Download Cell-cell fusion assay

Valentina Bordei
Cell-cell fusion assay
Principle
Fusion of cells expressing viral coat glycoproteins with cells that express viral receptors and a
reporter gene is used to mimic coated virus-cell fusions.
Objective
The aim of the procedure is to study virus-cell fusion processes in order to gain more insight into
the glycoprotein-mediated infection mechanism of some viruses.
Materials & methods
•
cell populations transfected with a plasmid coding for the coat protein of the virus and a
polymerase that binds a specific promoter (e.g.T7)
•
T7 Polymerase is one of a group of very active phage polymerases, that use a
short double-stranded promoter sequence to initiate transcription at a specific
base in the template, and then transcribe single or double stranded DNA with
good fidelity until the end of the DNA bottom strand is reached;
•
in case the cells cannot be transfected by conventional methods, a
recombinant vaccinia virus (mild virus from the pox family) encoding for the
polymerase is used
•
cell populations transfected with the receptor and a reporter gene situated under the control
of the specific promoter. The most commonly used reporter genes code for firefly luciferase
or β-galactosidase.
Procedure
All cell populations (target and effectors) are incubated over night to allow the expression of
plasmid/ vaccinia encoded proteins. Equal amounts of target and effector cells are mixed on plates
and cell fusion activity is assayed by measuring the activity of the reporter gene product in
detergent-treated cell lysates.
Specific example
The procedure was used to study the receptor activity of GLUT-1 for the HTLV-1 Env protein. The
target gene populations expressed the potential receptor along with the lacZ gene linked to the T7
promoter (PT7-lacZ reporter); the effector cell population expressed HTLV-1 Env and T7 RNA
polymerase encoded by a recombinant vaccinia virus.
Cell fusion is monitored by activation of the β-galactosidase reporter gene in response to the
interaction of the T7 RNA polymerase with the T7 promoter in the cytoplasm of fused cells.
Effector (Env-expressing) cells were prepared by infection with recombinant vaccinia viruses
encoding either Env63 or Unc63(fusion defficient mutant) . Expression of PT7-lacZ reporter was
accomplished by infecting the cells with a recombinant vaccinia virus vTF7-3 encoding lacZ under
T7 promoter. All cell populations (effectors and targets) were incubated overnight at 31 °C to allow
expression of the vaccinia-encoded proteins. Duplicate samples containing 105 target cells and 105
effector cells (expressing the indicated Env protein) were mixed in 96-well microtiter plates and
incubated at 37 °C for 2–3 h. The cell fusion activity was assayed by measuring β-galactosidase
activity in detergent-treated cell lysates.
Figure 1: Schema of assay function for a T7 polymerase coding effector cell and a vTF7-3
lacZ expressing target cell (Modified from Sakamoto et all., 2003)
References
Sakamoto. T, Ushijima. H, Okitsu. S, Suzuki. E, Sakai. K, Morikawa. S, Müller. W, (2003):
Establishment of an HIV cell–cell fusion assay by using two genetically modified HeLa cell lines
and reporter gene, Journal of Virological Methods
Jin.Q, Agrawal. L, VanHorn-Ali. Z, Alkhatib.G (2006): Infection of CD4+ T lymphocytes by the
human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1Evidence using
antibodies specific to the receptor's large extracellular domain, Virology
http://genetics.mgh.harvard.edu/szostakweb/resources/Public%20Protocols/transcription/index.html
http://www.stanford.edu/group/virus/pox/2000/vaccinia_virus.html