Measuring the subcellular compartmentalization of viral infections by protein complementation assay.
Cell Line
Cell Nucleus
/ metabolism
Cytoplasm
/ metabolism
Green Fluorescent Proteins
/ metabolism
HIV Infections
/ metabolism
HeLa Cells
High-Throughput Screening Assays
/ methods
Humans
Nuclear Envelope
/ metabolism
Nuclear Pore
/ metabolism
Ribonucleoproteins
/ metabolism
Virus Diseases
/ metabolism
Virus Replication
/ drug effects
subcellular compartments
technological development
viruses
Journal
Proceedings of the National Academy of Sciences of the United States of America
ISSN: 1091-6490
Titre abrégé: Proc Natl Acad Sci U S A
Pays: United States
ID NLM: 7505876
Informations de publication
Date de publication:
12 01 2021
12 01 2021
Historique:
entrez:
6
1
2021
pubmed:
7
1
2021
medline:
11
5
2021
Statut:
ppublish
Résumé
The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.
Identifiants
pubmed: 33402530
pii: 2010524118
doi: 10.1073/pnas.2010524118
pmc: PMC7812837
pii:
doi:
Substances chimiques
Ribonucleoproteins
0
Green Fluorescent Proteins
147336-22-9
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Déclaration de conflit d'intérêts
Competing interest statement: J.F., S.N., and N.J.A. are inventors on a patent application describing the measurement of protein complementation upon nuclear import as described in this paper.
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