BIOLOGY OF HUMAN PAPILLOMAVIRUSES 
Laimonis A. Laimins, Ph.D., Assistant Investigator 
Dr. Laimins and his colleagues are studying the 
biology of human papillomaviruses (HPVs) . Areas of 
interest include 1) transformation of human epithe- 
lial cells, 2) regulation of viral gene expression, and 
3) propagation of HPV in culture. 
Papillomaviruses are small DNA viruses that infect 
epithelial cells and induce hyperplasias in most 
mammals, including humans. More than 60 differ- 
ent types of HPVs have been identified to date, and 
one-third of these are specific for genital epithe- 
lium. A subset of these viruses (notably types 16, 
1 8 , and 31) are the etiological agents of cervical and 
other anogenital cancers. Papillomavirus infection 
occurs through microwounds of the epithelium, 
which allow entry into basal cells, where viral ge- 
nomes are established as episomes. The full life cy- 
cle of HPV requires epithelial stratification, since 
amplification of viral genomes and capsid synthesis 
occur only in the terminally differentiated cells of 
the upper epithelial layers. This tight coupling of 
the viral life cycle to differentiation and the inability 
to duplicate these features in tissue culture may ex- 
plain why these viruses have not previously been 
propagated in culture. Study of HPVs is also compli- 
cated by the fact that only small amounts of virus can 
be obtained from patients. Most studies of HPVs 
have therefore involved the use of cloned viral ge- 
nomes isolated from biopsies of cervical cancers. 
Transformation 
The mechanisms by which the oncogenic HPVs 
immortalize human keratinocytes and alter their dif- 
ferentiation capabilities are being examined in vi- 
tro. Using human foreskin and cervical keratino- 
cytes, the laboratory has demonstrated that two 
early gene products, E6 and E7, are necessary for 
high-frequency transformation in vitro. These 
genes are specifically retained and expressed in 
high-grade neoplasias and cancers in vivo. Although 
the E7 gene product can by itself extend the life 
span of transfected cells, the presence of the E6 pro- 
tein greatly increases the rate of immortalization. 
The E6 and E7 gene products also alter the differen- 
tiation properties of epithelial cells. In an in vitro 
system for epithelial differentiation, referred to as 
the raft system, epithelial cell lines transfected with 
HPV- 1 8 E6 and E7 were observed to exhibit morpho- 
logical changes similar to those seen in cervical 
neoplasias in vivo. Stratified rafts of cell lines that 
express the E7 gene product alone exhibit minimal 
changes in differentiation and resemble histological 
cross sections of biopsies of normal human keratino- 
cytes. Only with the addition of the E6 protein is 
epithelial differentiation dramatically altered in the 
raft system. 
The E7 gene product transforms cells through its 
interaction with the cell cycle regulatory protein, 
retinoblastoma. Dr. Laimins and his colleagues have 
demonstrated that the E7 protein exists as a multi- 
meric complex that is coordinated by the presence 
of zinc atoms. Similarly, the E6 protein interacts 
with the cellular p53 protein. The laboratory has 
demonstrated that the binding of E6 protein de- 
creases the half-life of p53 by two- to threefold in 
vivo. Both cell cycle regulatory proteins, retinoblas- 
toma and p53, have been implicated in transcrip- 
tional control, suggesting a mechanism by which 
their normal function is altered through the binding 
of E7 and E6. Studies in this laboratory have demon- 
strated that p53 specifically represses TATA- 
containing promoters but does not affect initiator- 
mediated transcription. The E6 protein was found to 
abrogate this repression. 
Regulation of Viral Gene Expression 
Dr. Laimins and his colleagues have identified the 
cis sequences and trans factors that are important for 
the tissue-specific expression exhibited by HPVs. An 
HPV region of ~ 1 kb in length — the upstream regu- 
latory region (URR) — has been found to contain sev- 
eral enhancer elements. One of these enhancers, the 
C enhancer, depends solely on cellular factors for 
activity and is a major determinant of the host range 
of HPV infection. This enhancer is responsible for 
the initial activation of viral expression following 
infection and is the major regulator of HPV tran- 
scription in cervical cancers. The binding of the 
ubiquitous AP- 1 factor together with a novel kera- 
tinocyte factor, KRF- 1 , are necessary for C enhancer 
function. Furthermore, it has been observed that oc- 
tamer proteins bind to sequences that overlap the 
KRF- 1 -binding site and act as repressors of HPV 
expression. The interaction of these factors in a 
stratified epithelium activates viral expression in a 
differentiation-specific manner. 
A second enhancer in the URR is responsive to the 
virally encoded transcriptional activator E2. In low- 
grade lesions where viral genomes are maintained as 
episomes, E2 is the major regulator of viral expres- 
sion and is thought to act as a repressor of E6/E7 
transcription. In contrast, in high-grade neoplasias 
and cancers, viral sequences are frequently found 
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