Biology Reference
In-Depth Information
DE
FG
HI
111
151
257
298
349
359
1
505
L50
C175 C185
S282
C428
a part of H16. E70 epitope
S-S bonding
S-S bonding
a part of H16.V5 epitope
S-S bonding
Fig. 1.
Amino acid residues associated with L1 functions. Numbers repre-
sent amino acid numbers of HPV16 L1. DE, FG, and HI are HPV16 capsid
surface loops.
17
C175 (cysteine at aa175), C185, and C428 are required for
the normal assembly of L1-capsids.
16
L50 and S282 are located in conforma-
tionally dependent epitopes for MAbs H16.E70
39
and H16.V5.
41
We have studied a series of HPV16 L1 mutants having nucleotide
substitution of serine for cysteine and identified cysteine residues
required for the intercapsomeric disulfide bonds
16
(Figs. 1 and 2).
HPV16 L1 comprises 505 amino acids (aa), including 12 cysteines.
The nuclear extracts from sf 9 cells expressing the HPV16 L1 mutants
have been examined for the presence of intermolecular bonding by
SDS-PAGE with or without a reducing agent. Wild-type (WT) capsid
is separated into two bands of L1-trimers and L1-dimers in the non-
reducing SDS gel. C175S (substitution for cysteine at aa175) and
C185S do not form the dimer and C428S does not form the trimer
or the dimer.
Figure 2 shows structures formed by the mutants (photos taken
under a transmission electron microscope). WT forms typical L1-capsids
with a diameter of 55 nm. C175S forms mainly heterogeneous rod-
shaped tubular structures. C185S forms capsid-like particles mostly
smaller than WT, with diameters of 40 to 55 nm. C428S contains only
capsomeres, indicating that lack of trimerization and dimerization
does not affect capsomere formation. The three cysteines are thought
to be localized in the loops displayed on the surface by the X-ray
crystallographic analysis of the capsid-like structures composed of
truncated L1 of HPV16.
17,18
Although it is not clear which pairs of cysteines would make the
intercapsomeric disulfide bonds for correct assembly of capsids, C428
may participate in intercapsomeric bonding of all capsomeres, and
C175 and C185 may be involved in bonding the complex of three and
two capsomeres, respectively. Two types of junctions of capsomeres
