HUMAN PAPILLOMAVIRUSES 
Laimonis a. Laimins, Ph.D., Assistant Investigator 
Dr. Laimins is studying the molecular biology of 
human papillomaviruses (HPVs). Areas of interest 
include 1) transformation of human epithelial cells, 
2) structure and function of viral gene products, 3) 
regulation of viral gene expression, and 4) trans- 
genic mouse models for HPV-induced malignancy. 
Papillomaviruses are small DNA viruses that are 
responsible for a wide range of benign tumors in 
humans and other animal species. Subtypes of the 
HPVs are the presumed etiological agents of carci- 
nomas of the cervix and penis. HPV types 16 and 18 
are found in more than 90% of all cervical tumors, 
suggesting that papillomaviruses may play a caus- 
ative role in the development of these malignan- 
cies. In the majority of malignancies, some or all 
copies of the HPV genomes are found integrated 
into the host chromosome, while in benign lesions 
the virus remains episomal. In the integrated state 
only a subset of all viral gene products is expressed. 
One model for HPV-induced malignancy proposes 
that integration is an activation event for transfor- 
mation. 
I. Transformation and Transforming Proteins. 
Dr. Laimins and his colleagues are using primary 
human keratinocytes to study HPV transformation 
in vitro. Previous studies in rodent fibroblasts have 
shown that both the E6 and E7 gene products have 
independent transformation activities, but only 
one gene is necessary for the transformation of 
immortalized rodent fibroblasts. Studies in human 
keratinocytes, the natural host cells for HPV infec- 
tion in vivo, have demonstrated that both E6 and 
E7 are required for high-frequency transformation 
of keratinocytes. The ability to immortalize and 
alter differentiation in vitro has been used as a cri- 
terion for transformation. 
An in vitro system for the differentiation of epi- 
thelial cells involving growth of cells on collagen at 
an air-to-liquid interface has been used by Dr. 
Laimins and his colleagues to assay for altered dif- 
ferentiation in vitro. This system has been used to 
duplicate the histological changes seen in cervical 
cancers in vivo. A common characteristic of cervical 
tumors is a lack of epithelial differentiation, and 
cancers can be thought of as composed of cells that 
do not undergo terminal differentiation. The E7 
gene product alone can, at a low frequency, immor- 
talize keratinocytes but has little effect on differen- 
tiation. High-frequency transformation, as indicated 
by an altered pattern of differentiation, appears to 
require the synergistic action of both the E6 and E7 
gene products. 
Recombinant baculoviruses have been used to 
synthesize large quantities of the E6 and E7 open 
reading frames in insect cells in order to under- 
stand the molecular mechanisms by which HPV E6 
and E7 gene products act to transform cells. HPV- 
18 E6 protein synthesized by recombinant bac- 
uloviruses has been localized to the nucleus of 
infected cells and appears to be a DNA-binding pro- 
tein. In addition, Dr. Laimins and his colleagues 
have determined that E6 binds zinc and is thus a 
member of the zinc finger family of proteins. How- 
ever, E6 is an unusual zinc finger protein, as it has 
30 amino acid residues separating finger motifs, as 
opposed to the usual 15 residues. It also has been 
shown that the stability of the E6 protein is deter- 
mined, at least in part, by its amino-terminal amino 
acids. 
II. Regulation of Viral Gene Expression. 
Dr. Laimins and his colleagues are characterizing 
the cis and trans elements responsible for regula- 
tion of HPV expression. Three viral enhancer ele- 
ments have been identified, and two were found to 
be responsive to papillomavirus trans-acting fac- 
tors. The third element functions as a constitutive 
enhancer and requires only cellular factors for 
function. The first enhancer (IE2) is responsive to a 
papillomavirus E2 protein, while the second en- 
hancer (IE6) is responsive to the E6 gene product. 
The constitutive enhancer functions in a wide vari- 
ety of cell types but exhibits a cell-type preference 
for epithelial cells. The cellular factors involved in 
the action of this enhancer are being characterized. 
The IE2 enhancer is probably the principal en- 
hancer active in benign lesions, where the virus ex- 
ists as an episome. In the integrated state, E2 ex- 
pression is disrupted and the constitutive enhancer 
becomes most active. This change in transcriptional 
patterns may play an important role in the progres- 
sion of the lesion to a malignant state. The E2 pro- 
tein has been overproduced in baculovirus-infected 
cells, and the biochemistry of this protein is being 
studied in in vitro systems. In additional studies, 
the laboratory has shown that viral transactivators 
from herpes simplex virus (HSV) may also activate 
Continued 
235 
