Biology Reference
In-Depth Information
the DGNNV capsid protein were first constructed to examine the
involvement of the N-terminus in assembly of the virus.
4
The
mutant
N52 (truncating 1-52 N-terminal residues) deleted all pos-
itive charges at the N-terminus. The mutant
∆
N35 (truncating 1-35
N-terminal residues) deleted 13 positively charged residues out of
15, while the mutant
∆
N25 (truncating 1-25 N-terminal residues)
removed nine Lys/Arg. The main difference between
∆
∆
N25 and
∆
N35 is the number of triple-Arg:
∆
N25 retains one triple-Arg while
∆
N35 has none. The
∆
N52 and
∆
N35 did not produce detectable
VLPs while
N25 maintained the formation of VLPs, suggesting that
the residues between the 25th and 35th residues are essential for
forming VLPs. Among the 10 residues, the role of triple-Arg at the
29-31 residues in forming VLPs is noticeable. The density of
∆
N25
VLPs is 1.34 g/cm
3
, which is the same as the native particles. Further
deletions within 25 N-terminal residues were performed to examine
their functions in VLP formation. There is a triple-Arg located at
23-25 residues. With triple-Arg at both 23-25 and 29-31 residues,
the mutants of deleting 4 (
∆
N16) N-terminal residues
retained the formation of VLPs. These VLPs were very similar in
shape to the VLPs of the full-length coat protein, but they were
slightly smaller. The particle diameters of
∆
N4) and 16 (
∆
N25 were 26 and
24 nm, respectively. The quantities of VLPs from the N-terminal dele-
tion mutants of
∆
N4 and
∆
∆
N4,
∆
N16, and
∆
N 25 are the same as that of the
full-length capsid protein.
Expression of C-terminus Deletion Mutants
C288) was ini-
tially constructed where the site of Asn/Ala resembles the autocleav-
age sequence in alphanodavirus. The
A mutant of deleting 289-338 C-terminal residues (
∆
C288 mutant produced a
31.5 kDa protein, but no VLPs were found. The mutants of deleting
328-338 (
∆
C335) C-terminal residues
produced slightly smaller particles than those of native virus parti-
cles, while
∆
C327) and 336-338 (
∆
∆
C292,
∆
C296, and
∆
C308 did not produce VLPs.
4
The particle diameters of
C335 were 24 nm and 26 nm,
respectively. The particles with short truncations (
∆
C327 and
∆
∆
N4 and
∆
C335)
