Biology Reference
In-Depth Information
The coxsackievirus and adenovirus receptor (CAR) is used by
many adenovirus, CAdV-2 included [
4
,
5
]. Regarding its expres-
sion in the CNS, early studies showed that its expression peaks
during development and drops after birth [
6
]. It has been sug-
gested that CAR could regulate axonal growth through interaction
with members of the extracellular matrix [
7
]. Its role in some AdVs
entry is relatively well characterized where it mainly acts as a pri-
mary docking factor as its cytoplasmic tail was shown dispensable
for infection. Secondary receptors such as integrins will allow viri-
ons to recruit the intracellular machinery necessary for their endo-
cytosis. Once in the cell, acidifi cation during endosomal progression
will trigger conformational changes in the capsid and subsequent
endosome lysis. Members of the motor protein family will then be
recruited by cytoplasmic virions to reach the nucleus and inject
their genome [
8
]. CAdV-2, however, does not seem to use second-
ary receptors. Its cell tropism is absolutely dependent on CAR [
5
].
We showed that the molecular mechanisms behind CAdV-2 neu-
rotropism and axonal transport were due to the innate ability of
CAR in neurons to be endocytosed and axonaly transported
towards the cell body [
9
] (
see
Note 1
).
Numerous viruses are using molecules found at synaptic termi-
nals such as peripheral nerve endings to enter and spread in the
CNS [
10
]. Visualizing and characterizing their intracellular traf-
fi cking can help to better understand their pathogenesis but also
key pathways involved in neuronal homeostasis.
2
Materials
2.1 Virus and Protein
Production
1. Phosphate buffer saline.
2. Cesium Chloride (CsCl) dissolved in PBS.
3. Ultra clear centrifuge tubes 14 × 95 mm.
4. Needles 21 G × 1½ and Syringes.
5. Slide A Lyser dialysis cassette 10,000 MWO.
6. Glycerol.
7.
Escherichia coli
strain BL21.
8. LB (Luria Broth) supplemented with ampicillin (100
μ
g/mL).
9. Isopropyl
-
D
-1-thiogalactopyranoside (IPTG) dissolved in
water at 1 M and stored in aliquots at −20 °C.
10. Protino Ni-TED 1000 kit.
11. His-tagged TEV protease.
12. Ni
2+
-agarose beads.
13. Cy5 and Cy3 mono-reactive dyes.
14. NAP5 column.
β
