Münger K

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105‐RB). Similar to the E7 protein of HPV‐16, the E7 proteins of HPV‐18, HBV‐6b and HPV‐11 were found to associate with p105‐RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV‐16 and HPV‐18) formed complexes with p105‐RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105‐RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV‐1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105‐RB. The amino acid sequences of the HPV‐16 E7 protein involved in complex formation with p105‐RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105‐RB. Furthermore, the HPV‐16 E7‐p105‐RB complex was detected in an HPV‐16‐transformed human keratinocyte cell line.

Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses

More than 120 human papillomaviruses (HPVs) have now been identified and have been associated with a variety of clinical lesions. To understand the molecular differences among these viruses that result in lesions with distinct pathologies, we have begun a MS-based proteomic analysis of HPV–host cellular protein interactions and have created the plasmid and cell line libraries required for these studies. To validate our system, we have characterized the host cellular proteins that bind to the E7 proteins expressed from 17 different HPV types. These studies reveal a number of interactions, some of which are conserved across HPV types and others that are unique to a single HPV species or HPV genus. Binding of E7 to UBR4/p600 is conserved across all virus types, whereas the cellular protein ENC1 binds specifically to the E7s from HPV18 and HPV45, both members of genus alpha, species 7. We identify a specific interaction of HPV16 E7 with ZER1, a substrate specificity factor for a cullin 2 (CUL2)-RING ubiquitin ligase, and show that ZER1 is required for the binding of HPV16 E7 to CUL2. We further show that ZER1 is required for the destabilization of the retinoblastoma tumor suppressor RB1 in HPV16 E7-expressing cells and propose that a CUL2–ZER1 complex functions to target RB1 for degradation in HPV16 E7-expressing cells. These studies refine the current understanding of HPV E7 functions and establish a platform for the rapid identification of virus–host interactions.

Functions of human papillomavirus proteins

Cervical cancer is the second leading cause of deaths from cancer among women worldwide with approximately 500 000 deaths annually. Epidemiologic studies have implicated a sexually transmitted agent as a cause of cervical cancer, and molecular virology studies over the past 10 years have established a strong association between specific human papillomavirus (HPV) types and certain anogenital carcinomas, including cervical cancer (reviewed in zur Hausen and Schneider 1987). Over 65 different HPV types have now been described, and each is associated with a specific clinical entity (DeVilliers 1989). Approximately 20 or 25 HPVs have been associated with anogenital lesions; these HPVs have been further classified as either “low-risk” or “high-risk” types based on the preneoplastic character of the clinical lesions with which they are associated. Low-risk HPVs such as HPV-6 and HPV-11 are generally associated with venereal warts or condyloma acuminata which only rarely progress to malignancy. The high-risk HPVs include HPV-16 and HPV-18 and these are associated with squamous intraepithelial neoplasias which are potentially precancerous. In the cervix, they are associated with cervical intraepithelial neoplasia, or CIN. These CIN lesions are considered preneoplastic in that a small percentage of high-grade CIN lesions will progress to cervical cancer. Approximately 70% of human cervical cancers contain either HPV-16 or HPV-18 DNA (zur Hausen and Schneider 1987). Indeed, HPV-16 and HPV-18 DNA were originally isolated from human cervical carcinoma tissues (Durst et al. 1983; Boshart et al. 1984). Other high-risk HPVs, including types 31, 33, 35, 39, 45, 51, and 52, have subsequently been identified and have also been associated with CIN lesions and with invasive cervical carcinomas. All together, approximately 85% of cervical cancers can be shown to contain DNA of one of the high-risk HPV types (Riou et al. 1990).

Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins.

