2.2 BMV virus production
The VLP particles were produced and purified from the native virus as
previously reported.[42] The BMV native virus was
amplified through plant infection to produce VLPs. First, barley plants
(Hordeum vulgare ) were grown under controlled conditions. Then,
young barley leaves were inoculated with the BMV virus. Once the leaves
showed the infection pattern, they were harvested and stored at -20°C to
be later liquefied in a blender (Osterizer) with virus extraction buffer
(0.5 M sodium acetate, 80 mM magnesium acetate, pH 4.5). The resulting
macerated material was filtered through cheesecloth, then the
flowthrough was mixed with chloroform 1:1 v/v. The mixture was
centrifuged at 10,000 rpm, 4°C, for 20 min. Since two phases formed in
the centrifuge bottle; the upper phase (aqueous phase) was collected and
stirred at 4°C overnight. Next, the aqueous extract containing the BMV
virions was ultracentrifuged on a 5 mL sucrose cushion in a 32 mL
centrifuge tube at 32,000 rpm, 4°C for 2 h. After centrifugation, the
supernatant was discarded and a pellet containing the BMV virions was
resuspended in a virus suspension buffer (50 mM sodium acetate, 8 mM
magnesium acetate, pH 4.5). Then, the BMV virion suspension was poured
into a previously prepared centrifuge tube with a sucrose density
gradient (10-40%) and centrifuged at 30,000 rpm, 4°C for 2 h. After
centrifugation, the tube was examined under white light to confirm the
presence of the blue phase containing the viruses. The blue phase was
then recovered and diluted 1:4 v/v in a virus suspension buffer. The
dilute blue phase was centrifuged at 32,000 rpm, 4°C for 3 h to
concentrate the virions in a pellet. After centrifugation, the
supernatant was discarded and the pellet containing the virions was
resuspended in virus suspension buffer and stored at -70°C.