Biology Reference
In-Depth Information
recruit a variety of cellular RNA molecules to satisfy the highly
charged amino-terminal portions of the subunit. The peak I parti-
cles were irregular because the coat protein had recruited RNA mol-
ecules too small to satisfy the requirements for forming an icosahedral
lattice of the correct size.
The RNA2 of DGNNV includes a single ORF that encodes a 338
amino acid protein of 37,085 Da, the putative capsid protein. The
recombinant clone is the first attempt to express nodavirus VLPs in
prokaryote
E. coli
. The molecular weight of the recombinant full-
length capsid protein on the VLPs was the same as the native virus
coat protein. Electron microscopy revealed that the features of VLPs
resemble the native virus. VLP formation from various viral capsid
proteins expressed in
E. coli
has been reported in other types of viruses
elsewhere. Two visible bands appeared at a density of 1.27 g cm
−3
(Fraction I) and 1.34 g cm
−3
(Fraction II). The concentration of
Fraction I was approximately 5% of Fraction II, estimated by UV
absorbance (A280). The molecular mass of the proteins (~37 kDa) in
both fractions was apparently the same as the native virus in SDS-
PAGE. Heterogeneous VLPs existed in both fractions. Electron
microscopy of VLPs in Fraction I showed that the small particles were
about 23 nm in diameter, a few of which were irregular. In contrast,
Fraction II consisted of equal amounts of intact and stain-permeable
particles with a diameter of about 30 nm. Comparing these parti-
cles revealed that the heavy VLPs had a similar size and geometry to
the native piscine nodavirus and Sf 21-expressed VLPs. Interestingly,
the surfaces of both native particles and VLPs are rougher than that
of the insect nodavirus flock house virus.
3
Similarly, two types of
Dicentrarchus labrax
encephalitis virus have been reported.
VLPs from N-terminus Deletion Mutants
The coat protein of DGNNV consists of a high percentage of basic
residues in the N-terminal region, nine Arg and six Lys (Lu and Lin,
1999; AF245004, GenBank). The N-terminal basic arm is involved in
binding the negatively charged phosphate backbone of the RNAs
located inside the viral capsid. Several N-terminal deletion mutants of