The E6 and the E7 proteins of the oncogenic human papillomavirus types 16 and 18 can stably associate with p53 and the retinoblastoma protein, respectively. The E6‐p53 interaction results in the accelerated degradation of p53 in vitro via the ubiquitin‐dependent proteolysis system. In this study we demonstrate that a fusion protein consisting of the N‐terminal half of the HPV‐16 E7 protein and the full length HPV‐16 E6 protein promotes the in vitro degradation of the retinoblastoma protein. This indicates that the property of the HPV‐16 E6 protein to stimulate the degradation of p53 can be targeted to other proteins. Unlike the HPV‐16 or HPV‐18 E6 protein, the E6 proteins of HPV‐6 and 11 do not bind to p53 and consequently do not target p53 for degradation. Analogous E7‐E6 fusion proteins using the E6 proteins of HPV‐6 and HPV‐11, however, also have the ability to promote the degradation of the retinoblastoma protein, indicating that the property to target associated proteins for degradation is shared by the anogenital specific HPV E6 proteins.

c-myc and pRB: Role in TGF-β1 Inhibition of Keratinocyte Proliferation

The TGF-beta s are potent inhibitors of proliferation of most cell types in culture and in vivo. Previous studies have demonstrated that TGF-beta inhibition of skin keratinocyte proliferation involves suppression of c-myc transcription. Evidence derived from use of expression plasmids for certain DNA viral oncoproteins has suggested that the retinoblastoma gene (RB) may be involved in this process. Transient expression of pRB, like TGF-beta 1, in skin keratinocytes represses expression of a human c-myc reporter plasmid, and the same c-myc promoter region (TCE) is required for repression by either TGF-beta 1 or pRB. We showed here that proliferation and c-myc expression in a cell line lacking normal pRB (DU145 human prostate adenocarcinoma cells) are not inhibited by TGF-beta 1. Oligonucleotides containing the TCE were found to bind to a cellular protein of approximately 106 kD (termed p106) in Southwestern assays, utilizing extracts from both the skin keratinocytes and DU145 cells. TCE binding to p106 was diminished by TGF-beta in TGF-beta-sensitive skin keratinocytes but not in TGF-beta-insensitive SV40-transformed keratinocytes. These data support the hypothesis that pRB is required for TGF-beta 1 suppression of c-myc transcription and suggest the involvement of a cellular factor(s) in addition to pRB in the TGF-beta 1 pathway for inhibition of c-myc transcription and growth inhibition.

Functional and Sequence Similarities Between HPV16 E7 and Adenovirus E1A

Recent molecular and epidemiological data have established a strong association between certain human papillomaviruses (HPVs) and some types of human anogenital cancers. More than a dozen different HPV types have now been isolated from epithelial tumors of the genital region. In general, benign genital condyloma acuminata have been associated with the presence of HPV types 6 and 11, whe reas precancerous lesions such as moderate to severe cervical dysplasia and carcinoma in situ, as well as invasive cervical carcinoma, have been associated with HPV types 16, 18, 31, 33, and 35 (zur Hausen and Schneider 1987). Of the HPVs which have been associated with anogenital malignancies, HPV-16 has been detected most frequently (> 60%) in biopsies from cervical carcinoma.

Human Papillomavirus E6 and E7 Oncogenes

Likemost otherDNA tumor viruses, papillomaviruses encodemultiple oncogenes. In epithelial cells and transgenic mice, the E6 and E7 genes of the high-risk HPV display a number of growth promoting activities, including the ability to cooperate to immortalize primary cultured human keratinocytes. The E6 and E7 proteins affect cell growth by binding to and modulating the activity of cellular proteins involved in growth control, including p53, retinoblastoma family members, and PDZ domain-containing proteins. These same cellular proteins are also modulated by DNA tumor viruses of entirely different virus families, suggesting that there are relatively few intracellular growth regulatory pathways that viruses target to facilitate virus replication. In general the E6 and E7 proteins from low-risk HPV display reduced ability to perturb these cellular targets. Thus, these in vitro measuresof activity reflect theunderlyingbiologyofcervical carcinogenesis.Strikingly, repression of HPV E6 and E7 expression in cervical cancer cells inhibits the growth of these cells, validating the viral oncogenes as potential therapeutic targets in HPV-associated precancerous and cancerous lesions.