The Interaction between Human Papillomavirus Type 16 El and E2 Proteins is Blocked by An Antibody to the N‐Terminal Region of E2

Abstract

El could be blocked by a monoclonal antibody that bound E2 in the region of amino acids 18-41 of E2 whereas a monoclonal antibody reactive with a nearby part of the molecule (amino acids 2-17) only partially blocked this interaction. These results suggest that a region in the N-terminus of E2 around amino acids 18-41 is a site of interaction with the E l protein.Keywords. Papillomavirus; HPV16; E l ; E2; protein interactions.The papillomaviruses are a group of over 70 types of doublestranded DNA viruses that can infect a variety of animal species. Viral infection is associated with the formation of benign epithelial tumours and, depending on the viral type, has been linked with malignant carcinoma. Human papillomavirus type 16 (HPV16) is a high risk type that is frequently associated with progression to malignancy in the human cervix (zur Hausen and Schneider, 1987).A number of early open reading frames have been defined for HPV16. The gene product of the E2 open reading frame is a 365-amino-acid protein, divided into three functional domains. The N-terminal trans-acting domain is separated from a C-terminal DNA-binding domain by a proline-rich hinge (Gin and Yaniv, 1988).The E2 protein has an important regulatory function as a trans-acting protein in the control of papillomavirus P,, promoter. This promoter regulates expression of HPV16 E6 and E7 proteins, which are involved in cellular transformation (Munger et al., 1992). Although HPV16 E2 can trans-activate heterologous promoters such as the simian virus 40 (SV40) early promoter, it has a trans-repressing effect on the HPV16 P , promoter. This promoter region contains several sites for cellular trans-activators such as Spl and AP1 and is activated in the absence of E2 (Cripe et al., 1990). The repressive effect of E2 on the P,, promoter is likely to be due, at least in part, to E2 DNA binding causing displacement of other transcription factors sucfi as Spl and TFIID from the promoter region (Tan et al., 1992(Tan et al., , 1994.The replicative function of the E2 gene product has been extensively studied in bovine papillomavirus type 1 (BPVl), as DNA from this type can become established as a stable, replicated plasmid in murine and bovine fibroblasts (Lambert, 1991). The ability of viral DNA to replicate in this system is dependent on the presence of the gene products of the viral E l and E2 open reading frames (OW) in association with other cellular factors (DiMaio and Settleman, 1988). Similarly, replication of HPVll DNA in a squamous carcinoma cell line requires the presence of both E l and E2. Using the same system, it has been possible to replicate the origin of HPV16 but only in the presence of HPVll E l and HPV16 E2 expressed from strong heterologous promoters (del Vecchio et al., 1992).The nuclear-localised early protein E l shares some biochemical characteristics with SV40 large T antigen, including helicase and ATPase activities (Clertant and Seif, 1984;Sun et al., 1990). The SV40 large T antigen is the only viral protein required for SV40 replicati...

Expression of human papillomavirus 16 E2 protein in Schizosaccharomyces pombe delays the initiation of mitosis

“…SDS ± PAGE and Western blots were performed on Protan nitro-cellulose (Schleicher and Schuell) as described by Harlow and Lane (1988). E2 was detected with the mouse monoclonal antibody TVG261 (Hibma et al, 1995). Secondary antibodies were HRP-conjugated antimouse or anti-rabbit and detected with ECL.…”

Regulation of human papillomavirus type 16 DNA replication by E2, glucocorticoid hormone and epidermal growth factor.

“…Protein concentrations were determined using the Bio-Rad protein assay and equal amounts were run on PAGE and transferred to a nitrocellulose membrane. HPV-16 E2 protein was detected using a pool of the anti-E2 monoclonal antibodies TVG 261 and TVG 271 (Hibma et al, 1995), followed by incubation with rabbit anti-mouse biotin conjugate (DAKO) and avidin peroxidase HPV DNA replication HPV DNA replication conjugate (DAKO). Blots were developed using the Amersham ECL system according to the manufacturer's instructions.…”

“…The full-length E2 protein is able to bind to the E1 protein through its N terminus and recent studies have identified two widely separate regions of the E2 protein which are essential for this binding Piccini et al, 1995 ;Hibma et al, 1995). To date, one detailed analysis of regions of the HPV-16 E2 protein important for viral DNA replication has been reported (Sakai et al, 1996).…”

The ATP-binding and ATPase activities of human papillomavirus type 16 E1 are significantly weakened by the absence of prolines in its ATP-binding domain

“…E. eoli TB1 cells bearing these recombinant vectors, pGEXElwt and pGEXE1Pro, were cultured in 1 M-sorbitol + 2.5 mM-betaine in LB medium and induced with 0.3 mM-IPTG when the optical densities of the cultures at 600 nm were 0.8-1-2, to express GSTElwt and GSTE1Pro respectively. The inductions were carried out at room temperature for 16 h. Bacteria were harvested and the fusion proteins purified according to the method described by Hibma et al (1995). Essentially, the bacterial pellets were resuspended in 20 ml of lysis buffer (50 mM-Tris-HC1, 10 mM-2-mercaptoethanol, 100 mM-NaC1, 1 mM-EDTA, 20 mM-MgC12, 1 mM-azide and 10% glycerol), freeze-thawed and sonicated for three rounds of 30 s each.…”

“…Interaction with E2-binding assays were performed as described previously (Hibma et al, 1995). Essentially, GST, GSTElwt and GSTE1Pro immobilized on glutathione Sepharose resin were incubated with bacterially expressed and purified native HPV-16 E2 protein (gift from M. Hibma, University of Otago, New Zealand) in binding buffer (20 mN-Tris-HC1 pH 8, 70 mM-KC1, 2mM-MgC12, 20 gM-zinc acetate, 0.2 mM-DTT, 6% glycerol, 0.1% Tween 20, 2% BSA, 2 lag calf thymus DNA) for 1 h at 4 °C.…”