Biology Reference
In-Depth Information
uloviruses in insect sf 9 cells. Subsequently, HPV16, HPV11, and
HPV33 were shown to yield self-assembled L1/L2-capsids in the sim-
ilar way. 9,10 These particles can be easily purified by a CsCl-equilibrium
density-gradient centrifugation. They are morphologically indistin-
guishable, under an electron microscope, from the authentic HPV
virions extracted from the lesions. 6,11
Recently, Leder et al . 12 introduced another method to produce
HPV16 L1/L2-capsids, using an expression plasmid with codon-
modified L1 and L2 genes. The modification, which entails certain
nucleotide substitutions without changing the amino acid sequences
in the two proteins, greatly enhanced the expression levels of L1 and
L2 in human 293T cells transfected with the expression plasmid.
Similarly, L1 and L2 of HPV6, 18, 31, and 52 were found to be effi-
ciently expressed and to yield L1/L2-capsids in the cells transfected
with expression plasmids having the modified L1 and L2 genes (our
unpublished data).
It turned out that HPV L1 and L2 mRNAs transcribed from the
unmodified genes are extremely unstable in mammalian cells trans-
fected with expression plasmids. Collier et al . 13 and Oeben et al . 14
mapped the cis elements that cause the mRNA instability to the 5
500-
nucleotide regions of L1 and L2 open reading frames. It is likely that
the L1 and L2 mRNAs transcribed by RNA polymerase II in the nuclei
are degraded by an unidentified cellular function, which presumably
accounts for the lack of HPV capsid production in the cells transfected
with the authentic capsid genes. In the vaccinia vector system, L1 and
L2 mRNAs are not degraded, probably because they are transcribed by
the viral RNA polymerase in the cytoplasm, and insect cells lack the
putative degradation function. The instability of the mRNAs and the
resulting suppression of capsid protein expression appear to be related
to the lifecycle of HPV, because L1 and L2 are expressed from the
authentic genes in differentiating keratinocytes (our unpublished data).
Disulfide Bonds Required for Capsid Assembly
McCarthy et al . 15 showed that reducing agents cause disassembly of
HPV11 L1-capsids to the level of the capsomeres, indicating that the
capsid assembly is dependent on intercapsomeric disulfide bonds.
Search WWH ::




Custom Search