Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZP HPV -1 and AZP HPV -2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZP HPV -2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZP HPV -2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.The papillomaviruses are double-stranded DNA viruses that induce benign proliferative squamous epithelial and fibroepithelial lesions (warts and papillomas) in their natural hosts. They have been isolated from a variety of animal species, and Ͼ100 human papillomavirus (HPV) types have been identified and fully sequenced so far (reviewed in reference 13). A subgroup of HPVs classified as "high-risk" viruses, including HPV types 16,18,31, 35, 39, 45, 51, 52, 58, and 59, has been found to be associated with the development of cervical cancer (1, 28). Each year, about 500,000 such infections at the uterine cervix undergo malignant conversion, making cervical cancer the second most common malignancy in women worldwide (17). About 90% of such tumors contain high-risk HPVs, with HPV-16 and -18 being the most prevalent. The incidence shows no evidence of declining, and current treatment options are limited (http://www.boehringer-ingelheim.ca/research/res _area_humpap.asp). Therefore, effective antiviral therapies/ treatments for this widespread and troublesome disease are clearly needed.The papillomavirus proteins required for viral DNA replication are the viral E1 and E2 proteins (reviewed in reference 9). The E1 protein is a 70-to 80-kDa nuclear phosphoprotein possessing DNA helicase activity (reviewed in reference 29). Sequence-specific binding of E1 to the viral origin of replication is most likely mediated by the papillomavirus E2 protein (2, 6, 26, 30, 31); E2 bound to the origin recruits E1 to the origin, which results in initiation of the replication process. The 43-to 50-kDa E2 protein comprises two well-conserved functional domains linked by a hinge domain (reviewed in reference 14). The amino-terminal domain of E2 is necessary for direct association with the E1 protein. The carboxyl-terminal portion of E2 binds a 12-base-pair palindromic DNA...