Biology Reference
In-Depth Information
COS-1 and 293T. The transfection of the cells with the L1 and L2
expression plasmids together with a reporter plasmid with the SV40
replication origin (pSV plasmid) produces the L1/L2-capsids contain-
ing reporter DNA replicating in the nuclei of the cells. When secreting
alkaline phosphatase is used as a reporter, the resulting pseudovirions
provide a highly sensitive neutralization assay method for serum anti-
bodies against HPV16 L1.
In the above-mentioned pseudovirion production, L2 is required
for efficient encapsidation of DNA.
22,23
The other viral nonstructural
proteins including E2 are not required to enhance the production of
the pseudovirions.
23
The specific DNA sequences required for pack-
aging, such as packaging signals, have not yet been found.
We developed a method for encapsidating plasmid DNA into
HPV16 L1/L2-capsids in a cell-free system.
24
The L1/L2-capsids
that have been disassembled to the level of capsomeres with reducing
agent 2-mercaptoethanol (2-ME) (5%) are mixed with purified pSV2
plasmid DNA expressing a reporter gene, and then 2-ME is removed
by dialysis to allow the capsomeres to reassemble into L1/L2-capsids.
Some of the reassembled capsids package the plasmid to form pseudoviri-
ons. The pseudovirions produced by reassembling in the cell-free system
are morphologically similar to the original L1/L2-capsids.
Infection with the pseudovirions can be monitored by the expres-
sion of the reporter gene. The pseudovirions provide tools to examine
antibodies for their capability to inhibit HPV infection and to study
early events in the infection, such as cell binding and internalization.
However, when cells are infected with the pseudovirions at a low mul-
tiplicity of infection (MOI), the expression is usually difficult to
detect. The autonomous replication of the pSV2 reporter plasmid
mediated by SV40 T-antigen in particular cells, such as COS-1 and
293T, is required for efficient detection of the infected cells.
Interaction of HPV Capsids with Cell Surface
Cellular Attachment Receptor for HPV
Viral infection is supposed to start from the attachment of virions to
the cell surface. The adsorption of HPV appears to occur from the
