The Upstream Regulatory Region of Human Papillomavirus Type 31 Is Insensitive to Glucocorticoid Induction

Abstract

The upstream regulatory region (URR) of various types of human papillomaviruses (HPVs) has been shown to contain functional glucocorticoid response elements (GREs), including HPV type 11 (HPV11), HPV16, and HPV18. Glucocorticoids have been demonstrated to induce the transcriptional activity of the early promoters of these HPV types. Although it has been assumed that the URR of HPV31 contains at least one GRE, no functionality has been demonstrated. We attempt to show here inducibility of the URR of HPV31 by the synthetic glucocorticoid dexamethasone (dex). By sequence analysis we identified three potential GREs in the URR of HPV31. Gel shift analysis indicated that each of these three sites has the potential to be a functional GRE. However, constructs containing the full-length URR, 5 deletions of the URR, and an internal fragment of the URR containing all three putative GREs were only weakly inducible by dex. Linker scanning mutants, whereby each potential GRE was replaced individually, in double combination, or in triple combination by a unique polylinker, had no effect on dex inducibility. Replacement of each of the three HPV31 GREs with the GRE of HPV18 failed to induce a response to dex. Placement of the HPV18 GRE into the URR of HPV31 in a region similar to its location in the HPV18 URR was also unable to result in a strong dex induction of the HPV31 URR. These data suggest that the lack of dex inducibility is due to the overall context of the HPV31 URR and may be dependent on the requirements of the major early promoter for transcriptional activation. Human papillomaviruses (HPVs) are small, doublestranded DNA viruses that specifically infect mucosal and cutaneous epithelium. Over 100 types of HPV have been identified, with roughly one-third infecting the genital epithelium. With the identification of HPV as the etiologic agent of cervical cancer (74, 76), HPV types that infect the genital epithelium are separated based on their associated risk. High-risk types, most commonly HPV type 16 (HPV16) and HPV18, are frequently found in high-grade cervical lesions and invasive cancer. Intermediate-risk types, including HPV31, HPV33, and HPV35, are associated with all grades of cervical lesions and are often associated with invasive cancer. Low-risk HPV types, most frequently HPV6 and HPV11, are commonly associated with low-grade cervical lesions and are rarely found in invasive cancer (35).The genomes of all of the genital HPV types are approximately 8 kb in length and are similarly organized into early, late, and noncoding regions. The noncoding region, or upstream regulatory region (URR), contains cis-enhancer elements to many cellular and viral factors. Binding of these factors to the URR modulates both viral replication and viral gene transcription (33). Binding sites have been identified for the viral proteins E1 and E2 (39,56,66,68), as well as for the cellular factors AP1 (12,25,30,34), Oct-1 (25), YY1 (30), Sp1 (25), NF1 (21), TEF-1 (30), and CDP/Cut (48, 55), among others.Steroid hormones have been sugg...

Induction of the Upstream Regulatory Region of Human Papillomavirus Type 31 by Dexamethasone Is Differentiation Dependent

“…Bromberg-White and Meyers have previously shown that the URR of HPV31 is not inducible by the synthetic glucocorticoid dexamethasone (dex) in monolayer culture (10). In the present study, we demonstrate that suspension of the HPV31b-positive 9E cell line in the semisolid medium methylcellulose results in a dex-inducible activation of a luciferase promoter construct driven by the URR of HPV31 that was not seen in monolayer culture.…”

“…The full-length URR of HPV31 or HPV18 was cloned into the pGL2-Basic luciferase reporter vector as described previously (10,40). All other constructs were created as described previously (10,40).…”

“…HFK cells were maintained in KGM-2 (BioWhittaker, Walkersville, Md.). Deficient E medium was used to culture 9E cells for transfection experiments as described previously (10).…”

“…Advances in the organotypic (raft) culture system have allowed for the growth of differentiating epithelium in vitro and have made it possible to provide all of the factors necessary for the complete viral life cycle (10,32,33,35). However, preparation of raft cultures can be time consuming, requiring up to 12 days in culture prior to analysis of late viral events, which can limit the rapid analysis of multiple constructs.…”

Research Article Synergic effect of oral contraceptives, GSTP1 polymorphisms, and high-risk HPV infection in development of cervical lesions.

“…Later, mutational analysis has demonstrated that inactivation of this GRE within the promoter-proximal portion of the HPV-18 LCR abolished the dexamethasone response of the E6/E7 promoter (Butz and Hoppe-Seyler, 1993). Some additional progesterone/GREs have also been found within HPV-11, HPV-16, and HPV-31 LCR (Chen et al, 1996;Bromberg-White and Meyers, 2002). So, if a significant variation in estrogen/progesterone metabolism might be a consequence of GST polymorphism-and estrogen-upregulated HPV E6/E7 oncogene expression, cancer risk would also be altered in HPV-positive patients who use oral contraceptive methods and present some of the GST described polymorphisms.…”