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J Virol, February 1998, p. 1071-1077, Vol. 72, No. 2
Department of Microbiology-Immunology,
Northwestern University Medical School, Chicago, Illinois 60611
Received 23 June 1997/Accepted 27 October 1997
Human papillomavirus (HPV) E2 proteins regulate viral replication
by binding to sites in the upstream regulatory region (URR) and by
complex formation with the E1 origin recognition protein. In the
genital HPV types, the distribution and location of four E2 binding
sites (BS1 to BS4) which flank a single E1 binding site are highly
conserved. We have examined the roles of these four E2 sites in the
viral life cycle of HPV type 31 (HPV31) by using recently developed
methods for the biosynthesis of papillomaviruses from transfected DNA
templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA
93:3062-3067, 1996). In transient assays, no single site was found to
be necessary for replication, and mutation of the early
promoter-proximal site (BS4) led to a fourfold increase in replication.
Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with
expression vectors revealed that E1 stimulated replication of HPV31-wt
as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast,
increased expression of E2 decreased replication of these genomes.
Replication of the HPV31-BS4 mutant genome was not further increased by
cotransfection of E1 expression vectors but was stimulated by E2
coexpression. In stably transfected normal human keratinocytes,
mutation of either BS1, BS3, or BS4 resulted in integration of viral
genomes into host chromosomes. In contrast, mutation of BS2 had no
effect on stable maintenance of episomes or copy number. Following
growth of stably transfected lines in organotypic raft cultures, the
differentiation-dependent induction of late gene expression and
amplification of viral DNA of the BS2 mutant was found to be similar to
that of HPV31-wt. We were unable to find a role for BS2 in our assays
for viral functions. We conclude that at least three of the four E2
binding sites in the URRs of HPVs are essential for the productive
viral life cycle. The specific arrangement of E2 binding sites within
the URR appears to be more important for viral replication than merely
the number of sites.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Differential Requirements for Conserved E2 Binding
Sites in the Life Cycle of Oncogenic Human Papillomavirus Type
31
and
*
Corresponding author. Mailing address: Department of
Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0648. Fax: (312)
503-1339. E-mail: LAL{at}merle.acns.nwu.edu.
Present address: Abbott Laboratories, Abbott Park, IL 60064.
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