Interfering / metabolism
Transcription Factors / antagonists & inhibitors
Transcription Factors / genetics*
Transcription Factors / metabolism
Vacuolar Proton-Translocating ATPases / antagonists & inhibitors
Vacuolar Proton-Translocating ATPases / genetics
Vacuolar Proton-Translocating ATPases / metabolism
Vero Cells
Virion / genetics*
Virion / metabolism
Virus Replication / genetics

Substances

CHMP2A protein, human
CHMP4A protein, human
DNA-Binding Proteins
Endosomal Sorting Complexes Required for Transport
RNA, Small Interfering
Transcription Factors
Tsg101 protein
Vacuolar Proton-Translocating ATPases
ATPases Associated with Diverse Cellular Activities
VPS4A protein, human